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Dysregulation of integrin-linked kinase (ILK) signaling during colorectal carcinogenesis : modulation… Marotta, Anthony 2002

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DYSREGULATION OF INTEGRIN-LINKED KINASE (ILK) SIGNALING DURING COLORECTAL CARCINOGENESIS: MODULATION OF SIGNALING PATHWAYS B Y NON-STEROIDAL ANTI- INFLAMMATORY DRUGS (NSAID) B Y : Anthony Marotta B.Sc , University of British Columbia, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Faculty, of Graduate Studies Department of Medicine We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A A p r i l 2002 © Anthony Marotta, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date Apr]'I 19* 2-OQ ABSTRACT One of the most common events involved in the development of human colon cancer is the mutation of the adenomatous polyposis coli (APC) gene. This protein through its interactions with Axin and GSK3p, serves to regulate the cytosolic levels of p-catenin. Mutation of the A P C gene, which impairs complex formation, results in the stabilization of p-catenin. Stabilization is believed to coincide with the translocation of p-catenin to the nucleus, where it up-regulates a number of genes implicated in oncogenesis. Interestingly, stable over-expression of the integrin-linked kinase (ILK) in rat intestinal epithelial cells has been demonstrated to modulate p-catenin sub-cellular localization and function. However, the significance of this finding in human colorectal carcinogenesis is unclear. To determine if ILK signaling was disrupted in colorectal carcinogenesis, this signaling pathway was characterized during various stages of development beginning with the earliest lesion, the adenomatous polyp. The results from these studies demonstrated that ILK was significantly overexpressed and exhibited an increased phosphotransferase activity in polyps resected from patients diagnosed with familial adenomatous polyposis. Changes in ILK activity reflected changes on downstream targets, predominantly GSK3p. In addition to this, dramatic increases in ILK immunoreactivity were observed in all abnormal crypts from sporadic polyps, when compared with the normal appearing crypts, within the same resected specimens. To delineate whether these changes in ILK signaling could be generalized for colon cancer, this signaling nexus was also i i investigated in both primary lesions as well as secondary deposits within regional lymph nodes. The results from these studies demonstrate that ILK was significantly hyperexpressed in malignant acini from either the primary or secondary site in relation to the normal crypts within the same lesion. Furthermore, over-expression of the ILK protein coincided with an increase in the M B P phosphotransferase activity of the immunoprecipitated ILK in colon cancer in approximately 63% of the primary lesions examined. In addition to this, the data indicated that there was a direct correlation between the protein expression of ILK and the protein levels of Lef-1 in the cases of colon cancer that were analyzed. As aspirin and sulindac have been demonstrated to elicit chemopreventative effects in colon cancer, I tested whether non-steroidal anti-inflammatory agents targeted the ILK signaling nexus in vivo. Both of these drugs inhibited the serum-induced activation of ILK and PKB, modulated serine-9 phosphorylation on GSK3p, and down-regulated Tcf-4 transcriptional activity. In addition to this, sulilndac was shown to also inhibit another protein kinase that is known to influence p-catenin, protein kinase CK2. Furthermore, the data demonstrated that over-expression of ILK, PKB or CK2 in a cell culture system, inhibited NSAID mediated apoptosis. In conclusion, dysregulation of the ILK signaling nexus appears to be an early event during the development of colon cancer and it is possible that selective inhibition of this kinase might be an important chemopreventative/chemotherapeutic strategy in the colon. i i i TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables xi List of Figures xii List of Abbreviations xv Dedication xviii Acknowledgements xix Chapter 1. General Introduction 1.1. Cancer 1 1.2. Colon Cancer 1 1.2.1. Epidemiology 2 1.2.2. Pathogenesis 7 1.2.3. Clinical Features 12 1.2.4. Familial Syndromes 12 1.2.5. Colorectal Carcinogenesis 14 1.2.5.1. Wnt/Wingless Signal Transduction 14 Cascade 1.2.5.2. DNA Mismatch Repair Genes (MMR) 19 1.2.5.3. DNA Methylation 20 1.2.6. Molecular Carcinogenesis of Colorectal 20 Cancer 1.2.6.1. The Adenomatous Polyposis Coli 21 (APC) Gene 1.2.6.1.1. p-catenin 27 1.2.6.1.2. Axin 32 1.2.6.1.3. Glycogen synthase kinase (GSK)- 33 3p iv 1.2.6.2. The Monomeric G protein, Ras 36 1.2.6.2.1. Overview of Ras/Mapk Signaling „ 36 Cascade 1.2.6.2.2. Ras-lts Relevance to Cancer 43 1.2.6.2.3. Ras and Lipid Signaling 45 1.2.6.3. The p53 protein 45 1.2.6.3.1. p53 and Cell Cycle Arrest 48 1.2.6.3.2. p53 and Apoptosis 49 1.2.7. The PI-3 Kinase Signaling Nexus 49 1.2.7.1. Phosphatidylinositol (PI)-3 Kinase 51 1.2.7.1.1. Class I - Phosphatidylinositol (PI)- 52 3 Kinases 1.2.7.1.2. Class II - Phosphatidylinositol 53 (PI)-3 Kinases 1.2.7.1.3. Class III - Phosphatidylinositol 54 (PI)-3 Kinases 1.2.7.2. Phosphatidylinositol (PI)-3 Kinase and 54 Signaling 1.2.7.3. Phosphoinositide-Dependent Protein 58 Kinase (PDKj-1 1.2.7.4. Integrin-Linked Kinase (ILK) 59 1.2.7.4.1. ILK Genetics 60 1.2.7.4.2. Structure of ILK 61 1.2.7.4.3. ILK Function 65 1.2.7.5. Protein Kinase B (PKB) 69 1.2.7.5.1. PKB Genetics 70 1.2.7.5.2. PKB Structure 71 1.2.7.5.3. PKB Regulation 72 1.2.7.5.4. PKB - Downstream Targets 76 1.2.7.5.5. PKB and Cancer 78 v 1.2.7.6. Protein Tyrosine Phosphatase and 79 Tensin Homologue (PTEN) 1.2.8. Apoptosis 81 1.2.9. Protein Kinase CK2 82 1.2.10. Treatment Strategies for Colon Cancer 84 1.2.10.1. Mechanism of Action for NSAIDs 85 1.2.11. Rationale and Research Objectives 94 1.2.11.1. Rationale 94 1.2.11.2. Objectives 97 Chapter 2. Materials and Methods 2.1. General Materials 98 2.1.1. Chemical Reagents 98 2.1.2. Laboratory Supplies 101 2.1.3. Plasmids "V 102 2.1.4. Antibodies 102 2.1.5. Human Colonic Tissue 103 2.2. General Methods 104 2.2.1. Preparation of Human Tissue Samples 104 2.2.2. Evaluation of Protein Concentration 104 2.2.3. Electrophoresis and Western Blotting 105 2.2.3.1. SDS-Polyacrylamide Gel 105 Electrophoresis 2.2.3.2. Immunoblotting 106 2.2.3.3. Stripping Immunoblots for Reprobing 107 2.2.4. Immunoprecipitation 108 2.2.5. Immune Complex Kinase Assays 108 2.2.6. Mono Q Fractionation 109 2.2.7. Protein Kinase Assays 109 2.2.8. In vitro Kinase Assays 110 vi 2.2.9. Immunohistochemistry 110 2.2.10. Immunocytochemistry 111 2.2.11. Cell Culture 112 2.2.10.1. MTS Cell Viability Assay 112 2.2.10.2. Transfections 113 2.2.10.2.1. Tcf4 Reporter Assay 113 2.2.10.2.2. Over-expression Studies 113 2.2.11. Bacterial Transformations 114 2.2.11.1. Plasmid Preparation 114 2.2.11.2. DNA Yield 115 2.2.12. Treatment Protocols 115 2.2.12.1. Stimulation Studies 115 2.2.12.2. Enzymatic Activation - Inhibitor 115 Studies 2.2.12.3. Transcriptional Assays 116 2.2.12.4. Cell Viability Assays 116 2.2.12.5. Preparation of Inhibitors 116 2.3. Statistical Analysis 117 Chapter 3. Characterization of Signaling Modules within Polypoid Lesions. 3.1. General Introduction 118 3.1.1. Introduction to ILK 119 3.1.2. Investigation of the ILK Signaling Nexus in 120 FAP patients 3.1.3. Introduction to the Map kinase Signaling 132 Nexus 3.1.4. Investigation of Erk1 and Erk2 in FAP 133 Patients 3.1.5. Introduction to Protein Kinase CK2 136 vii 3.1.6. Investigation of Protein Kinase CK2 in patients with FAP 3.1.7. Investigation of the ILK Signaling Module in Sporadic Polyposis 3.2. Summary of Results 3.3. General Conclusions and Future Directions Chapter 4. Investigation of Signaling Modules in Colorectal Cancer 4.1. General Introduction 147 4.1.1. ILK - Clinical Perspectives 148 4.1.2. ILK Expression is Dysregulated in Sporadic 148 Cases of Colon Cancer 4.1.3. ILK Activity is Increased in Colonic Cancers 151 4.1.4. ILK Expression In Regional Lymph Nodes 163 4.2. Summary of Results 169 4.3. General Conclusions and Future Directions 169 Chapter 5. Modulation of Signaling Cascades by NSAIDs 5.1. General Introduction 171 5.2. ILK and NSAIDs 173 5.3. Effects of NSAIDs on Tcf4-Mediated Gene 182 Transcription 5.3.1. Specific Effects of NSAIDs onTcf4-Reporter 182 Activity 5.3.2. Effects of NSAIDs on the Expression of 186 Cyclin D1 137 141 145 145 v i i i 5.3.2.1. A S A Modulates Cyclin D1 Expression - Independent of Effects of A S A on Cell Viability 5.3.2.2. Sulindac Modulates Cyclin D1 Expression - Independent of Effects of Sulindac on Cell Viability 5.3.2.3. Metabolic Metabolites of Sulindac Inhibit Cyclin D1 Expression -Independent of Effects of These Agents on Cell Viability 5.3.2.4. Effects of NSAIDs on the Sub-cellular Distribution of p-catenin 4. PKB and NSAIDs 5.4.1. Sulindac Inhibits PKB activity 5.4.2. The Effects of NSAIDs on PKB Activity Correlate with a Decrease in Ser473 Phopshorylation 5. Introduction to protein kinase CK2 5.5.1. The Effects of NSAIDs on protein kinase CK2 6. NSAID Mediated Cell Death - Cox-2 Dependency 5.6.1. Differential Effects of NSAIDs on Cell Viability 5.6.1.1. Effects of A S A on Cell Viability 5.6.1.2. Effects of Sulindac on Cell Viability 5.6.1.3. Effects of Sulindac-Metabolites on Cell Viability 5.6.2. Sulindac Sulfide-Mediated Apoptosis 5.7. Effects of Protein Kinases on NSAID mediated 234 apoptosis 5.7.1. Over-expression of ILK modulates effects of 234 sulindac on cell survival 5.7.2. Over-expression of PKB modulates effects 239 of sulindac on cell survival 5.7.3. Over-expression of CK2 modulates effects of 244 sulindac on cell survival 5.8. Summary of Results 248 5.9. General Conclusions 249 Chapter 6. Conclusion 6.1. Summary of Results 250 6.2. Future Directions 252 Chapter 7. References 7.1. List of References 254 x LIST OF TABLES Table 1. The Effects of NSAIDs on in vitro ILK activity 174 xi LIST OF FIGURES Figure 1. The Annual Incidence Rates of Common Malignancies in 3 British Columbia. Figure 2. The Annual Mortality Rates of Common Malignancies in 5 British Columbia. Figure 3. The Chromosomal Instability Pathway 8 Figure 4. The Hypermutability Pathway 10 Figure 5. The Wnt Signaling Cascade 16 Figure 6. The Adenomatous Polyposis Coli (APC) Gene 23 Figure 7. The APC/p-catenin Signaling Nexus 30 Figure 8. The Ras/Mapk Signaling Cascade 41 Figure 9. The PI-3 kinase Signaling Pathway 55 Figure 10. Structure of the Integrin-Linked Kinase (ILK) 63 Figure 11. Mechanism of Action for Non-Steroidal Anti-Inflammatory 87 Drugs Figure 12. Increased ILK/MBP Phosphotransferase Activity in Polyps 122 from Patients Diagnosed with FAP. Figure 13. Increased MBP Phosphotransferase Activity Correlates 125 With Elevated Expression of the ILK Protein Figure 14. Effects of ILK on Downstream Targets in FAP 129 Figure 15. Examination of Erk1 and Erk2 Expression/Activity in FAP 134 Figure 16. Increased CK2 Activity in FAP Patients 138 xii Figure 17. Over-expression of ILK in Sporadic Polyps 142 Figure 18. Over-expression of ILK in Sporadic Colorectal Cancers 149 Figure 19. ILK Activity is Enhanced in Colorectal Cancers 153 Figure 20. Effects of ILK on Downstream Targets 158 Figure 21. Positive Correlation Between ILK Expression and the 161 Levels of Lef-1 in Colorectal Cancers. Figure 22. ILK Expression in Positive Lymph Nodes 165 Figure 23. ILK Expression is Significantly Increased in Colorectal 167 Cancers Figure 24. Inhibition of ILK Signaling with NSAID Administration 177 Figure 25. Modulation of ILK by NSAIDs is associated with changes 180 in the phosphorylation status of GSK30. Figure 26. NSAIDs Inhibit Tcf-4/Lef-1 Mediated Gene Transcription 184 Figure 27. Administration of A S A Leads to a Down-regulation of 188 Cyclin D1 - Independent of Effects of A S A on Cell Viability Figure 28. Administration of Sulindac Leads to a Down-regulation of 191 Cyclin D1 - Independent of Effects of Sulindac on Cell Viability Figure 29 . Administration of Sulindac Sulfide results in a Down- 195 regulation of Cyclin D1 - Independent of Effects of Sulfide on Cell Viability xii i Figure 30. Administration of Sulindac Sulfone results in a Down- 197 regulation of Cyclin D1 - Independent of Effects of Sulfone on Cell Viability Figure 31. The Effects of NSAIDs on the Sub-Cellular Distribuition of 200 p-catenin Figure 32. Sulindac inhibits PKB in vitro 205 Figure 33. Inhibition of PKB with NSAID Administration 208 Figure 34. Sulindac inhibits protein kinase CK2 in vitro 212 Figure 35. NSAIDs inhibit the biochemical activation of protein kinase 215 CK2 Figure 36. Effects of A S A on Colon Cancer Cell Viability: Influence of 219 Cox-2 Expression Figure 37. Effects of Sulindac on Colon Cancer Cell Viability: 222 Influence of Cox-2 Expression Figure 38. Effects of Sulindac Sulfide on Colon Cancer Cell Viability: 226 Influence of Cox-2 Expression Figure 39. Effects of Sulindac Sulfone on Colon Cancer Cell Viability: 228 Influence of Cox-2 Expression Figure 40. Effects of NSAIDs on Caspase Pathway 232 Figure 41. ILK suppresses effects of NSAIDS on cell viability 237 Figure 42. PKB suppresses effects of NSAIDS on cell viability 242 Figure 43. CK2 suppresses effects of NSAIDS on cell viability 246 xiv LIST OF ABBREVIATIONS Abbreviation Definition A P C Adenomatous polyposis coli A T P Adenosine Triphosphate C D K Cyclin dependent kinase CIN Chromosomal instability CK1 Casein kinase 1 CK2 Casein kinase 2 C O X Cyclooxygenase C R E B cAMP response binding element DNA Deoxyribonucleic acid DR5 Death domain E G F Epidermal growth factor E R K Extracellular regulated kinase FAP Familial adenomatous polyposis F G F Fibroblast growth factor G A P GTPase activating protein G D P Guanine diphosphate G N E F Guanine nucleotide exchange factor G S Glycogen synthase GSK3p Glycogen synthase kinase G T P Guanine triphosphate HBX Hepatitis B X protein H N P C C Hereditary non-polyposis colon cancer HPV Human papilloma virus IGF Insulin-like growth factor IGF-BP3 Insulin-like growth factor binding protein 3 IKK I Kappa kinase IL-1 p Interleukin 1 X V IRS Insulin receptor substrate kDa Kilodalton Lef Lymphocyte enhancer factor LOH Loss of heterozygosity MAPK Mitogen activated protein kinase MBP Myelin basic protein M C R Mutation cluster region MEK M A P K / E R K kinase MIN Microsatellite instability M M A C Mutated in multiple advanced cancers M M P Matrix metalloproteinase NSAID Non-steroidal anti-inflammatory drugs PAK p21 activated kinase P A R P Poly-ADP ribosylating protein P D G F Platelet derived growth factor PI-3K Phosphatidylinositol-3 kinase PIP3 Phosphatidylinositol phosphate PKB Protein kinase B P K C protein kinase C PLCy Phospholipase C gamma P M Plasma membrane PP2A Protein phosphatase 2A PTEN Phosphatase and tensin homologue mutated on chromosome 10 R A C Related to A and C kinase RB Retinoblastoma protein RTKs Receptor tyrosine kinase S A M P Serine-Alanine-Methionine-Proline repeat SH2 Src homology 2 SH3 Src homology 3 SHIP SH2 containing 5 phosphatase xvi S R F Serum response factor STAT Signal transducers and activators of transcription Tcf T-cell factor TEY Threonine-Glutamate-Tyrosine TGFp Transforming growth factor beta THR Threonine TIMP3 Tissue inhibitor TNF Tumor necrosis factor TYR Tyrosine xvii DEDICATION I would like to dedicate this thesis to my wonderful mother and father. Mom and Dad, there are not too many people that would do what you have done for their children. To be willing to compromise so much in your own lives just to give your children the opportunity to pursue their own endeavors. You taught us to dream, and you gave us the courage to fulfill our goals. And when times were difficult and I wanted to give up, your words of wisdom gave me the courage to overcome all. You taught us about respect, and you always offered your love. And at this time, I would like to thank you from the bottom of my heart for all that you have done for me, and I hope that one day I can became all that you are. Love always, your son Anthony x v i i i ACKNOWLEDGEMENTS To my Ph.D. supervisor, Dr. Salh: I would like to take this opportunity to thank you for not only taking me on as a graduate student in your laboratory but as well, I would like to thank you for your continued support throughout the course of this project. When times were rough, you were always there to lend a supporting hand. Thank you for being more of a friend than a supervisor. To my Ph.D co-supervisor, Dr. Owen: I would like to thank you for all of your great advice and specifically for teaching me histology as well as collecting all of the clinical samples and evaluating the clinical data with me. To my supervisory committee; Drs. Duronio and Dedhar; thank you for your critical input on this project. To all of my colleagues; especially Raj Hundal and Ken Parhar, thank you for all the great times. Raj, I will never forget the times we spent cruising. And to Ken, thanks for all of those late nights at the Combo. Nothing like those Pancake and Eggs. To Kinetek Pharmaceuticals Inc, especially Jas and Arthur, thanks for all of those stimulating conversations. Finally, to my girlfriend Rosanna; thank you for being so patient and understanding. I am so fortunate to have found someone as special as you are. xix Chapter 1. General Introduction 1.1 Cancer Cancer is a disease that plagues Western society. It is estimated that over one million individuals will be diagnosed with cancer each year and more than half-a-million people will die from this disease in the United States alone. Although considerable insight into the etiology of this disease has improved both the diagnosis and treatment of some forms of cancer, for example female breast cancer, there is still an urgent need to improve our understanding of the disease in general. This will undoubtedly facilitate identifying novel strategies for earlier detection of cancers but as well may provide alternative strategies for therapeutic intervention. 1.2 Colon Cancer Colon cancer has served as a paradigm in understanding the etiology of cancer in general; however, to date, it has had very little impact on the overall survival rates associated with this particular malignancy. Currently, colon cancer is the second leading cause of cancer-associated mortality within the Western world. With an estimated 130, 000 new cases per year and over 57, 000 deaths, colorectal cancers account for 10% of all cancer-related deaths in the United States (Goss and Groden, 2000). 1 1.2.1 Epidemiology It is estimated that 50% of the North American population will be diagnosed with colorectal cancer by the age of 70, with the peak incidence for this malignancy being between 60 to 79 years (Landis et al., 1998). It is estimated that less than 20% of the cases occur before the age of 50. Early onset of the disease can be attributed to either preexisting ulcerative colitis (Ekbom et al., 1990; Lashner et al., 1990; Nugent et al., 1991) or one of the hereditary colorectal cancer syndromes (Cranley et al., 1986; Haggitt et al., 1985). Environmental factors including diet and lifestyle are thought to play an important role in the development of this disease. It is believed that physical inactivity and higher consumption of red meat are contributing factors. The role of excessive lipid intake or the protective effects of increased dietary fiber in this disease remains somewhat controversial; prospective studies have not supported either of these (Giovannucci et al., 1995; Giovannucci and Willett, 1994; Le Marchand et al., 1997; Thun et al., 1992; Whittemore et al., 1990). In fact, present data most strongly support a benefit of higher consumption of folic acid, a component of fruits and vegetables, in relation to cancer risk (Song et al., 2000b; Su and Arab, 2001; Willett, 2000). 2 Figure 1. T h e A n n u a l Incidence Rates of C o m m o n Mal ignanc ies in Br it ish C o l u m b i a . This figure was obtained on the B C Cancer Agency website (http://bccancer.bc.ca) 3 (a|B3s 6o|) 000 '001 Jad ajej pazipjepuejs 36y 4 Figure 2. T h e A n n u a l Mortality Rates of C o m m o n Mal ignanc ies in Brit ish C o l u m b i a . This figure was obtained on the B C Cancer Agency website (http://bccancer.bc.ca). 5 (eie^s 6o|) 000'001 Jsd siPJ pazjpjepuejs s6v 1.2.2. Pathogenesis There are at least two main pathogenic pathways implicated in the development of colorectal cancers. Although, the underlying genetic aberrations involved in the development of this disease are distinct, both of these pathways converge on a common pathological entity. The Chromosomal Instability (CIN) pathway, which accounts for approximately 85% of the cases of sporadic cancer mimics the hereditary condition, familial adenomatous polyposis (FAP) (Jen et al., 1994). Colorectal cancer is initiated through the mutation of the adenomatous polyposis coli (APC) gene in these patients. Progression of the disease requires additional mutations of cellular proto-oncogenes like K-Ras and tumor suppressor genes like p53 (see Figure 3). In the hypermutability pathway, a pathway that is implicated in the 15% of the cases, patients are predisposed to colon cancer as they inherit germline mutations in the genes that govern DNA mismatch repair. This pathway is known to mimic the hereditary condition hereditary non-polyposis colon cancer (HNPCC). As a result of the mutation in these genes, other genes that contain microsatellite repeats within their sequence become mutated. These genes include TGFp and the pro-apoptotic protein Bax amongst others (see Figure 4). In addition to these, mutations in the p-catenin gene have been reported to occur at a higher frequency than mutations in the A P C gene in this pathway (Akiyama et al., 2000). 7 Figure 3. T h e C h r o m o s o m a l Instability Pathway. One of the most common events involved in the initiation of familial adenomatous polyposis (FAP) and 85% of the cases of human colon cancer is the mutation of adenomatous polyposis coli (APC) gene. Other chromosomal aberrations include mutations in K-Ras, an unknown gene located on Chr.18 and p53. The genes involved in mediating the metastatic process are yet to be elucidated. 8 Chromosomal Instability Pathway Normal Colonic Epithelium Early Adenoma Intermediate Adenoma Late Adenoma Primary Cancer Metastatic Cancer Figure 4. The Hypermutability Pathway. Although mutations in the A P C gene are known to account for the majority of tumors arising within the colon, approximately 15% of the cases arise due to microsatellite instability. This pathogenic pathway mimics the hereditary condition, hereditary non-polyposis colon cancer (HNPCC). It is well documented that mutations in the genes that regulate DNA mismatch repair (MMR) result in a hypermutable state. As a result genes with microsatellite repeats are susceptible to inherent errors in DNA replication. 10 Hypermutability Normal Colonic Epithelium Unknown Tumorigenic Stages Primary Cancer Metastatic Cancer MMR Gene i TGF-P, B A X e t c r ? 1 ? 1.2.3. Clinical Features Patients can remain relatively asymptomatic for many months to years. Some of the most common symptoms include fatigue, weakness, iron deficiency anemia, occult bleeding, crampy left-lower quandrant discomfort, and prominent disturbances in bowel function, such as bleeding, diarrhea and constipation. Interestingly, colorectal cancers located in the rectum or in the sigmoid region of the colon appear to be more infiltrative at the time of diagnosis than proximal lesions. As a result, these patients tend to have a somewhat poorer prognosis (reviewed in Chapter 18 of Robbins - Pathological Basis of Disease). All colorectal cancers initially spread by direct extension from the mucosa through the basement membrane into adjacent structures, such as the muscularis mucosae and the muscularis propria. These cancers continue to spread and eventually metastasize to secondary sites through either the lymphatic system or blood vessels. Some of the most common sites of metastasis include regional lymph nodes, liver, lungs and bones. In general, the most important prognostic indicator, is the extent of tumor infiltration at the time of diagnosis (reviewed in Chapter 18 of Robbins - Pathological Basis of Disease). 1.2.4. Familial Syndromes Although the autosomal dominant familial syndromes are very uncommon, they have provided a number of clues into the molecular basis of colorectal 12 cancer. There are two main familial conditions: familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC). Patients with familial adenomatous polyposis inherit a mutated copy of the A P C gene from one of their parents (Kinzler et al., 1991); a gene that is located on chromosome 5q21 (Cao et al., 1999). It is postulated that somatic mutation of the second wild-type allele predisposes these patients in the second to third decade of life to the development of numerous adenomas (Goss and Groden, 2000). These adenomas are the precursor lesions of most cases of colorectal cancer (Kinzler et al., 1991). Histologically, the vast majority of these adenomas are of the tubular form; occasionally some may exemplify villous morphology. Tubular adenomas are generally connected to the normal epithelium through a narrow stalk and are comprised of dysplastic cells arranged in straight tubular glands. They can exist independently or in multiples. On the other hand, villous adenomas are sessile outgrowths composed of narrow, finger-like projections arranged on a delicate connective tissue stroma (reviewed in Chapter 18 of Robbins - Pathological Basis of Disease). Patients diagnosed with hereditary non-polyposis colon cancer (HNPCC) are also predisposed to colon cancer at an earlier stage in life; however, these patients typically develop cancers independent of the adenomatous stage. The genetic defect in these patients involves mutation or silencing of genes that are critical in preserving the integrity of the genome; these are the DNA mismatch repair (MMR) genes. Loss of DNA MMR function leads to a hypermutable state; this defect appears to specifically target simple repetitive DNA sequences, 13 termed micro-satellite regions (Kinzler and Vogelstein, 1997). These sequences have been identified in the coding regions of a number of genes involved in a myriad of functions which include: transforming growth factor (TGF)-p type II receptor, the insulin-like growth factor (IGF)-II receptor, T-cell factor (Tcf)-4, p-catenin, Bax and DNA MMR enzymes (reviewed in Boland et al., 2000). Mutation of the latter is thought to be instrumental in the development of the disease via this route. 1.2.5. Colorectal Carcinogenesis As outlined above, colorectal carcinogenesis involves the successive inactivation of tumor suppressor genes like A P C and p53 and the activation of proto-oncogenes like K-Ras (Kinzler and Vogelstein, 1996). One of the earliest and most common events in this process involves the loss of functional A P C , a component of the Drosophila Wnt/Wingless signal transduction cascade. It is important to note that additional mechanisms including defects in DNA mismatch repair function and DNA methylation have been identified in colonic carcinogenesis. 1.2.5.1. Wnt/Wingless Signal Transduction Cascade The classical Wnt/Wingless signal transduction cascade, which was initially identified for its role in Drosophila development (Cadigan and Nusse, 1997), appears to play a central role in the pathogenesis of human cancers (Barker et al., 2000; Bienz and Clevers, 2000; Chen and McCormick, 2001; 14 Uthoff et al., 2001). The components involved in this signaling module includes: the Wnt ligand (to date there are sixteen different Wnt ligands); the Wnt receptor, Frizzled; the cytoplasmic protein, Dishevelled (DSH); the adenomatous polyposis coli (APC) protein; the axis inhibition protein, Axin; Armadillo; the T-cell factor (Tcf) and Shaggy/Zeste-white 3 (see Figure 5). 15 Figure 5. The Wnt S igna l ing C a s c a d e . Wnt stimulation results in the activation of the seven transmembranous G-protein coupled serpentine receptor, Frizzled. Activation is believed to coincide with the translocation of Dishevelled from the cytosol to the plasma membrane. Interestingly, both CK1 and CK2 have been reported to directly phosphorylate Dishevelled (Dsh). Dsh is believed to stabilize Armadillo (the Drosophila homolog of the human protein, p-catenin) by binding directly with Axin, thus inhibiting its degradation. Stabilization is believed to coincide with the translocation of Armadillo into the nucleus. Within the nucleus, Armadillo binds to the T-cell factor (Tcf) family of transcription factors; Tcf proteins possess DNA binding activity whereas Armadillo serves as a transcriptional activator. When complexed, Tcf/Armadillo stimulates the transcription of a number of Wnt responsive genes. 16 Notably, all the vertebrate homologs of this pathway have been identified and will be discussed in more detail in the proceeding sections. In the absence of Wnt stimulation, the Axin multi-protein complex containing A P C , Axin and Shaggy (the Drosophila homolog pf vertebrate GSK3p) regulates the levels of unbound cytoplasmic Armadillo, which is equivalent to the vertebrate protein p-catenin (Behrens et al., 1998; Fagotto et al., 1999; Hart et al., 1998; Kishida et al., 1999; Zeng et al., 1997). Complex formation leads to the hyperphosphorylation of the Armadillo protein by Shaggy; this is essential for the subsequent degradation of Armadillo by the ubiquitin-mediated proteasomal apparatus (Aberle et al., 1997; Ikeda et al., 1998). When cells are stimulated with Wnt ligands, the seven transmembrane G-protein coupled serpentine receptor, Frizzled, is activated. Activation is believed to coincide with the translocation of Dishevelled from the cytosol to the plasma membrane (Axelrod et al., 1998; Boutros et al., 2000). The precise mechanism by which Frizzled mediates its effects through Dishevelled are currently unclear; whether Dishevelled interacts directly with Frizzled or intermediary signaling proteins are involved in conveying the signals from Frizzled to Dishevelled remains to be elucidated. In any case, Dishevelled appears to stabilize Armadillo by binding directly with Axin, thus inhibiting its degradation (Kishida et al., 1999; Smalley et al., 1999). More recently, PKB has been reported to phosphorylate and inhibit GSK3p in response to Wnt; however, only in the presence of Dishevelled (Fukumoto et al., 2001). This phosphorylation would theoretically coincide with the stabilization of Armadillo. Stabilization is believed to coincide 18 with the translocation of Armadillo into the nucleus, which is facilitated through its interactions with the T-cell factor (Tcf) family of transcription factors (Behrens et al., 1996; Molenaar et al., 1996). Tcf proteins possess DNA binding activity whereas Armadillo serves as a transcriptional activator. When complexed, Tcf/Armadillo stimulates the transcription of a number of Wnt responsive genes. In the absence of Wnt, negative regulators like the Groucho family, that bind to Tcf directly, control the activity of Tcf (Cavallo et al., 1998; Roose et al., 1998). It is worth noting that not all Wnt signals are conveyed through Armadillo, for example, planar polarity signaling in the eye. 1.2.5.2. DNA Mismatch Repair Genes (MMR) Although the pathogenesis of the majority of colorectal cancers can be attributed to chromosomal instabilities (CIN), approximately 15% of all the cases arise as a result of microsatellite instabilities (MIN). The latter can be attributed to defects in the DNA mismatch repair system. After DNA synthesis is completed, base-pairing mistakes that have escaped the editing function of DNA polymerases are targeted for repair. The human mismatch repair enzymes (MMR), 'the caretakers' of the human genome, bind to these mismatches and repair the defects. This requires excision, resynthesis and ligation. To date, four of these proofreading genes have been identified: hMSH2 which is located on Chromosome 2p22; hMLH1 that is located on Chromosome 3p21; hPMS1 which is located on Chromosome 2q31-33 and 19 hPMS2 that is located on Chromosome 7p22 (Kinzler and Vogelstein, 1997). Inactivation of this repair system permits these mismatches, which occur at random during DNA synthesis, to be stably passed on to progenitor cells reviewed in (Boland et al., 2000). 1.2.5.3 DNA Methylation DNA methylation is believed to be a critical event in regulating the expresssion of certain genes; it serves as a mechanism to switch certain genes "on" or "off. Interestingly, embryogenesis cannot be completed when the expression of methyltransferase, the enyzme directly responsible for transferring methyl moieties onto adenine and cytosine, is reduced. It has been noted that changes in the pattern of DNA methylation, such as the hypomethylation of oncogenes and hypermethylation of tumor suppressor genes play a role in the development of cancer. Interestingly, these epigenetic changes have been documented to result in the inactivation of hMLH1 (a MMR gene), the tumor suppressor genes (p16 I N K 4 a , p 1 5 I N K 4 b , A P C , BRCA1 and Rb) and genes implicated in metastasis including the inhibitor of matrix metalloproteinase, TIMP3 (Esteller et al.,2001). 1.2.6. Molecular Carcinogenesis of Colorectal Cancer Virtually all cancers arise due to alterations at the genetic level. Colorectal carcinogenesis has served as a model system for studying cancer and has provided several clues into the general mechanisms involved in carcinogenesis. 20 One of the first steps involved in initiating this process within the colon is the mutation of A P C . A P C is a protein that is conserved amongst different species. A P C and the proteins it interacts with will be discussed in the subsequent section (Kinzler and Vogelstein, 1996). Following this, the key factors implicated in the development of colon cancer will be discussed. 1.2.6.1. The Adenomatous Polyposis Coli (APC) Gene The A P C gene, which is located on chromosome 5q21, encodes for a protein that is 2843 amino acids in length and approximately 300 kDa in mass (Goss and Groden, 2000). The A P C protein possesses no extensive homology to any other known proteins and has several functional domains throughout the entire length of the protein. The first 170 amino acids of this protein mediate homodimerization through the formation of a helical rods (Goss and Groden, 2000). Mutated or truncated A P C is able to associate with wild-type A P C through these homodimerization domains, providing a mechanism for a dominant negative effect. Mutations in the 5 end of the A P C coding sequence (prior to codon 169) result in an attenuated form of FAP (Lynch et al., 1995; Nagase et al., 1992; Spirio et al., 1993; Wu et al., 1998b). This is referred to as Hereditary-flat adenoma syndrome. Typically, these patients develop fewer adenomas, which eventually do progress to colorectal cancer. The A P C protein also contains a series of armadillo repeats between amino acids 435-766 (Goss and Groden, 2000); these domains are believed to mediate protein-protein interactions, p-catenin interacts with A P C protein at two 21 putative locations; the first site contains three 15 amino acid repeats located between amino acids 1020-1169 (Morin et al., 1997). The second site contains seven 20 amino acid repeats and is located between residues 1342-2075 (Morin et al., 1997). Interestingly, phosphorylation of critical serine/threonine residues located within this region by GSK3p is believed to increase the affinity between A P C and p-catenin (Rubinfeld et al., 1996), Within this region, there are also three conserved sequences of approximately 20 amino acids in length containing a Ser-Ala-Met-Pro motif ( 'SAMP repeats') that are implicated in Axin binding. Interestingly, the majority of the mutations that occur in A P C are located between amino acids 1342-2075; this is known as the mutation cluster region (MCR). Furthermore, the majority of the oncogenic mutants result in a truncated protein that does not contain the 'SAMP repeats', but does contain some of the p-catenin binding repeats (Behrens et al., 1998). Perhaps the interaction of A P C with Axin is more critical for A P C ' s tumor suppressor role than its interaction with p-catenin. Mutations that occur within the 'SAMP ' region of A P C are generally associated with a more severe disease phenotype in humans. The extreme C-terminal residues of A P C are important for mediating interactions with the cytoskeleton; A P C binds microtubule bundles (Munemitsu et al., 1994; Smith et al., 1994). More recently, A P C has been demonstrated to interact with EB1 (Askham et al., 2000), a protein with unknown function as well as Asef, a guanine-nucleotide exchange factor (GNEF), for the G protein Rac (Kawasaki et al., 2000). Rac has been demonstrated to control the actin cytoskeleton by promoting membrane ruffling (Mohri etal . , 1999). 22 Figure 6. Structure of the A P C Protein. The A P C gene encodes for a protein that is approximately 300 kDa in mass with several functional domains. The first 171 amino acids (homodimerization domain) are involved in mediating interactions between A P C proteins. Amino acids 453-766 are also involved in protein-protein interactions; there are several Armadillo repeats that reside within this region, p-catenin interacts with the A P C protein at two putative locations, amino acids 1020-1169 and 1342-2075. The majority of the oncogenic mutations occur between amino acids 1342-2075 (the mutation cluster region). These residues are also critical in mediating interactions with the Axin protein. The extreme C-terminal residues of A P C are important for mediating interactions with the cytoskeleton; A P C binds with EB1, a member of the E B / R P family of tubulin binding proteins. 23 p-catenin Binding Domains N terminus 1 1020 1169 1342 IV' i v l I* 171 453 766 1020 1169 1342 Homo- Armadillo dimerization Repeats Domain Membrane Phospholipids Cytoskeletal Interacting Domain 24 The A P C protein not only serves to regulate the levels of p-catenin but also has been demonstrated to play a role in cell growth, cell adhesion and mitotic spindle assembly (Nathke, 1999). In addition to these, the A P C protein has been documented to mediate cell migration (Nathke et al., 1996). A P C is a phosphoprotein that has consensus phosphorylation sites for a number of protein serine/threonine kinases. These include GSK3p, MAPK, cyclin-dependent kinases (CDKs), protein kinase A, protein kinase CK1 and CK2 as well as the calmodulin kinase. A P C has been documented to be hyperphosphorylated during the M-phase of the cell cycle at the C-terminus (Bhattacharjee et al., 1996; Bhattacharya and Boman, 1995). Mutation of A P C results in a C-terminal truncated protein that lacks these phosphorylation sites in addition to the EB1 binding site suggesting that A P C ' s tumor suppressing activity might also be attributable to its effects on the microtubule network (Askham et al., 2000). Under normal circumstances, the effects of A P C on cell growth are more than likely mediated through its effects on the cytosolic levels of p-catenin. A P C is pre-phosphorylated by GSK3p (Rubinfeld et al., 1996); as a result the phosphorylated form of A P C associates with Axin/conductin (Kishida et al., 1998; Yamamoto et al., 1998), GSK3p and p-catenin. Complex formation results in the phosphorylation of p-catenin by GSK3p; this is a critical step in the degradation of the former (Aberle et al., 1997; Hart et al., 1999; Kitagawa et al., 1999). Mutation of A P C leads to impaired complex formation resulting in the stabilization of p-25 catenin expression. This stabilization is believed to parallel the translocation of p-catenin into the nucleus, which is facilitated through its interaction with Tcf-4 transcription factor (Behrens et al., 1996; Huber et al., 1996; Korinek et al., 1997; Morin et al., 1997; Porfiri et al., 1997). Within the nucleus, this complex acts as a transcriptional activator up-regulating the expression of a number of genes that have been implicated in oncogenesis (Brabletz et al., 1999; He et al., 1999; He et al., 1998; Shtutman et al., 1999). Dysregulation of this nexus is deemed to be a critical initial step in colorectal carcinogenesis (Sparks et al., 1998). In the rare genetic condition, familial adenomatous polyposis, the molecular defect occurs at the level of A P C (Kinzler et al., 1991). Furthermore, mutations in A P C have been documented in approximately 80% of all cases of sporadic colorectal cancer (Fearon et al., 1990). These mutations result in either no protein production or the expression of a truncated protein that can no longer efficiently facilitate the phosphorylation and subsequent degradation of p-catenin (Bienz, 1999; Kinzler and Vogelstein, 1996; Peifer and Polakis, 2000). The other 15% of the cases harbor mutations in p-catenin, at the sites regulated by GSK3p (Aberle et al., 1997; Polakis, 1999). Dysregulation of this nexus results in the up-regulation of Tcf-4-dependent gene transcription in colon cancer. This phenomenon has also been documented for human gastric and pancreatic cancer (Caca et al., 1999). 26 1.2.6.1.1. p-catenin p-catenin, the vertebrate homolog of Drosophila armadillo protein (a protein involved in cell fate and segment polarity) plays a central role in cell adhesion and the Wnt/Wingless signaling pathway (Barth et al., 1997; Behrens, 1999; Cadigan and Nusse, 1997; Peifer et al., 1991; Peifer and Wieschaus, 1990; Willert and Nusse, 1998). The human p-catenin gene is located on Chromosome 3p21 (Bailey et al., 1995; Kraus et al., 1994; Trent et al., 1995; van Hengel et al., 1995) and encodes for a protein of approximately 88 kDa in mass. The amino terminal region of p-catenin may not only function in transcriptional activation but plays a key regulatory role in the degradation of itself. The amino terminus of p-catenin contains specific serine and/or threonine residues that are phosphorylated by GSK3p; phosphorylation of these residues results in the degradation of p-catenin by the ubiquitin-mediated proteasomal pathway (Aberle et al., 1997; Hart et al., 1999; Kitagawa et al., 1999; Polakis, 1999). The central region of the p-catenin protein is highly conserved among species; it consists of 12 armadillo repeats. These repeats are important in mediating protein-protein interactions; p-catenin interacts with E-cadherin, a-catenin, A P C , Tcf-4/Lef1 and Axin/conductin through these domains (Behrens, 1999; Ben-Ze'ev and Geiger, 1998; Willert and Nusse, 1998). The carboxyl terminal region of p-catenin has been demonstrated to be important in transcriptional activation. Within epithelial cells, p-catenin resides at either the membrane or within the cytosol. At the level of the cell membrane, p-catenin is present in adheren junctions, where it links E-cadherin via a-catenin to the actin cytoskeleton (Ben-2 7 Ze'ev and Geiger, 1998). This interaction may play a role in regulating cell proliferation. The APC/Axin/GSK3p complex controls the cytosolic levels of p-catenin. In the absence of growth promoting signals, p-catenin is complexed with A P C , Axin and GSK3p. Complex formation results in the hyperphosphorylation of p-catenin by GSK3p. In turn, the hyperphosphorylated form of the p-catenin protein binds to the F-box protein, p-TrCP, a subunit of the SCF-type E3 ubiquitin ligase complex (Maniatis, 1999). This renders p-catenin sensitive to proteasomal degradation (Hart et al., 1999; Kitagawa et al., 1999). Interestingly, the majority of the activating mutations that occur in p-catenin in human cancers affect the GSK3p phosphorylation sites (Aberle et al., 1997). These mutations are thought to stabilize the cytosolic levels of the former; stabilization is believed to coincide with its translocation into the nucleus, which is facilitated through binding with the T-cell factor (Tcf)-4/Lymphocyte enhancer factor (Lef)-1 family of transcription factors (Behrens et al., 1996; Huber et al., 1996; Korinek et al., 1997; Morin et al., 1997; Porfiri et al., 1997). Alone these sequence specific DNA binding proteins are unable to activate transcription; however, when complexed with p-catenin (the transcriptional activator), these complexes activate the transcription of a number of different genes; genes involved in cell growth and the inhibition of apoptosis (Korinek et al., 1997; Morin et al., 1997). Furthermore, many of these have been implicated in oncogenesis. p-catenin/Tcf-4 responsive genes include cyclin D1, a protein involved in regulating the G1/S transition of the cell cycle (Shtutman et al., 1999; Tetsu and McCormick, 1999); c-myc, a transcription 28 factor that regulates the expression of cyclin D1 (He et al., 1998); matrilysin/MMP-7, a matrix metalloproteinase involved in regulating tumor cell metastasis (Brabletz et al., 1999); peroxisome proliferator-activated receptor PPAR5, a nuclear receptor believed to be involved in colonic maturation/differentiation (He et al., 1999); the transcriptional protein, Tcf1 (Roose et al., 1999); gastrin (Koh et al., 2000) and the multi-drug resistance gene (Yamada et al., 2000). 29 Figure 7. The APC/p-catenin Signaling Nexus. The A P C protein, which is commonly mutated in human colon cancer, plays a central role in the regulation of p-catenin. This is achieved through complex formation between A P C , Axin, p-catenin and GSK3 . Upon complex formation, G S K 3 is known to phosphorylate p-catenin. This is essential for the ubiquitination of p-catenin and its subsequent degradation. Mutation of A P C results in a C-terminally truncated protein that is unable to form a complex with Axin, p-catenin and GSK3 . This results in the stabilization of p-catenin expression within the cytosol. Stabilization is believed to coincide with its translocation into the nucleus where it binds with Tcf-4/Lef-1 (T-cell factor/lymphoid enhancing factor) resulting in the upregulation of a number of genes involved in mediating cell proliferation and tissue remodeling. 30 U N S T I M U L A T E D S T I M U L A T E D C Y T O P L A S M Target Gene 31 1.2.6.1.3. Axin Axin, a protein involved in inhibiting axis formation in vertebrates (Zeng et al., 1997), acts as a negative regulator of Wnt signaling (Hart et al., 1998). Ax in l , a putative tumor suppressor gene, is present on Chromosome 16p13.3 and is closely related to the protein axil/conductin (Axin2), which is present on Chromosome 17 between q23-q24 (Mai et al., 1999). The Axin protein, a component of the Wnt signaling cascade, is one of the key proteins involved in the ternary complex that regulates the cytosolic expression of p-catenin (Behrens et al., 1998; Hart et al., 1998; Ikeda et al., 1998; Kishida et al., 1998; Yamamoto etal . , 1998). The Axin protein has several functional domains that are important in mediating protein-protein interactions. In fact, Axin is the only protein in the ternary complex that contains separate binding sites for A P C , p-catenin and GSK3p. The amino terminal region of Axin that mediates binding with the A P C protein has significant homology to the G-protein signaling (RGS) family of proteins (Behrens et al., 1998; Cadigan and Nusse, 1997; Hart et al., 1998). The central region of Axin is important in mediating interactions between GSK3p and p-catenin (Behrens et al., 1998; Ikeda et al., 1998). Interestingly, GSK3p has been demonstrated to phosphorylate Axin; this is believed to be associated with an increased affinity of Axin for p-catenin (Yamamoto et al., 1999a). Furthermore, Axin-mediated GSK3p-dependent phosphorylation of p-catenin in vitro and in vivo and the subsequent degradation of the latter has been reported to be 32 independent of A P C binding (Behrens et al., 1998; Hart et al., 1999; Ikeda et al., 1998). More recently, Axin has been demonstrated to homodimerize as well as interact with other proteins involved in Wnt signaling, mainly protein phosphatase 2A (PP2A) and Dishevelled (Fagotto et al., 1999; Hsu et al., 1999; Ikeda et al., 2000; Ratcliffe et al., 2000; Sakanaka and Williams, 1999; Smalley et al., 1999; Strovel et al., 2000; Yamamoto et al., 2001). Thus, it would appear that Axin plays a pivotal role in regulating Wnt's effects on p-catenin/Tcf-4 signaling. Interestingly, Axinl mutations have been documented in human hepatocellular cancers (Satoh et al., 2000) while Axin2 has been reported to be mutated in colorectal cancers arising due to defects in DNA mismatch repair (Liu et al., 2000). 1.2.6.1.4. Glycogen synthase kinase (GSK)-3p GSK3p, the vertebrate homolog of the Drosophila Shaggy protein, is located on Chromosome 3q13.3 and has an apparent protein mass of 47 kDa (Lau et al., 1999). GSK3p is a protein serine/threonine kinase involved in a myriad of functions including cell proliferation, apoptosis, gene expression and cell metabolism (Avruch, 1998; Cui et al., 1998; Grimes and Jope, 2001; Hoeflich et al., 2000; Morisco et al., 2001; Woodgett, 1994). Furthermore, this protein kinase, which has been reported to be involved in the Wnt signal transduction cascade, is of considerable medical importance. This kinase has been implicated in a number of diseases like non-insulin dependent diabetes mellitus, 33 Alzheimer's disease and cancer (Cohen et al., 1997; Grimes and Jope, 2001; Hanger et al., 1992; Kim and Kimmel, 2000). Until very recently, GSK3p was best defined for its role in insulin signaling. Under physiological conditions, GSK3p, a protein kinase with high basal activity, acts as a negative regulator of glycogen synthesis. GSK3p's effects on this particular pathway are mediated through glycogen synthase; GSK3p phosphorylates glycogen synthase and inhibits its enzymatic activity in the absence of insulin stimulation (Embi et al., 1980; Plyte et al., 1992). Upon stimulation of cells with insulin, GSK3p activity is inhibited in a phosphatidylinositol (PI)-3 kinase dependent manner. The insulin stimulated inhibition of GSK3p is associated with the phosphorylation of a single serine residue (Ser-9) on this particular protein kinase. This phosphorylation has been demonstrated to be mediated by protein kinase B (PKB), a downstream target of PI3-kinase (Cross et al., 1995; Cross et al., 1997; Moule et al., 1997; van Weeren et al., 1998). In addition to this, GSK3p has been implicated in the Wnt signal transduction cascade (Novak and Dedhar, 1999). This kinase forms a complex with Axin, A P C and p-catenin. Complex formation is essential for the degradation of p-catenin. Upon Wnt stimulation, GSK3p is phosphorylated and its kinase activity is inhibited. The underlying mechanism involved in the inhibition of this protein kinase; however, is unclear and may involve Axin and LRP-5 (Mao et al., 2001). Inhibition of GSK3p coincides with the stabilization of p-catenin expression. Interestingly, cross talk between the Wnt and insulin signaling pathway has been observed in that PKB has been demonstrated to 34 phosphorylate GSK3p when complexed with its Wnt binding partners. However, this occurred only with prolonged stimulation with Wnt. It is important to note that PKB-mediated inhibition of GSK3p does not mimic the Wnt response; a synergistic effect between Wnt-1 and PKB has been reported (Yuan et al., 1999). So the question as to how GSK3p is regulated in response to Wnt stimulation still exists. In this regard, P K C otherwise referred to as protein kinase C (Cook et al., 1996; Lavoie et al., 1999; Tsujio et al., 2000) and the integrin-linked kinase (ILK) have been documented to phosphorylate GSK3p on Ser9 and inhibit its protein kinase activity (Delcommenne et al., 1998). Perhaps, these protein kinases play a critical role in the Wnt signal transduction cascade. Unlike other protein kinases, the substrate specificity for GSK3p varies; this protein kinase will phosphorylate clustered serine residues spaced at 4 amino acid intervals provided that the most carboxy-terminal serine residue is pre-phophorylated as in the case of glycogen synthase. Phosphorylation of serine-656 by protein kinase CK2 is required for GSK3p phosphorylation of serine residues 640, 644, 648 and 652 on G S (Skurat and Roach, 1995). Other studies have demonstrated that GSK3p substrates like c-Jun (de Groot et al., 1993) and Tau (Hanger et al., 1992; Moreno et al., 1995; Sperber et al., 1995) do not require pre-phosphorylation. Instead, these substrates contain a conserved proline residue to the carboxyl side of the targeted serine/threonine residue; this proline is essential for phosphorylation by GSK3p. Interestingly, p-catenin, another GSK3p substrate, has a multi-serine motif; however, this substrate lacks prolines and does not require pre-phosphorylation. 35 Other GSK3p targets include: ATP-citrate lyase (Ramakrishna et al., 1990), an enzyme which converts citrate to Acetyl CoA; elF2B (Welsh et al., 1998; Welsh et al., 1997), a guanine nucleotide exchange factor essential for the initiation of protein synthesis; the adenomatous polyposis coli (APC) protein (Ikeda et al., 2000); Axin (Yamamoto et al., 1999a), a protein involved in regulating cell proliferation; p-catenin (Aberle et al., 1997; Kawahara et al., 2000), a transcriptional activator implicated in carcinogenesis and cyclin D1 (Diehl et al., 1998), the binding partner of the cyclin-dependent kinase (CDK)-4/6 . 1.2.6.2. The monomeric G protein, Ras The Ras family is a class of monomeric G proteins involved in conveying a variety of extracellular signals into a cell. Currently, three alternative isoforms have been identified; these include H-Ras (Harvey), N-Ras (Normal) and K-Ras (Kirsten). The latter has been demonstrated to be mutated in approximately 50% of cancers. 1.2.6.2.1. Overview of Ras/Mapk signaling cascade The K-Ras gene, that is located on chromosome 12p12, encodes for a ubiquitously expressed protein that is approximately 21 kDa in mass and is localized to the inner side of the plasma membrane. These monomeric G proteins are activated in response to a number of different stimuli including P D G F (platelet-derived growth factor) and E G F (epidermal growth factor); they aid in transducing signals from the external 36 environment into the cell. Ligand binding, in the case of the classical receptor tyrosine kinase, leads to receptor dimerization and the subsequent autophosphorylation on tyrosine residues (Heldin, 1995). These phosphorylated tyrosine residues are important in mediating protein-protein interactions and recruiting a wide variety of signaling proteins to the membrane. These include a number of SH2 (Src homology 2) containing proteins like p85 (the regulatory subunit of PI3-kinase, phosphatidylinositol-3 kinase), PLCy (phospholipase Cy), STAT proteins (Signal Transducers and Activators of Transcription) and the adaptor protein growth factor receptor binding-2, Grb-2 (Kouhara et al., 1997; Skolniketal . , 1993). Grb-2, as outlined above, is an adaptor protein that interacts not only with phosphorylated tyrosine residues through its one SH2 domain but with other proteins through its two SH3 (Src homology 3) domains; these motifs bind to proline-rich regions of other proteins. Grb-2 specifically binds to Sos (Son-of-sevenless), a guanine nucleotide exchange factor (GNEF) that stimulates the exchange of G D P for G T P on Ras; this exchange results in the activation of the latter (Bowtell et al., 1992; Chardin et al., 1993; Martegani et al., 1992; Shou et al., 1992; Wei et al., 1992). Activation of Ras is tightly controlled through intrinsic GTPase activity; this intrinsic activity is stimulated through the binding of a GTPase activating protein (GAP). GTPase catalyzes the conversion of G T P to G D P thus inhibiting Ras downstream signaling events. Interestingly, the majority of the mutations that occur in colon cancer affect this intrinsic GTPase activity. Ras binds to its G A P when mutated; however, the G A P protein is unable to 37 modulate the intrinsic GTPase activity. Thus, Ras is believed to exist in its active G T P bound state. In the GTP-bound state, Ras transmits its signals through its effector domain (N-terminal residues 32-40) to Raf, a serine/threonine protein kinase involved in the Ras/Mapk (mitogen-activated protein kinase) signal transduction cascade. The Raf family of protein kinases is comprised of three different isoforms; these are A-Raf (68 kDa), B-Raf (95 kDa) and Raf-1 (74 kDa). They share greater than 70% sequence homology in their kinase domain; however, the expression patterns of these kinases vary (Storm et al., 1990). Transcripts of A-Raf and B-Raf have been detected in only a few non-proliferating tissues like the kidney and the brain. On the other hand, Raf-1 appears to be ubiquitously expressed (Storm et al., 1990). These kinases have common structural characteristics; an N-terminal domain termed C R 1 ; the N-terminal region of Raf (residues 52-132) has been demonstrated to be important in mediating interactions with Ras in its GTP-bound state. A central domain, referred to as CR2 , that is heavily phosphorylated in vivo and a C-terminal catalytic (CR3) domain (Hall, 1994; Morrison et al., 1993; Pelech and Charest, 1995). All three isoforms have been demonstrated to phosphorylate and activate Mek1 and Mek2; although the relative ability of each of these members to catalyze the reaction varies (B-Raf > Raf1 > A-Raf). The precise mechanism by which the Raf family members are activated is presently unclear. Translocation of Raf-1 to the plasma membrane appears to be 38 an important factor in regulating the activation of this particular kinase. Moreover, the interaction between Raf-1 and Ras has been demonstrated to partially activate the latter (Moodie et al., 1993; Van Aelst et al., 1993; Vojtek et al., 1993; Warne et al., 1993). Several lines of evidence have suggested that protein kinase C (PKC) family members can activate Raf-1; Src tyrosine kinases have also been implicated. More recently, the 14-3-3 proteins have been suggested to play a key role in the activation of this kinase. Once activated, Raf/MEKK (Mapk/Erk kinase kinase) phosphorylates the dual specificity protein kinase, Mek1/2 (Mapk/Erk kinase) on Ser-218 and Ser-222. The Mek family members are considered to be dual specificity kinases in that they are able to phosphorylate tyrosine residues as well as serine and threonine residues (Alessi et al., 1994; Gomez and Cohen, 1991; Gotoh et al., 1994; Matsuda et al., 1992; Pages et al., 1994; Seger et al., 1994; Yan and Templeton, 1994; Zheng and Guan, 1994). Once activated, Mek1 and/or Mek2 phosphorylate the mitogen activated protein kinase (Mapk)/extracellular-regulated kinase (ERK)- land -2 at the Thr-Glu-Tyr (TEY) motif located just before the conserved kinase subdomain VIII 'APE ' region; this results in the activation of the latter (Anderson et al., 1990; Hanks et al., 1988; Rossomando et al., 1989; Rossomando et al., 1991). Phosphorylation of the T E Y motif has been documented to occur in an orderly fashion, tyrosine phosphorylation precedes threonine phosphorylation. Both phosphorylations are essential for maximal activation (Haystead et al., 1992; Robbins and Cobb, 1992). Erk1 (44 kDa) is phosphorylated on Thr-202 and Tyr-39 204, while Erk 2 (42 kDa) is phosphorylated on Thr-183 and Tyr-185 (Payne et al., 1991). These ubiquitously expressed extracellular-signal regulated protein kinases have been reported to be activated through a variety of external signals including receptor-tyrosine kinases, hematopoietic receptors and seven transmembrane domain/trimeric G protein-coupled receptors (Boulton et al., 1991; Cobb et al., 1991; Pelech et al., 1990). Furthermore, Erk1 and Erk2 have been shown to play a role in cell growth and differentiation as well as mediate apoptosis. Erk1 and Erk2 are proline directed kinases that can phosphorylate various proteins including the microtubule-associated protein (MAP)-2, myelin basic protein (MBP) and Rsk (ribosomal S6 kinase) and the transcription factor Elk1. Activation of Erk1 and Erk2 is believed to coincide with their translocation into the nucleus where they act upstream of and regulate the expression of many early growth response genes like c-Fos. 40 Figure 8. T h e Ras/Mapk S igna l ing C a s c a d e . The monomeric G-protein Ras is mutated in approximately 50% of the cases of human colon cancer. Growth factor stimulation results in receptor dimerization and the subsequent activation of the intrinsic receptor tyrosine kinases. As a result, the activated tyrosine kinases cross-phosphorylate intrinsic tyrosine residues on the cytoplasmic portion of the receptor. These phosphorylated tyrosine moieties serves as docking sites for SH2 containing proteins like Grb2. Grb2 interacts with Sos through its proline rich regions and stimulates the exchange of GTP for G D P on Ras, which results in the activation of the latter. Activated Ras in turn leads to the activation of Raf. Raf phosphorylates and activates Mek; Mek phosphorylates and activates Erk1/Erk2. These kinases phosphorylate the transcription factors, Elk-1 and Ets, resulting in gene transcription. 41 Interestingly, Mek1 and Mek2 have also been demonstrated to translocate to the nucleus. This translocation may be essential to allow for the phosphorylation of a small pool of constituitively nuclear, unphosphorylated Erk1 and Erk2. However, the duration of this translocation is tightly controlled. Mek1/2 possess an N-terminal nuclear export signal (NES) that is responsible for exporting them from the nuclear compartment to the cytosol (Fukuda et al., 1997). This rapid export may be an important factor in preserving the fidelity of the signal. 1.2.6.2.2. Ras-lts Relevance to Cancer Mutation of the Ras gene is one of the most prevalent abnormalities in human cancers; 90% of all pancreatic lesions and 50% colonic cancers harbor mutations within this gene (Bos, 1989). Frequent mutations in this gene have also been documented in tumors arising in the lung (30%) and in the thyroid (50%). However, the precise mechanism by which Ras promotes transformation and aids in tumorigenesis is currently unclear and is thought to involve the classical Ras/Mapk signaling nexus. Targeting this pathway has been of extreme interest to a number of different pharmaceutical companies (Adjei, 2001; Sebolt-Leopold, 2000; Tuveson and Fletcher, 2001). To assess the importance of this pathway, several independent laboratories have examined the influence of the different components within this cascade on properties like cell growth, cell differentiation, cell transformation, and sensitivity to chemotherapeutic agents. From these studies, we have learned that Ras-mediated transformation of rodent fibroblast cells is dependent on Mek 43 (Cowley et al., 1994); this transformation can be blocked with either a dominant-negative version of Mek or with a synthetic inhibitor that inhibits this kinase (Dudley et al., 1995). Since Mek is known to phosphorylate and activate Erk1 and Erk2, one could postulate that activation of these kinases may play an integral part in disease development. However, others have shown that oncogenic Ras is able to cause membrane ruffling and the cytoskeletal changes associated with transformation in cells where the activation of Erk1 and Erk2 is blocked (Lloyd et al., 1989; Sun et al., 2000). Thus, it would appear that the activation of Erk1 and Erk2 are not essential for Ras-induced cell transformation. To further support the latter, there has been an enormous interest in investigating the individual components within this pathway in the context of human cancers like pancreas and colon, where Ras mutations are common. An earlier study demonstrated that Erk1 and Erk2 were significantly over-expressed (10-20 fold) and hyperactive (5-10 fold) in all of the cases of breast cancer that were analyzed (Sivaraman et al., 1997). Our studies indicated that these kinases were over-expressed and hyperactive (up to 2.5 fold) in approximately 50% of the cases (Salh et al., 1999). In addition to this, one group demonstrated that although 90% of all pancreatic cancers are known to harbor oncogenic mutations in the Ras proto-oncogene, this did not correlate well with the activation of Erk1 and Erk2 (Yip-Schneider et al., 1999). These authors suggest that constitutive activation of Erk is not required for tumor maintenance or progression in K-ras transformed pancreatic cells. Yet another study demonstrated that MAPK activity was in fact decreased in colonic tumors when compared with the normal control 44 tissue (Attar et al., 1996). The data here are conflicting, but appear to suggest that mutations in the Ras gene do not directly coincide with changes in expression or activity of Erk1 and Erk2; two of the downstream targets in the Ras pathway. Based upon this, it is important to identify which signaling pathways are affected as a result of mutations in Ras. Identifying these proteins or pathways, which are dysregulated, could aid in novel strategies for intervention. 1.2.6.2.3. Ras and Lipid Signaling Lipids not only form an integral part of the cell membrane, but as well have been demonstrated to have important roles as secondary messengers in a number of signaling cascades like cell growth and cell survival. One of the major players involved in transmitting these signals is phosphatidylinositol (PI)-3 kinase. Activation of PI3-kinase, which has been reported to be required for Ras-mediated transformation (Rodriguez-Viciana et al., 1997), is believed to play an important role in oncogenesis. This will be discussed in more detail in subsequent sections. 1.2.6.3. The p53 protein The p53 gene, that is located on 17p13.1, is the single most common target for genetic alterations in human cancers. Chromosomal losses at 17p have been documented in 70-80% of the cases of colon cancer. More importantly, these mutations have been demonstrated to occur infrequently in colorectal adenomas (Shivapurkar et al., 1997), suggesting that mutations in this gene 4 5 occur late in colorectal carcinogenesis. Furthermore, the p53 protein has been demonstrated to be the target of a number of virally expressed proteins associated with neoplasia. For instance, both the hepatitis B X (HBX) protein and the human papilloma viral (HPV) E6 protein have been shown to bind to and inhibit p53 function; inactivation of p53 is probably important for viral replication (Hall et al., 1996; Prives and Hall, 1999). The p53 gene encodes for a protein with an apparent mass of 53 kDa that is predominantly nuclear in localization. This short-lived (approximately 20 minutes in half-life), multifunctional protein possesses 4 highly conserved regions that are important in mediating its function (Buschmann et al., 2000; Haupt et al., 1997; Midgley and Lane, 1997). The N-terminal region of the p53 protein possesses two transcriptional activation domains located between residues 1-63 and 60-97. This region is also subject to phosphorylation by a number of different protein kinases including casein kinase (CK)-1, DNA (DNA-PK) protein kinase and Ataxia telangectasia (ATM) kinase (Meek, 1994). Phosphorylation of p53 by DNA-PK, a protein kinase activated in response to DNA double-stranded breaks, is believed to stabilize the expression of the former (Jayaraman and Prives, 1999). In addition, the ATM kinase, which is mutated in Ataxia telengiectasia (AT), has been demonstrated to phosphorylate p53 in response to ionizing radiation (Banin et al., 1998). Interestingly, dephosphorylation of p53 by the protein phosphatase (PP)-5 has been demonstrated to alleviate the p53 G1-induced cell cycle arrest (Zuo et al., 1998). 46 The transcriptional activation domain is separated from the DNA binding domain by a series of proline residues reminiscent of an SH3 binding domain. The majority of missense mutations that occur in p53 occur within this region; these mutations alter the ability of p53 to bind to DNA. DNA binding is largely facilitated through the tetramerization domain of p53 (Jeffrey et al., 1995). Mutated p53 is able to associate with wild-type p53 through its tetramerization domains, providing a mechanism for a dominant negative effect. The C-terminal region, which interacts with single-stranded DNA, is composed of predominantly basic residues and subject to a number of post-translational modifications including acetylation and phosphorylation. Both protein kinase CK2 and protein kinase C (PKC) have been demonstrated to phosphorylate p53 on the C-terminal side, on Ser-392 and Ser-371, 376 and 378, respectively (Ko and Prives, 1996). The biochemical relevance of these covalent modifications is currently unclear and may facilitate DNA binding. As previously mentioned, the p53 protein acts as a transcriptional activator and has been reported to regulate the expression of as many as 100 other genes that are implicated in a variety of biological functions including proliferation, DNA repair and apoptosis (Tokino et al., 1994). These targets include: p2l/Waf1/Cip-1 (a cell cycle inhibitor), GADD45 (a protein involve in DNA repair), MDM2 (an inhibitor of p53 function), P C N A (a protein expressed during the cell cycle), cyclin G, 14-3-3c (an adaptor protein), Bax (a pro-apoptotic protein), B C L - X L (an anti-apoptotic protein) and IGF-BP3 (an inhibitor of IGF receptor signaling) amongst 47 others (Barak et al., 1993; el-Deiry et al., 1993; Kastan et al., 1992; Ko and Prives, 1996; Miyashita and Reed, 1995; Morris et al., 1996). 1.2.6.3.1. p53 and cell cycle arrest The cell cycle is a highly coordinated process, which is subject to a number of control checkpoints; these checkpoints are critical in ensuring both the fidelity of DNA replication and chromosomal segregation. Additionally, these checkpoints are activated in response to DNA damage to allow for not only DNA repair but for the transcription of the genes involved in the repair process, itself. Moreover, a number of these checkpoint proteins have been demonstrated to be mutated or inactivated in human cancers. These losses result in genomic instability, which is a consequence of dysregulated cell cycling. Under normal conditions, progression through the cell cycle is a highly regulated process. It is interdependent upon the relative expression of cell cycle inhibitors like p21 Waf-1 and the activity of the cyclin-dependent family of protein kinases (CDKs). These inhibitors of cycling mediate their effects by specifically inhibiting the activity of the CDKs. Interestingly; p53 has been demonstrated to play a pivotal role in inducing a cell cycle arrest both at the level of G1/S and G2/M as well as the mitotic spindle assembly checkpoint. The cell cycle arrest at the level of G1 is the best understood. In response to DNA damage, the p53 protein induces a G1 arrest by binding to the promotor region of the p21/Waf1 gene and upregulating its expression. p21 preferentially inhibits the activity of 48 cyclin D/CDK4/6 complexes and thus blocks the phosphorylation of downstream targets (el-Deiry et al., 1993). 1.2.6.3.2. p53 and apoptosis The p53 protein is not only important in ensuring the fidelity of DNA replication, but has also been demonstrated to play an extremely important role in deciding the fate of a cell. P53 has been reported to regulate a number of different biological processes including cell turnover and embryonic development. The p53 protein has been demonstrated to play a critical role in inducing apoptosis. This process is believed to dependent upon the extent of DNA damage present. If this damage is irreparable, the p53 protein, through its transcriptional activity, upregulates the expression of the pro-apoptotic genes likes Bax and IGF-BP3. Elevated expression of Bax and IGF-BP3 is believed to facilitate the p53-mediated apoptotic response (Ko and Prives, 1996). Additionally, p53 has been shown to regulate the expression of the TNF death receptors (Fas/APO-1 and Killer/DR5) (Fukazawa et al., 1999; Sheikh and Fornace, 2000). Apoptosis will be covered in detail in a later section. 1.2.7. The PI3-kinase Signaling Nexus One of the first pieces of evidence implicating PI3-kinase in the development of cancer was the observation that the regulatory subunit of PI3-kinase (p85) associated with oncogenic proteins like the polyoma middle T antigen and v-src (Fry, 1994; Kaplan et al., 1986; Whitman et al., 1985). These 49 interactions were associated with the activation of PI3-kinase. Additionally, PI3-kinase has been demonstrated to be activated by another oncogenic protein, Ras (Fukui and Hanafusa, 1989; Sugimoto et al., 1984). In addition to this, others have shown that transformation by Ras is dependent upon PI3-kinase activity (Rodriguez-Viciana et al., 1994; Rodriguez-Viciana et al., 1997; Rodriguez-Viciana et al., 1996). GTP-loaded Ras has been demonstrated to directly interact with catalytic subunit of PI3-kinase (p110). Recent findings further support the involvement of PI3-kinase in development of cancer. These include the identification of the retroviral oncogene, p3k (v-p3k) of the avian sarcoma virus 16 (ASV-16) that encodes for the catalytic subunit of PI3-kinase (Chang et al., 1997), the amplification of PIK3CA gene in human cervical cancers (Shayesteh et al., 1999), and the isolation of an oncogenic mutant of p85 that participates with the v-raf oncogene to transform mammalian fibroblast (Jimenez et al., 1998). PI3-kinase has also been demonstrated to play a role in the de-differentiation of gastric cancers (Kobayashi et al., 1999). Bakin et al. have shown that PI3-kinase is required for TGF-p (transforming growth factor beta)-mediated epithelial to mesenchymal transition and cell motility in mammary epithelial cells (Bakin et al., 2000). Other studies have shown that PI3-kinase pathway may be involved in the progression to hormonal independence in the case of both breast and prostate cancers (Nakatani et al., 1999b) as well as conferring a cytoprotective effect in response to conventional chemotherapeutic agents like cisplatin (Tachiiri et al., 2000). 50 1.2.7.1. Phosphatidylinositol (Pl)3-kinase The PI3-kinase enzymes are a group of ubiquitously expressed proteins that have been demonstrated to be essential for a plethora of biological responses including cell survival, cell proliferation, glucose transport, actin polymerization and membrane ruffling (Vanhaesebroeck and Waterfield, 1999). PI3-kinase family members catalyze the transfer of the y-phosphate from A T P to the 3-OH position of the inositol ring of phosphatidylinositol forming lipid products like phosphatidylinositol 3-phosphate (PI3-P), phosphatidylinositol 3,4-bisphosphate (PI3,4-P 2) and phosphatidylinositol 3,4,5-triphosphate (PI3,4,5-P3). These phospholipid intermediates are believed to be critical in not only regulating protein-protein, protein-lipid interactions through binding to pleckstrin homology (PH) domains but as well, in regulating the activation of protein kinases and protein phosphatases. The levels of these intermediates are regulated by two lipid phosphatases. The phosphate moiety at the 5' position of the inositol ring is targeted by the SH2 containing 5' -inositol phosphatases (SHIP), whereas the 3' position is targeted by the recently identified tumor suppressor protein, the phosphatase and tensin (PTEN) homologue (Stambolic et al., 1998; Wu et al., 1998c). The role of PTEN in the development of cancer will be discussed below. The PI3-kinase family can be divided into three separate groups based upon their regulation; they are Class I, Class II and Class III. 51 1.2.7.1.1. Class I - Phosphatidylinositol (PI)-3 kinases The Class I family of PI3-kinases have been demonstrated to be activated by a variety of external signals which mediate their effects through receptor tyrosine kinases (RTKs), receptors coupled to tyrosine kinases, G-protein linked receptors and in some cases through the monomeric G-protein Ras (Vanhaesebroeck and Waterfield, 1999). Activation involves their translocation from the cytosol to the plasma membrane where they are believed to come into contact with their substrates. Interestingly, the class I PI3-kinases have not only been demonstrated to be the only class that is capable of promoting the activation of protein kinase B (PKB) in cell-based assays, but as well are sensitive to PI3-kinase inhibitors like LY294002 and Wortmannin. Moreover, this class of PI3-kinases can be further divided into Class la and lb, based upon the regulatory/adaptor proteins that they interact with. Class la PI3-kinases exist as a heterodimer; they are made up of a catalytic subunit with an apparent molecular mass of approximately 110 kDa. The regulatory/adaptor proteins that they interact with govern the substrate specificity and activity of this class (Vanhaesebroeck and Waterfield, 1999). The Class la PI3-kinases are very diverse in mammals; there are at least three different catalytic subunits: p110a, p110p and p110s (each of these are encoded by a separate gene). There are seven different regulatory binding partners that are generated by expression and alternative splicing of three different genes; these include p85a, p85p and p55y. Activation of this class is believed to play a major role in cell signaling. 52 The regulation of Class lb, is quite distinct from that of Class la. Too date, the only catalytic subunit identified in this class is p110y. This particular kinase has been demonstrated to be activated by the py-subunit of trimeric G-proteins and to interact with the adaptor protein, p101. The p101 protein does not appear to possess any homology to any other protein. More recently, PI3-kinase y has been demonstrated to interact with and to be activated by the a subunit of G-proteins (Vanhaesebroeck and Waterfield, 1999). 1.2.7.1.2. Class II - Phosphatidylinositol (PI)-3 kinases The class II PI3-kinase family members are a group of ubiquitously expressed enzymes that have been demonstrated to be activated by insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and monocyte chemotactic peptide-1 (MCP-1) as well as integrins (Brown et al., 1999; Zhang et al., 1998). This class of PI3-kinases has been demonstrated to contain a C2 domain related to those of P K C family members (Virbasius et al., 1996); this family includes PI3-kinase-C2 a, p and 8 (Vanhaesebroeck and Waterfield, 1999). Unlike the Class I PI3-kinases that are predominantly cytosolic, the members of this class are predominantly localized to the plasma membrane (Arcaro et al., 1998). Interestingly, the Class II PI3-kinases cannot utilize PI(4,5)P 2 as a substrate and are also insensitive to PI3-kinase inhibitors (Domin et al., 1997) (Vanhaesebroeck and Waterfield, 1999). 5 3 1.2.7.1.3. Class III - Phosphatidylinositol (PI)-3 kinases The class III PI3-kinases are widely expressed enzymes that are highly homologous to the yeast protein, Vps34p (vesicular protein-sorting protein). Unlike other PI3-kinase classes, the class III PI3-kinases are only able to use phosphatidylinositol (PI) as a substrate (Vanhaesebroeck and Waterfield, 1999). At present, very little information regarding the regulation of this class has been acquired. 1.2.7.2. Phosphatidylinositol (PI)-3 kinase and Signaling PI3-kinases have been demonstrated to be activated by a variety of extracellular signals. This section will briefly highlight the activation of the class la PI3-kinases in response to growth factors like insulin, E G F and P D G F and discuss some of the downstream targets of Pl-kinase (Vanhaesebroeck and Waterfield, 1999). Ligand binding to its receptor results in receptor dimerization and the activation of the intrinsic receptor tyrosine kinases. The kinases phosphorylate tyrosine residues located on the cytoplasmic side of the receptor; these phosphorylated moieties serve as docking sites for multiple SH2-containing proteins like the regulatory subunit (p85) of PI3-kinase. 5 4 Figure 9. The PI3-kinase Signaling Pathway. Insulin stimulation results in the activation of the receptor tyrosine kinases and the subsequent phosphorylation of the insulin receptor substrate (IRS). The IRS protein serves as a docking site for PI3-kinase. Recruitment of PI3-kinase to the membrane results in the phosphorylation of PI(4)P and PI(4,5)P 2 at the 3' position of the inositol ring. These phosphorylated phosphoinositide moieties recruit PH containing proteins like PDK1, ILK and PKB to the membrane. Translocation of PKB to the cell membrane creates a conformational change in the protein rendering the Thr-308 site on PKB accessible to PDK-1. Phosphorylation of PKB on Ser473 by ILK is believed to be dependent upon phosphorylation at the 308 site. As a result, activated PKB phosphorylates and inhibits GSK3 . Furthermore, PKB has been demonstrated to inhibit apoptosis by specifically phosphorylating a number of the components involved in this pathway. 55 56 Activation of the class la PI3-kinases involves their translocation from the cytosol to the plasma membrane where PI3-kinase interacts with the receptor through its two SH2 domains located on the C-terminal side of the p85 regulatory subunit (Kapellerand Cantley, 1994). The p85 protein is believed to play a central role in the activation of PI3-kinase. This protein possesses: an N-terminal SH3 domain that has been shown to be important in the binding of the adaptor protein She, a BH (breakpoint cluster region homology) domain that is highly homologous to the GTPase activating protein (GAP) domain, two proline rich regions that facilitate interactions with non-receptor tyrosine kinases like src, lyn, fyn, abl and Ick, and two SH2 domains separated by an inter-SH2 (iSH2) domain. The iSH2 domain is involved not only in mediating the binding of p85 to p110 but as well is believed to participate in the binding of PI(4,5)P2. Phosphorylation of Ser608 within the iSH2 domain has been demonstrated to be associated with decreased lipid kinase activity (Fry, 1994; Vanhaesebroeck and Waterfield, 1999; Wymann and Pirola, 1998). Recruitment of PI3-kinase to the membrane brings it in close proximity with its lipid substrates. Class la PI3-kinases preferentially bind with PI(4,5)P2 and phosphorylate the 3-hydroxyl group of the inositol ring. These phosphorylated lipid products are believed to be important in mediating interactions with PH-containing proteins and have been implicated in the activation of protein kinases including phosphoinositide-dependent protein kinase (PDK)-1, integrin-linked kinase (ILK)-1 and protein kinase B (PKB). Two lipid 57 phosphatases, SHIP and PTEN negatively regulate PI3-kinase signaling as outlined above. 1.2.7.3. Phosphoinositide-dependent protein kinase (PDK)-1 PDK-1 is a ubiquitously expressed protein serine/threonine kinase that was identified by its ability to phosphorylate Thr308 of P K B a in vitro. PDK-1 has an apparent molecular weight of 63 kDa; it possesses an N-terminal catalytic domain that is similar to protein kinase A (PKA), protein kinase B ( P K B ) and protein kinase C (PKC) family and a C terminal PH domain. The latter is believed to be critical in regulating its activity. PDK1 has been described as a converging point for a number of different pathways. PDK1 has been demonstrated to activate a variety of kinases involved in distinct signaling networks; these include PKB, SGK, PKC^ , PKCs, p70 S6 kinase, p90 ribosomal S6 kinase (p90 Rsk) and p21-activated kinase (PAK) (Balendran et al., 2000; Balendran et al., 1999b; Biondi et al., 2001; King et al., 2000). PDK1 is believed to activate its substrate kinases by one of two different mechanisms; either directly or indirectly. PDK1 directly phosphorylates PKB at Thr-308 within the activation loop whereas it phosphorylates p70S6 kinase on the C-terminal side; phosphorylation of the C-terminal residues creates a conformational change that is required for the activation of this kinase. Although a number of the PDK1 downstream targets have been identified, very little information regarding the regulation of PDK1 activity itself has been acquired. 58 Several studies have attempted to delineate how PDK1 activity is regulated in vivo. It has been suggested that this kinase is constitutively active in cells since unstimulated cells were found to possess a high basal activity; however, a number of recent studies demonstrate that PDK1 activity is tightly regulated. The PH domain of PDK1 is thought to be involved in the regulation of its kinase activity. Binding of the PH domain to P IP 3 at the level of the cell membrane creates a conformational change in the protein that exposes the activation loop; this change results in its activation (Anderson et al., 1998). However, the precise mechanism by which P IP 3 modulates the intrinsic catalytic activity of PDK1 is currently unclear. It has been suggested that PDK1 is activated through autophosphorylation on Ser-247; mutation of this site has been demonstrated to abolish its kinase activity. More recently, PDK1 has been shown to be activated through tyrosine phosphorylation in response to oxidative stress. As well, PDK1 has been reported to be phosphorylated and activated in vitro by src and abl tyrosine kinases. However, whether this pathway can bypass the requirement of PI3-kinase in the activation of PDK1 remains to be determined. 1.2.7.4. Integrin-linked kinase (ILK) The integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that is approximately 59 kDa in mass. ILK was initially identified through its interactions with the p1 integrin subunit using the yeast-two hybrid system (Hannigan et al., 1996). This study revealed that ILK was one of three proteins that were capable of interacting with this integrin subunit; the other 59 two proteins were ICAP-1 (a protein that is phosphorylated in response to cell-extracellular matrix interactions) and prothrombin inhibitor (a member of the serpin family of intracellular protease inhibitors). Furthermore, ILK was demonstrated to co-immunoprecipitate with the p3 subunit and to be localized within focal adhesions. It has been suggested that ILK interacts with highly conserved threonine residues located within the cytoplasmic domains of the p1 and p3 subunits. Mutation of these sites has been demonstrated to impair the interaction between ILK and the integrin subunits. 1.2.7.4.1. ILK Genetics The ILK gene is highly conserved amongst species; homologues of ILK have been identified in human, mouse, Drosophila and in Caenorhabditis elegans. The human ILK gene has been localized to chromosome 11 p15.5-p15.4; a site that exhibits loss of heterozygosity (LOH) in certain types of tumors. Therefore, dysregulation of ILK signaling might be important in human carcinogenesis and modulating its activity with selective inhibitors may be of therapeutic interest. One recent report has demonstrated that administration of an inhibitor to ILK reduced tumor growth in SCID mice explanted with human colon cancer cells (Tan et al., 2001). More recently, a novel isoform of ILK, termed ILK-2 has been cloned from the human HT-144 melanoma cell line. This kinase has been demonstrated to be expressed in highly invasive tumor cell lines. Furthermore, expression of this isoform has been demonstrated to be regulated by T G F beta 1 (Janji et al., 2000). 60 1.2.7.4.2. Structure of ILK The ILK protein is approximately 59 kDa in mass and contains three structural motifs. There are four ankyrin repeats located in the N-terminal region of ILK; these repeats are critical in mediating interactions with a protein termed PINCH through its five LIM domains. Furthermore, these ankyrin motifs are believed to facilitate the redistribution of ILK to focal adhesions within cells. These four ankyrin repeats are followed by a phosphoinositide-binding domain that is similar to a PH domain within other proteins. However, it is unclear whether ILK activity in vivo is regulated through binding of phosphoinositide moieties. In this regard, exogenous addition of PI(3,4,5)P 3 has been demonstrated to modulate ILK activity in vitro and that integrin- and growth factor-dependent stimulation of ILK can be inhibited by PI3-kinase inhibitors (Delcommenne et al., 1998). Taken together, these data suggest that ILK activity may be dependent upon PI3-kinase. The C-terminal region of ILK contains a readily identifiable kinase domain. It is worth noting however, that there are differences between the primary structure of ILK and other protein kinases. Amino acid variations are present within subdomains I, VIB and VII. The typical sequence found within subdomain I, VIB and VII are G X G X X G , HRD and DFG. In ILK they are N E N H S G , P R H and MAD respectively. It is worth noting that other protein kinases lacking all three of these conserved regions have been identified; these include Mik1 and Vsp15p (Dedhar et al., 1999). However, ILK does possess the typical A P E sequence that flanks the activation loop; amino acid substitution at this site (E359K) generates a dominant-negative or inactive 61 version of ILK (Dedhar et al., 1999). The C-terminal region of ILK also contains an integrin-binding domain that is required for integrin-binding and the translocation of ILK to focal adhesions. 62 Figure 10. Structure of the Integrin-Linked Kinase (ILK). ILK is a protein serine/threonine kinase that was identified through its interactions with the p1 integrin subunit. The first 164 amino acids contain 4 ankyrin binding motifs that are important in mediating protein-protein interactions. Amino acids 179-215 contain a PH-like domain, while the kinase domain spans amino acids 186-452. The p1 integrin binding domain is located between amino acids 309-452. 63 Kinase Catalytic Domain N terminus 1 186 11 C terminus 452 164 179 215 309 452 4 Ankyrin PH-like p1 cytoplasmic Repeats domain binding domain 1.2.7.4.3. ILK Function Over the past five years, a wealth of information regarding the regulation of and function of ILK has emerged. ILK has been demonstrated to play a critical role in diverse signaling pathways and possess a number of oncogenic properties. One of the first studies to emerge after the initial identification of ILK, demonstrated that stable over-expression of ILK in rat intestinal epithelial cells resulted in anchorage-independent cell cycle progression (Radeva et al., 1997). Under normal conditions, when cells detach from their extra-cellular matrix, they undergo a process described as anoikis. In addition to the effects of ILK on the cell cycle, Radeva et al. showed that over-expression of this kinase inhibited matrix-detachment induced apoptosis. The observed effects of ILK on the cell cycle correlated with elevated expression of cyclin D1, the activation of CDK4 and the hyper-phosphorylation of the retinoblastoma (Rb) protein; all of which are required for progression from the G1 to the S phase of the cell cycle. Cell cycle progression was associated with phosphorylation of p27; phosphorylation of this cell cycle inhibitor is believed to alter its CDK inhibitory activity (Radeva et al., 1997). A subsequent study, examined the influence of ILK on extra-cellular matrix assembly and tumor formation. This study demonstrated that over-expression of ILK was associated with the up-regulation of fibronectin (Fn) matrix assembly (Wu et al., 1998a); Fn is a major component of the extra-cellular matrix that is deposited during wound healing and may aid in invasion and metastasis by allowing cells to attach and move. To further support this notion that ILK may be involved in enhancing cell motility, over-expression of ILK was demonstrated 65 to lead to a decrease in the level of E-cadherin, a transmembrane protein that is critical in forming contacts between cells. Down-regulation of E-cadherin has been demonstrated to be associated with the progression of colon cancer. Furthermore, Wu et al., 1998a showed that over-expression of ILK enhanced tumor formation in athymic nude mice. Taken together, these findings demonstrated that ILK played a pivotal role in cell growth and tumorigenesis. Another group examined the influence of ILK on invasion as well as the p-catenin and Tcf-4/Lef1 pathway since p-catenin had been proposed to regulate the level of E-cadherin (Novak et al., 1998). Novak et al. reported that over-expression of ILK in both intestinal and mammary epithelial cells not only resulted in an enhanced invasive phenotype, but as well promoted the translocation of p-catenin into the nucleus. This translocation accompanied the activation of Tcf-4/Lef-1 dependent gene transcription. They suggested that ILKs oncogenic properties might, in part be achieved through the activation of Tcf-4/Lef-1. Based upon these findings, it is plausible that ILK may be a component of the Wnt signaling nexus. One of the central components that negatively regulates the Wnt signaling pathway is GSK3p. This kinase has been demonstrated to be regulated in a PI3-kinase-dependent fashion. Since the ILK protein possesses a phosphoinositide binding domain, it is likely that ILK is also activated in a PI3-kinase-dependent manner. In this regard, it has been reported that both fibronectin and insulin could activate ILK and that this activity was blocked with the PI3-kinase inhibitors 66 wortmannin and LY294002. Based upon this, it was concluded that ILK activity was dependent upon PI(3,4,5)P3(Delcommenne et al., 1998). Subsquently, this group examined the potential downstream targets of ILK. Since ILK has been shown to induce the translocation of p-catenin to the nucleus and activate Tcf-4-dependent gene transcription, a process that is negatively regulated by GSK3p, it was postulated that perhaps ILK might regulate GSK3P activity. This group showed both in vitro and in vivo that ILK could phosphorylate and inhibit GSK3p; a kinase regulated by PKB. They also showed that ILK could activate PKB in vivo and phosphorylate PKB on Ser473 in vitro (Delcommenne et al., 1998). They postulated that ILK acts as a proximal effector of PI3-kinase signals regulating the activities of both PKB and GSK3p. However, controversy exists over whether ILK can directly phosphorylate PKB on Ser473. A study by Lynch et al. suggests that ILK might function as an adaptor protein and regulates phosphorylation on Ser473 by an indirect mechanism (Lynch et al., 1999). This will be discussed in further detail in the proceeding section. Over the past three years, a number of different studies have examined the influence of ILK upon both PKB and GSK3p. One study showed that ILK could specifically activate the transcription factor AP-1 ; activation was demonstrated to be dependent upon GSK3p. GSK3p can phosphorylate c-jun within the C-terminus; this inhibits the DNA-binding activity of c-jun. Activation of AP-1 occurred independently of PKB (Troussard et al., 1999). In addition to this, the authors showed that ILK could control the expression of the matrix metalloproteinase (MMP)-9 via GSK3p and AP-1 . Furthermore, they 67 demonstrated that a selective inhibitor of ILK (termed KP-SD1) could inhibit MMP-9 and AP-1 promoter activities as well as inhibit invasion through matrigel (Troussard et al., 2000). Similarly, ILK was also shown to enhance C R E B (cAMP-responsive-element binding protein) transactivation and binding of the C R E B protein to the cyclin D1 promoter in a PI3-kinase/PKB- and GSK3p-dependent manner (D'Amico et al., 2000). This pathway has been implicated in Wnt signaling since the C R E B site was shown to collaborate with Lef-1 site in regulating the expression of genes like WISP-1. The activation of ILK has been demonstrated to be dependent upon lipid products involved in the PI3-kinase signaling nexus. One of the key enzymes involved in negatively regulating this pathway is the lipid phosphatase P T E N . PTEN has been demonstrated to be mutated and inactivated in a number of human cancer syndromes like breast and prostate. Interestingly, two independent studies demonstrated that expression of wild-type PTEN in either human glioma cells or prostate carcinoma cells resulted in a decrease in ILK activity; this is likely explained by a decrease in the level of PI(3,4,5)P3 (Morimoto et al., 1999; Persad et al., 2000). Additionally, one of these studies showed that transfection of either the wild-type P T E N or a dominant-negative version of ILK in PTEN-mutant prostate carcinoma cells was not only associated with a decrease in the phosphorylation of PKB on Ser473 but as well resulted in a G1 cell cycle arrest and enhanced apoptosis (Persad et al., 2000). In this regard, over-expression of ILK has been shown to inhibit suspension-induced apoptosis (anoikis) in mammary epithelial cells. Additionally, this group demonstrated that 68 transfection with either the kinase-dead version of ILK or treatment with KP-SD1 promoted apoptosis in two human breast cancer cell lines (Attwell et al., 2000). This is believed to be largely due to ILK's effects on PKB. More recently, administration of a selective inhibitor to ILK (KP-SD1), or transfection with the kinase-dead version of ILK has been reported to attenuate Tcf-4-dependent transcription in colon cancer cell lines harboring A P C mutations (Tan et al., 2001). Moreover, down-regulation of transcription was accompanied by an increase in the levels of E-cadherin, a protein involved in regulating cell-to-cell contacts. Down-regulation of the latter is believed to facilitate the progression from the adenomatous to the cancer stage. Furthermore, administration of K P -SD1, led to a significant reduction in tumor growth in a xenograft mouse model. Taken together, it appears that the oncogenic protein ILK could have an important role in the progression of cancer and modulating its activity could be of extreme importance in the treatment of human cancer syndromes. 1.2.7.5. Protein kinase B (PKB) The protein kinase B (PKB) family is a group of protein serine/threonine kinases that have been shown to function downstream of the PI3-kinase and have been implicated in a myriad of biological functions including gene transcription (Brunet et al., 1999; Guo et al., 1999), protein synthesis (Jefferies et al., 1997; Redpath et al., 1996; Scott et al., 1998), glucose metabolism (Barbieri et al., 1998; Cong et al., 1997; Cross et al., 1995; Kohn et al., 1996; Tanti et al., 1997), cell proliferation (Ahmed et al., 1997; Brennan et al., 1997; Diehl et al., 69 1998; Li and Sun, 1998; Sun et al., 1999) and cell survival (Ahmed et al., 1997; Blume-Jensen et al., 1998; Crowder and Freeman, 1999; del Peso et al., 1997; Dolcet et al., 1999; Dudek et al., 1997; Eves et al., 1998; Gautreau et al., 1999; Gerber et al., 1998; Gibson et al., 1999; Gold et al., 1999; Kauffmann-Zeh et al., 1997; Kelley et al., 1999; Kennedy et al., 1997; Kontos et al., 1998; Kulik et al., 1997; Leverrier et al., 1999; Liu et al., 1999; Parry et al., 1997; Songyang et al., 1997; Yao and Cooper, 1995; Yao and Cooper, 1996). Furthermore, P K B is believed to play a pivotal role in the development of some forms of cancers based upon the discovery of an oncogenic form of PKB (Bellacosa et al., 1991). 1.2.7.5.1. Protein kinase B (PKB) Genetics Over ten years ago, two independent lines of research led to the discovery of one of the human homologues of the v-akt oncogene. The first group cloned the cellular homologue of v-akt and called it c-Akt (Bellacosa et al., 1991; Staal et al., 1977). While two other independent groups cloned the same cDNA in an attempt to identify novel protein kinases that were related to P K A (protein kinase A) and protein kinase C (PKC); the novel kinase was called R A C or related to A and C kinases (Coffer and Woodgett, 1991; Jones et al., 1991). Since this time, Akt homologues have been identified in a number of different species including birds, insects, nematodes, slime mold and yeast (Andjelkovic et al., 1995; Chang et al., 1997; Chen et al., 1993; Franke et al., 1994; Meili et al., 1999; Paradis and Ruvkun, 1998; Tanaka et al., 1999). 7 0 To date, three major P K B mammalian isoforms have been identified; these are Akt1 /PKBa, Akt2/PKBp and Akt3 /PKB Y (Altomare et al., 1995; Brodbeck et al., 1999; Jones et al., 1991; Nakatani et al., 1999a). All three P K B proteins are encoded by separate genes; P K B a was mapped to chromosome 14q32, while PKBpand PKBy were mapped to chromosome 19q13.1-13.2 and 11q43-44, respectively (Altomare et al., 1995; Masure et al., 1999; Murthy et al., 2000). The P K B a gene is proximal to the immunoglobulin-heavy chain locus; this region is disrupted in human T-cell leukemias and lymphomas and has been demonstrated to be amplified in gastric carcinomas (Staal, 1987). On the other hand, the gene that encodes for PKBp is in close proximity to genes encoding for T G F p l , carcinoembryonic antigen (CEA) and several other proteins involved in DNA repair. This gene has been demonstrated to be amplified in both mammary carcinomas and some ovarian cancer cells as well as human pancreatic ductal adeoncarcinomas (Bellacosa et al., 1995; Cheng et al., 1996; Miwa et al., 1996; Ruggeri et al., 1998; Thompson et al., 1996; Yuan et al., 2000). While PKBy, has been demonstrated to be over-expressed and active in breast cancer cell lines and tumors that lack the estrogen receptor as well as in prostate cancer cell lines that are androgen-insensitive (Nakatani et al., 1999b). 1.2.7.5.2. Protein kinase B (PKB) Structure All three mammalian PKB isoforms are similar in structure, the first 100 amino acids on the N-terminal side contain a pleckstrin homology (PH) domain that make up a major part of the amino-terminal regulatory domain (residues 1-71 147). The PH domain is important in mediating protein-protein and protein-phospholipids interactions as well as in recruiting PKB to the plasma membrane (Datta et al., 1995; Mayer et al., 1993; Musacchio et al., 1993). Adjacent to the PH domain is a short glycine rich region that bridges the PH domain with the central kinase catalytic domain (residues 148-411). The last 70 amino acids within the C-terminus (residues 412-480) are believed to contain a hydrophobic regulatory domain (Bellacosa et al., 1991). All three isoforms contain a conserved threonine (Thr308) and serine (Ser473) that together with the PH domain are critical for PKB activation. This will be covered in detail below. 1.2.7.5.3. Protein kinase B (PKB) Regulation The PKB family is subject to multiple levels of regulation inside cells. Several studies demonstrate that PKB is a proximal effector of the lipid kinase, PI3-kinase. These studies include the observation that the activation of PKB was dependent upon tyrosines Y740 and Y751 in the P D G F receptor. These sites are required for the binding of the p85 regulatory subunit of PI3-kinase (Franke et al., 1995). Additionally, the activation of P K B could be inhibited with the PI3-kinase inhibitors LY294002 and wortmannin and that a constitutively activated version of the p110 catalytic subunit of PI3-kinase could activate PKB (Burgering and Coffer, 1995; Cross et al., 1995; Franke et al., 1995). PI3-kinase is known to phosphorylate phosphatidylinositol lipids on the 31 position of the inositol ring; these phospholipid moieties are believed to serve as docking sites for PH containing proteins like PKB. In this regard, Franke et al. 72 have shown that point mutations within the PH domain that decrease phospholipid binding, block the activation of PKB by growth factors (Franke et al., 1997). Moreover, Bellacosa et al. demonstrated that mutations within the PH domain that enhance phospholipid binding can superactivate PKB (Bellacosa et al., 1998). Other studies have demonstrated that PKB can bind to either PI(3,4)P 2 or PI(3,4,5)P3 with high affinity and specificity; however, binding to these phospholipid moieties is not sufficient for maximal activation of PKB. In this regard, PI(3,4)P 2 but not PI(3,4,5)P3 has been reported to modestly activate this kinase in vitro. The main function of the PH domain is to recruit PKB to the plasma membrane. Interestingly, in v-akt, a truncated viral group-specific antigen, gag, is fused with the full length Akt through a short 5' untranslated region. Normally, the viral gag protein is myristoylated at its N-terminus and targeted to the plasma membrane. This has serious consequences for the regulation and function of v-Akt since v-Akt is myristoylated and demonstrated to be localized at the plasma membrane, within the cytosol as well as in the nucleus (c-Akt is predominantly cytosolic). The translocation of PKB to the cell membrane more than likely places PKB in close proximity to the upstream kinase(s) that activates it. Alessi and colleagues have identified four different sites on P K B a that are phosphorylated in vivo; these are Ser124, Thr308, Thr450 and Ser473. For P K B a to be maximally acitvated, this kinase must be phosphorylated at two of these sites, Thr308 in the kinase domain and Ser473 in the hydrophobic carboxy-terminal regulatory domain. Ser124 and Thr450 appear to be constitutively 73 phosphorylated. The importance of Thr308 and Ser473 in the activation of PKB has been further substantiated through site-directed mutagenesis; mutation of these sites to non-phosphorylatable residues impairs the activation of this kinase, whereas mutations to acidic residues activates the kinase in the absence of growth factor stimulation (Alessi et al., 1997a; Alessi et al., 1997b). The kinase that directly phosphorylates P K B a on Thr308 is the phosphoinositide-dependent protein kinase (PDK)-1 (Alessi et al., 1997b). This kinase which is described in detail above, has been reported to phosphorylate P K B in a PI(3,4)P 2 and PI(3,4,5)P3 dependent manner. However, this kinase does not appear to be responsive to growth factor stimulation; PDK1 has been described as being constitutively active. It has been postulated that recruitment of P K B to the cell membrane creates a conformational change in the protein, which exposes the Thr308 site making it accessible to PDK1. In addition to Thr308, Ser473 phosphorylation is required for maximal activation of P K B a . A great deal of controversy exists as to which kinase in vivo is responsible for phosphorylating P K B a at this site. Earlier studies by Stokoe et al., Stephens et al. and Alessi et al. demonstrated both in vitro and in co-transfection experiments that PDK1 was incapable of phosphorylating P K B a at Ser473 (Alessi et al., 1997b; Stephens et al., 1998). It was suggested that a distinct kinase was responsible for this phosphorylation, hence the name PDK2. More recently, PDK1 has been reported to interact with the PDK1 interacting fragment (PIF) and phosphorylate both Thr308 and Ser473 (Balendran et al., 1999a). PIF has been identified as the carboxy terminal region of the protein 74 kinase C related kinase (PRK-2). However, one cannot rule out the possibility that other kinases like Mapkapk-2 and ILK are PDK2-like kinases that phosphorylate PKB at Ser473. In this regard, Delcommenne et al. and Persad et al. have demonstrated that ILK can phosphorylate PKB on Ser473 in vitro and in vivo; a process that is dependent upon PI3-kinase (Delcommenne et al., 1998; Persad et al., 2000; Persad et al., 2001). While others have shown that transfection with a dominant negative version of ILK or inhibiting ILK activity with a selective inhibitor (KP-SD1) blocks the phosphorylation at this site (Persad et al., 2000). Whether ILK's effects on Ser473 are direct or indirect will require further delineation. Certainly, Persad et al. have demonstrated that ILK can directly phosphorylate P K B at this site (Persad et al., 2001). As with other protein kinases, the activity of PKB is tightly regulated; this kinase is subject to multiple levels of regulation. For instance, the PH domain of P K B can function both as a positive and negative regulator of its activity. It is postulated the PH domain in it's unbound state, may create a conformational change in the protein rendering it inaccessible to its upstream activating kinases. The sub-cellular distribution of PKB might also play a role in the regulation of this kinase, since translocation to the cell membrane is required for its activation. An additional level of control appears to be the duration of PKB activation; both the phosphothreonine and phosphoserine residues have a relatively short half-life. This dephosphorylation event has been reported to be PP2A (protein phosphatase 2A)-dependent. Another mechanism that negatively regulates PKB occurs at the level of the cell membrane. Two lipid phosphatases appear to 75 antagonize PI3-kinase signaling, these are the SH2-containing 5-inositol phosphatase (SHIP) and the phosphatase and tensin homologue (PTEN). The latter will be discussed in detail below. 1.2.7.5.4. Protein kinase B (PKB) - Downstream Targets Protein kinase B has been implicated in regulating a myriad of biological functions. One of the first targets demonstrated to be phosphorylated by this kinase in response to insulin was G S K 3 a and G S K 3 p . The identification of these kinases combined with the observation that PKB was able to phosphorylate histone H2B (HH2B) aided in delineating the optimum substrate consensus sequence required for efficient phosphorylation by PKB. This sequence is defined as follows: Arg-Xaa-Arg-Yaa-Zaa-Ser/Thr-Hyb, where Xaa is any amino acid, Yaa and Zaa are small amino acids other than glycine and Hyb is a bulky hydrophobic residue. Although, a number of putative PKB substrates have been identified through protein database searches, whether PKB can phosphorylate these substrates requires further delineation. These substrates could be phosphorylated by other protein kinases that like P K B recognize similar consensus motifs. These kinases include PKA, p70 S6 kinase and p90 Rsk reviewed in (Vanhaesebroeck and Alessi, 2000). One of the first biological functions attributed to P K B was its role in insulin signaling. Insulin has been reported to activate PKB in a PI3-kinase-dependent manner. Activation has not only been reported to stimulate glucose transport by specifically recruiting glucose transporters (GLUT1 and GLUT4) to the cell membrane but as well has been demonstrated to promote glycogen synthesis by 76 inhibiting the kinases (GSK3a and GSK3p) that specifically regulate this process. Moreover, PKB has been reported to regulate glycolysis in response to insulin; PKB can phosphorylate and activate 6-Phosphofructose 2-kinase (PFK2). More recently, this kinase has received a great deal of attention for its role in promoting cell survival (Vanhaesebroeck and Alessi, 2000). One of the first studies to demonstrate that PKB was involved in mediating cell survival showed that IGF-I mediated cell survival could be inhibited by transfection with a dominant-negative version of PKB in cerebellar granule cells. In addition to this, the authors reported that transfection with either the wild-type P K B or a constitutive-active mutant could promote survival in the absence of IGF-I in these cells (Dudek et al., 1997). These initial observations were subsequently validated in a number of other studies, which reported that PKB could promote survival in a variety of cell lines. These include neuronal cells, hematopoietic cells, epithelial cells and endothelial cells. Furthermore, PKB has been shown to inhibit cell death in response to a number of different apoptotic stimuli including growth factor withdrawal, UV irradiation, DNA damage, matrix detachment, cell cycle discordance and treatment of cells with either anti-Fas or TGFp antibodies as well as paclitaxel (Page et al., 2000). To further validate the importance of this kinase in cell survival, a number of studies have shown that ceramide, a potent promoter of apoptosis, can inhibit the activity of PKB. More recently, PKB has been reported to be susceptible to proteolytic cleavage, mediated by one of the executioners of apoptosis, caspase-3 (Bachelder et al., 1999). However, just how PKB mediated its anti-apoptotic effects was unclear 77 and was suggested to potentially influence a number of the components involved in apoptosis. Putative PKB consensus phosphorylation sequences were identified in Bad, Bcl-2, Apaf-1, Caspases 7-9, and the caspase inhibitor X-IAP. The identification of these sequences raised the possibility that PKB mediates its anti-apoptotic effects by phosphorylating components of the cell death apparatus. In this regard, PKB has been demonstrated to phosphorylate and inhibit Bad (a member of the Bcl-2 oncogene family that promotes cell death), caspase-9 (Cardone et al., 1998; Fujita et al., 1999), transcription factors of the Forkhead family (Biggs et al., 1999; Brunet et al., 1999) and the k kinase (IKK) that regulates N F - K B signaling (Kane et al., 1999; Madrid et al., 2001; Pianetti et al., 2001; Romashkova and Makarov, 1999). Furthermore, PKB has also been demonstrated to inhibit the release of cytochrome c from the mitochondria (Kennedy et al., 1999). Thus, it appears that P K B may mediate its effects on apoptosis by specifically phosphorylating and inhibiting a number of the components involved in promoting death. 1.2.7.5.5. Protein kinase B (PKB) and Cancer One of the first lines of evidence implicating PKB in the genesis of cancer was the identification of a viral form of Akt; this fusion product has been demonstrated to be predominantly localized to the cell membrane. Moreover, over-expression of activated PKB has been shown to be sufficient to induce cellular transformation in NIH3T3 cells. The identification of the viral form of PI3-78 kinase and the discovery that PTEN is mutated in human cancer further implicates the PTEN/PI3-kinase/PKB pathway in the development of cancer. 1.2.7.6. Protein tyrosine phosphatase and tensin homologue (PTEN) PTEN/MMAC1 (protein tyrosine phosphatase and tensin homologue deleted on chromosome Ten/Mutated in Multiple Advanced Cancers) is a recently identified tumor suppressor gene that is located on chromosome 10q23 (Li and Sun, 1997; Steck et al., 1997). Somatic mutations of the PTEN gene are associated with a variety of malignancies including glioblastomas, melanomas, and carcinomas of the prostate, lung, breast, endometrium, kidney, head and neck (Chiariello et al., 1998; Li et al., 1997; Morimoto et al., 1999; Steck et al., 1997) . Furthermore, germline mutations in this gene have been identified in Cowden's disease and Bannayan-Zonana syndrome (Liaw et al., 1997; Marsh et al., 1998; Nelen et al., 1997; Stambolic et al., 2000; Tsou et al., 1998). Patients with C D are predisposed to the development of hamartomas in the skin, gastrointestinal tract amongst others; these patients are also susceptible to the development of breast and thyroid cancers (Marsh et al., 1998). On the other hand, patients with BZS have an elevated incidence of intestinal hamartomas; (an excessive focal outgrowth of cells native to the organ) however, this disease is associated with neurological abnormalities like mental retardation (Marsh et al., 1998) . The PTEN protein, which displays homology to tensin, a cytoskeletal protein that binds to actin and is localized to focal adhesions (Lo et al., 1994), 7 9 has been demonstrated to possess the active site consensus motif HCXXGXXR(S/T) found in all protein tyrosine phosphatases, suggesting that PTEN's anti-neoplastic properties might in part be achieved by inhibiting tyrosine kinase signaling. However, studies by two separate groups indicated that recombinant PTEN is weakly reactive towards phosphoproteins (Li and Sun, 1997; Myers et al., 1998). In particular, one report demonstrated that PTEN possessed lipid phosphatase activity and could dephosphorylate phosphoinositide phospholipids at the 3' position of the inositol ring and thus, antagonize PI3-kinase-dependent signaling (Maehama and Dixon, 1998). PTEN has been demonstrated to play a critical role in murine embryonic development (Suzuki et al., 1998), in cell proliferation (Ghosh et al., 1999; Graff et al., 2000; Kurose et al., 2001; Paramio et al., 1999; Ramaswamy et al., 1999; Sun et al., 1999), in apoptosis (Dahia et al., 1999; Davies et al., 1999; Stambolic et al., 1998; Tamura et al., 1999), in colony formation (Cheney et al., 1998; Furnari et al., 1997), in cell migration and invasion (Besson et al., 1999) as well as in tumor formation in nude mice (Cheney et al., 1998; Furnari et al., 1997). Furthermore, loss of PTEN has been reported to promote both hyperplasia and dysplasia in the skin, gastrointestinal tract and prostate as well as result in tumor formation (Di Cristofano et al., 1998). Thus, it appears that dysregulation of PTEN might confer a selective growth and anti-apoptotic advantage to cells based upon the fact that this phosphatase has been shown to block PI3-kinase/PKB-dependent signaling events. 80 1.2.8. Apoptosis Apoptosis, or programmed cell-death, is an extremely important process that is required not only for normal animal development but as well for maintaining tissue homeostasis. It has been suggested that malfunction or dysregulation of this process may be important in the establishment or development of a number of diseases, including neurodegenerative disorders and cancer. The process of apoptosis differs drastically from the other major form of cell death, necrosis. Apoptosis is characterized by a number of morphological and biochemical alterations which include membrane blebbing, cell shrinkage, chromatin condensation and D N A fragmentation. A variety of signals have been shown to initiate apoptosis. These include crosslinking of death receptors like T N F a and Fas, ultraviolet and ionizing radiation, serum deprivation, over-expression of certain oncogenes like c-myc or tumor suppressor genes like p53 and anti-neoplastic agents like non-steroidal anti-inflammatory drugs. Despite the heterogeneity of these signals and of the pathways that transduce these signals, the execution of apoptosis appears to be interdependent upon the activation of this evolutionary conserved interleukin-1 p (IL-lp)-like proteases referred to as cellular caspases. Since their initial discovery in 1993, over ten different human caspases have been cloned; these enzymes have been divided into two distinct groups: the initiator caspases like capase-8 and - 9 whose primary function is to activate downstream caspases, and the executioner caspases like caspase-3, -6 and - 7 that are responsible for dismantling cellular proteins. The latter mediate there 81 effects by specifically cleaving a number of different target proteins at sites down-stream of specific aspartic acid residues; these targets include the poly-ADP ribosylating protein (PARP), nuclear lamins, DNA-PK, Protein kinase Cs ( P K C 8 ) and the tumor suppressor proteins Rb, retinoblastoma protein; and A P C , adenomatous polyposis coli (Browne et al., 1998). More recently, both PKB (Bachelder et al., 1999) and p-catenin (Steinhusen et al., 2000) have been demonstrated to be susceptible to proteolytic cleavage by caspase-3. By cleaving proteins that are not only important in maintaining the structure of the cell, but as well that are important in repairing defective DNA or providing proliferative signals, the cell becomes committed to die. The final stage in this process, involves the clearance of apoptotic bodies by phagocytic cells like macrophages. 1.2.9. Protein kinase CK2 Protein kinase CK2 (formerly termed casein kinase II) is a ubiquitously expressed protein serine/threonine kinase that has been demonstrated to be up-regulated in transformed cell lines, rapidly proliferating tissues and a number of tumors of diverse etiology (Daya-Makin et al., 1994; Faust et al., 1996; Gapany et al., 1995; Guerra and Issinger, 1999; Issinger, 1993; Munstermann et al., 1990; Prowald et al., 1984; Schneider et al., 1986; Stalter et al., 1994). Furthermore, dysregulated expression of this enzyme is known to be oncogenic. Specifically, transgenic mice over-expressing the CK2a subunit develop 82 lymphomas (Kelliher et al., 1996; Landesman-Bollag et al., 1998; Seldin and Leder, 1995) as well as breast cancer (Landesman-Bollag et al., 2001). C K 2 a has been demonstrated to be expressed in both the cytosol and the nuclear compartments in eukaryotic cells and has been shown to phosphorylate well over one hundred different substrates implicated in a number of different biological functions including DNA replication, transcription and cell growth and metabolism (Pinna, 1997). Recently, this protein kinase has been added to the cascade of proteins involved in the Drosophila Wnt/Wingless signaling cascade. Specifically, it has been demonstrated to associate with and phosphorylate Dsh. Additionally, Song and colleagues have demonstrated that C K 2 phosphorylates p-catenin as well (Song et al., 2000a). Protein kinase CK2 exists as a heterotetramer, which contains two catalytic subunits, a or a (37-44 kDa) and two regulatory p subunits (24-28 kDa). More recently, CK2 has been reported to be active in the unbound state (Stigare et al., 1993). The general structure is a2p2or a a p 2 . Structurally, the two catalytic subunits are similar; however, different genes encode for them (Litchfield et al., 1990b). The p subunit itself is catalytically inactive; p has been demonstrated to stimulate the catalytic activity of the a subunit (Issinger, 1993; Pinna, 1990). CK2 can autophosphorylate on the a subunit; p is believed to enhance this autophosphorylation event. Furthermore, the p subunit is believed to stabilize the a subunit against heat denaturation and proteolysis as well as alter substrate specificity. 83 Protein kinase CK2 is characterized by the following biochemical properties; it is activated by polyamines (Chaudhry and Casillas, 1989; Leroy et al., 1997; Leroy et al., 1994); its enzymatic activity is inhibited by DRB (6-dichloro-1-beta-D-ribofuranosylbenzimidazole) (Egyhazi et al., 1982; Sehgal and Tamm, 1978; Zandomeni et al., 1986) and apigenin/chrysin (Critchfield et al., 1997); it utilizes both ATP and G T P (Cochet et al., 1982); it can bind DNA - the p subunit has 4 cysteine residues in a sequence which are reminiscent of a zinc finger (Pinna, 1990) and it phosphorylates serine or threonine residues immersed in acidic residues (substrate motif P-S/T-X-X-D/E (Hrubey and Roach, 1990; Litchfield et al., 1990a; Marin et al., 1992); both aspartic or glutamic acid can be located at position 3. Putative targets include: the cyclin-dependent protein kinase (CDK)-1 (cdc2); topoisomerase II; DNA ligase; c-Jun, c-Myb, serum response factor (SRF), Max and the large T antigen of the simian virus (SV)-40 (Pinna, 1997). More recently, CK2 has been shown to phosphorylate and stabilize E-cadherin expression and cell-to-cell contacts (Lickert et al., 2000). Interestingly, down-regulation of E-cadherin is believed to facilitate cell migration and invasion. 1.2.10. Treatment Strategies for Colon Cancer Improving our understanding of cancer in general will more than likely improve patient care; it could lead to novel strategies for earlier detection of these lesions as well as provide alternative treatment modalities. It should be noted, however, that an effective treatment strategy for one type of cancer like 84 breast might not be suitable for other types like colon. In addition to this, the treatment strategy might vary for a given type of cancer. For instance, the therapeutic regimen might differ for colonic tumors that arise as a result of microsatellite instability versus those that arise due to chromosomal instabilities. With time, we should be able to devise specific treatment strategies for each patient. 1.2.10.1. Mechanism of Action for NSAIDs Perhaps one of the most interesting epidemiological findings was the observation that people who routinely took aspirin had a reduced risk of dying from colon cancer. Since then, the protective effect of non-steroidal anti-inflammatory agents like aspirin on colon cancer has become widely recognized. Ingestion of aspirin is known to reduce the overall incidence and mortality of this disease in humans by almost 40-50% (DuBois, 1995; Gann et al., 1993; Kune et al., 1988; Peleg et al., 1994; Rosenberg et al., 1991; Suh et al., 1993; Thun et al., 1991; Tonelli et al., 1994; Waddell and Loughry, 1983). This notion is further supported from the observation that administration of sulindac, a non-sterodial anti-inflammatory drug (NSAID), leads to polyp regression in patients diagnosed with F A P as well as in animal models (Beazer-Barclay et al., 1996; Oshima et al., 1996; Stoneretal . , 1999). Non-steroidal anti-inflammatory drugs are believed to exert their anti-neoplastic effects by inhibiting prostanoid biosynthesis; they block the conversion 85 of arachidonic acid to prostaglandin H 2 (PGH 2 ) by targeting the cyclooxygenases (COX) enzymes. 86 Figure 11. Mechanism of Action for Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). NSAIDs are believed to be an effective means of treating patients diagnosed with Familial Adenomatous Polyposis (FAP); however, the precise mechanims by which these agents elicit their effects is unclear and thought to inhibit the conversion of arachidonic acid to prostaglandin H 2 by specifically inhibiting the cyclooxygenase enzymes. 87 Membrane Phospholipids' P h „ s p h o l i p a s e A, NSAIDS - Cyclooxygenase Cox-112 Prostaglandin H 2 Prostaglandin Prostaglandin D 2 Thromboxane A 2 Prostaglandin E 2 Prostaglandin 88 There are two different isoforms of Cox; Cox-1 has been demonstrated to be constitutively expressed in a number of cell types (Williams et al., 1997). Inhibition of this isoform leads to gastric ulceration (Boland et al., 2000). On the otherhand Cox-2, whose expression is inducible, has been demonstrated to be regulated by a variety of stimuli including growth factors, cytokines and hypoxia (Tsujii et al., 1998). These isoforms share greater than 60% sequence homology (Boolbol et al., 1996). Cox-2 has been shown to play a critical role in the inflammatory response (Smith et al., 1996). More importantly, Cox-2, which is not expressed in normal colorectal epithelium, has been reported to be over-expressed in a number of neoplastic tissues as well as some pre-malignant lesions such as polyps arising in FAP patients (Bamba et al., 1999; Sano et al., 1995). Not surprisingly, there has been an enormous effort to develop Cox-2 specific inhibitors and to determine whether or not they would be efficacious. Despite this effort, the precise mechanism by which Cox-2 aids in tumorigenesis is currently unclear; it may be involved in promoting angiogenesis. Additionally, there is a great deal of controversy regarding the mechanism(s) by which NSAIDs elicit their anti-cancerous effects (Giardiello et al., 1995; Prescott and White, 1996; Shift and Rigas, 1997; Smalleyand DuBois, 1997). There are several studies that have underscored the importance of Cox-2 in tumor development. In one particular study, the authors showed that Cox-2 deficient mice developed fewer adenomatous polyps in the background of A P C mutations; however polyp formation was not completely prevented (Oshima et al., 1996). Another group demonstrated that over-expression of Cox-2 in human 89 gastric cancers correlated with a higher degree of lymphatic invasion and metastasis (Murata et al., 1999). More importantly, a number of independent groups have demonstrated the importance of Cox-2 in angiogenesis; this process is critical for the growth of tumors beyond 2-3 mm in size (Oshima et al., 1996; Seed et al., 1997; Tsujii et al., 1998). Tsujii et al. showed that Cox-2 plays a critical role in the production of angiogenic factors like V E G F (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor). Additionally, they reported that these factors were primarily produced by the colonic epithelium (Tsujii et al., 1998). Moreover, these authors demonstrated that Cox-1 activity is essential for endothelial cell tubulogenesis. Specifically, treatment with indomethacin, a non-selective NSAID that preferentially inhibits Cox-1, led a significant reduction in tumor growth rate in a xenograft mouse model. A recent manuscript further supports the role of Cox-2 in angiogenesis. Lewis lung carcinoma (LLC) cells were implanted into either wild type, Cox-1 (-/-) or Cox-2 (-/-) mice to assess the effects of these molecules on tumor growth (Williams et al., 2000). There was a significant reduction in tumor growth in the Cox-2 (-/-) mice, whereas no differences were evident in the other two groups. Furthermore, treatment of wild-type mouse fibroblasts with Celecoxib, a Cox-2 specific inhibitor, was associated with a reduction in vascular endothelial growth factor (VEGF) levels. These data suggest that both Cox-1 and Cox-2 may be important in oncogenesis by specifically promoting angiogenesis; however, their roles in this process are quite distinct. Moreover, inhibition of either one or the other may not be as effective as a non-selective Cox inhibitor. However, it is not clear 90 whether inhibition of Cox-2 is sufficient for effecting tumor regression, additional mechanisms may be required. A number of recent studies indicate that NSAIDs may partly elicit their anti-neoplastic properties by mechanisms distinct from Cox. It is well established that treatment with NSAIDs results in apoptosis or programmed cell death in a number of different cell lines (Akashi et al., 2000). However, whether over-expression of Cox-2 alters the sensitivity of epithelial cells to these agents or whether inhibition of Cox-2 activity is involved in the apoptotic process is not well defined. One study demonstrated an inverse relationship between the expression of Cox-2 and the induction of apoptosis (Tsujii and DuBois, 1995). In this study, rat intestinal epithelial cells over-expressing Cox-2 were shown to be resistant to apoptosis. However, another report showed that exogenous expression of Cox-2 in a variety of cell lines resulted in a cell cycle arrest at the level of G 0 / G i (Trifan et al., 1999). Therefore, whether Cox-2 expression is involved in carcinogenesis by mediating cell growth or whether it acts as a suppressor, favoring a cell cycle arrest requires further investigation. To add to this uncertainty, it has been reported that one of the metabolites of sulindac, sulindac sulfone that lacks Cox-2 inhibitory properties, is able to promote apoptosis (Piazza et al., 1995). Sulindac is the pro-drug and lacks Cox-2 inhibitory properties. Sulindac is modified by colonic bacteria and the liver to either sulindac sulfide or sulindac sulfone (Thompson et al., 1995). Sulindac sulfide is the most active metabolite of sulindac and is concentrated in the colonic epithelium at concentrations that are at least 20-fold higher than those seen in the serum, in animal models (Duggan et 91 al., 1980). The observation that sulfone can mimic the anti-neoplastic effects of sulfide suggests that NSAIDs may not exert their effects by specifically inhibiting Cox-2 function (Rahman et al., 2000; Reddy et al., 1999; Thompson et al., 1997). It is well established that Cox-2 is over-expressed in both pre-malignant adenomas and colorectal cancers (Bamba et al., 1999; Dimberg et al., 2001; Fosslien, 2000; Lim et al., 2000; Maekawa et al., 1998; Sano et al., 1995; Shattuck-Brandt et al., 1999; Sinicrope et al., 1999; Smith et al., 2000); this elevated expression should theoretically alter prostanoid biosynthesis. If NSAIDs were to exert their anti-neoplastic effects by specifically inhibiting Cox-2, then one could hypothesize that prostaglandin biosynthesis would be altered with administration of these agents and that exogenous addition of the latter could alter the sensitivity of epithelial cells to these drugs. In support of this hypothesis, it has been demonstrated that both Cox-2 and P G E 2 were elevated in the C57BL/6J-Min mouse model, a model that mimics FAP (Boolbol et al., 1996). Administration of sulindac led to a reduction in the levels of Cox-2 and P G E 2 when compared to the normal animals. However, whether the anti-tumor effects of these agents are mediated through a reduction in prostaglandin biosynthesis was not established. One group demonstrated that the regression of pre-existing tumors by sulindac was independent of prostaglandin biosynthesis using the C57BL/6J-Min mouse model (Chiu et al., 1997). In this study, they showed that the effects of sulindac on tumor regression were independent of changes in prostanoid biosynthesis and that exogenous administration of arachidonic acid in the diet did not affect tumor size or number in response to sulindac. This work is 92 further supported by a number of other studies that demonstrated exogenous addition of P G s to cells lacking the ability to synthesize P G s did not reverse the apoptotic effect of NSAIDs (Hanif et al., 1996; Piazza et al., 1997a; Shift and Rigas, 1999). Moreover, another group demonstrated that the anti-proliferative effects of NSAIDs are independent of Cox-2. This was demonstrated using cyclooxygenase-null embryo fibroblasts (Zhang et al., 1999). Taken together, it would appear that modulation of Cox-2 activity by NSAIDs is independent of their effects on tumor regression. Currently, this issue remains unresolved. Recently, a number of other studies have shown that NSAIDs are able to modulate other biochemical processes and that inhibition of these may be relevant to understanding the mechanism of action of these agents. Specifically, administration of NS-398, a Cox-2 specific inhibitor, has been shown to promote apoptosis. This process was shown to be dependent upon cytochrome C and the subsequent activation of caspase-9 and caspase-3 (Li et al., 2001a; Li et al., 2001b). Furthermore, another group reported that NSAIDs are able to modulate N F - K B signaling by inhibiting the I kappa kinases or IKK (Yamamoto et al., 1999b). Furthermore, celecoxib, a Cox-2 specific inhibitor, was reported to inhibit the biochemical activity of protein kinase B (Hsu et al., 2000). These authors demonstrated that inhibition of PKB is independent of PI3-kinase, suggesting that NSAIDs inhibit PKB directly or block signaling proteins upstream of PKB, but downstream of PI-3K. Thus inhibition of PKB might be one of the mechanisms by which NSAIDs promote apoptosis. Additionally, another group demonstrated that NSAIDs favor ceramide generation. Ceramide has not only been implicated in 93 promoting apoptosis (Chan et al., 1998), but as well as been shown to inhibit PKB (Schubert et al., 2000). NSAIDs have also been shown to inhibit PPAR5 (He et al., 1999) and alter the Bax to Bcl -X L ratio (Zhang et al., 2000). Taken together, these studies indicate that alternative NSAID targets do exist, and understanding the mechanism of action of these drugs may aid in defining future therapy. It is worth noting that some new potential chemotherapeutic agents have been identified based upon their effects in animal models. These agents include: a-difluoromethylornithine (DMFO), Green tea extracts, curcumin, folic acid, mono- and dihydroxy vitamin D3, selenium, dietary flavonoids and ursodeoxycholic acid. All of these agents have the potential to prevent cancer; however, the efficacy of these drugs in polyp regression and prevention in humans remains to be established (Boland et al., 2000). 1.2.11. Rationale and Research Objectives 1.2.11.1. Rationale One of the earliest events involved in the initiation of human colon cancer is the mutation of the A P C gene. As a consequence of this mutation, it is believed that the degradation of p-catenin, a transcriptional protein, is impaired. This stabilization of p-catenin is believed to parallel its translocation into the nucleus where it binds to the Tcf-4/Lef-1 transcriptional apparatus and up-regulates the expression of a number of different genes that have an important role in not only mediating cellular proliferation but also tissue remodeling. 94 Interestingly, a recently published manuscript reported a negative correlation between the loss of A P C immunoreactivity and a nuclear p-catenin signal in colorectal adenomas. Based upon this information, the authors speculated that perhaps additional events such as the mutation of Axin or GSK3p, the kinase that regulates the expression of p-catenin, might be involved in the process. Therefore, whether mutations in A P C are sufficient for this observed phenomenon or whether additional signals are required warrants further investigation. Perhaps these additional events in the background of genetic mutations in A P C work together to promote tumorigenesis in the colon. In this regard, it has been demonstrated that stable over-expression of ILK in rat intestinal epithelial cells had similar consequences to mutations in the A P C gene on the sub-cellular distribution patterns of p-catenin (Novak et al., 1998). These authors reported that when the stable ILK over-expressing cell line was stimulated with serum, p-catenin was predominantly localized within the nuclear compartment. This translocation accompanied the activation of Tcf-4-dependent gene transcription. Since the re-distribution of p-catenin within the nucleus is a well-documented phenomenon in colorectal cancer and ILK was able to modulate the sub-cellular distribution of the former, I postulated that changes in the protein expression of and activity of ILK might occur in human colon cancer. To add further support to this hypothesis, PI3-kinase, an upstream activator of ILK has also been reported to be overactive in this disease. In addition to this, activated Ras has been reported to result in an increase in the cell surface expression of 95 the p i integrin subunit, which is has also been shown to interact with and be phosphorylated by ILK. Furthermore, it has been reported that increased levels of the integrin p3 subunit correlates with patient survival. Since ILK has been demonstrated to inhibit the enzymatic activity of GSK3p, I postulated that perhaps ILK-mediated inhibition of this protein kinase might serve as an additional mechanism in colorectal carcinogenesis by aiding in the stabilization of p-catenin. The chemopreventative effects of aspirin in colon cancer are widely recognized. Moreover, sulindac, a non-steroidal anti-inflammatory agent, is known to regress polyps in patients diagnosed with FAP as well as in animal models. NSAIDs are thought to elicit their anti-neoplastic effects by inhibiting the biochemical activity of the cyclooxygenase enzymes. However, a number of recent studies indicate that there are Cox-dependent and -independent mechanisms involved here. These include the observation that NSAIDs inhibit PPAR5 (He et al., 1999), alter the Bax-Bcl-X L ratio (Zhang et al., 2000), inhibit the I Kappa kinases (Yamamoto et al., 1999b), and more recently inhibit PKB activity (Hsu et al., 2000). Based upon this data, I postulated that if the ILK signaling nexus is altered in human colorectal carcinogenesis, then perhaps one of the mechanisms by which NSAIDs work is through the modulation of this signaling nexus. 96 1.2.11.2. Objectives One of the main objectives in these studies was to characterize the ILK signaling nexus, predominately ILK and GSK3p, during the development of human colon cancer. In order to perform this work, adenomas representing one of the earliest lesions during colorectal carcinogenesis, primary tumors and metastatic deposits within regional nodes were obtained. These specimens were assayed for protein expression by Western blotting and immunohistochemistry. In addition to this, the lysates from these samples were assayed for the biochemical activity of 3 different protein kinases: ILK, Mapk and protein kinase CK2. The expression and activity levels were compared to the corresponding adjacent colonic control tissue for each case. Based upon the data generated in the preceding studies, the influence of NSAIDs on key signaling proteins implicated in Wntsignaling were assessed. In particular, the effects of A S A and sulindac were tested on the biochemical activation of three putative protein kinases implicated in the Wnt signaling nexus, ILK, PKB and protein kinase CK2. Assessing the effects of NSAIDs, on colon cancer cell viability, as well as Tcf-Lef transactivation, complemented these studies. 97 Chapter 2. Materials a n d Methods 2.1. General Materials 2.1.1. Chemical Reagents Acetic acid Fisher Acetylsalic acid (ASA) Sigma Acrylamide ICN/Fisher Adenosine 5i-triphosphate disodium salt (ATP) Sigma Agar Difco-Fisher/VWR Ampicillin (D[-]-a-aminobenzylpenicillin) Sigma Anti-fade Molecular Probes Aprotonin Sigma [ r 3 2 P ] A T P Amersham/Pharmacia Bactotryptone Fisher/VWR Bactoyeast extract Fisher/VWR Bovine serum albumin (BSA) Sigma Bromophenol blue Fisher Coomassie Brilliant Blue G Fisher Coomassie Brilliant Blue R-250 Fisher DAPI Molecular Probes Dithiothreitol (DTT) BDH Dubellco's Modified Essential Medium (DMEM) Effectene Enhanced chemiluminescence kit (ECL) Ethanol Ethylene bis (oxyethylenenitrilo) tetraacetic acid (EGTA) Ethylene diamine tetraacetate disodium salt (EDTA) Fetal Bovine Serum Glutamine Glutaraldehyde Glycerol p-Glycerophosphate Glycine Histone H1 Hydrochloric acid (HCI) Lipofectamine Magnesium chloride (MgCI 2 6H 2 0) Manganous chloride (MnCI 2 4H 2 0 ) McCoy's 5A medium p-Mercaptoethanol Methanol Medium 199(M199) Modified Essential Medium (MEM) Gibco Gibco Amersham/Pharmacia Fisher/ICN Fisher/ICN Fisher/ICN HyClone Gibco BDH Sigma ICN/Fisher ICN/Fisher Kinetek Sigma Gibco Fisher Fisher Gibco Fisher Fisher Gibco Gibco MonoQ Pharmacia M O P S 3-[N-Morpholino]ethanesulfonic acid Sigma/ICN Myelin basic protein (MBP) Kinetek Nonidet P-40 (NP-40) Sigma NS-398 Calbiochem Penicillin-streptomycin Gibco/BRL Phenyl methylsulphonyl fluoride (PMSF) Fisher Phosphate buffered saline (PBS) Gibco Phosphoric acid ( H 3 P 0 4 ) Sigma Ponceau S concentrate Gibco Prestained S D S - P A G E standards BioRad Protein A Sepharose CL4B Pharmacia Qiagen kits Qiagen Inc. RPMI 1640 Gibco Skim milk Safeway Sodium acetate (NaOAc) BDH/VWR/Fisher Sodium azide Fisher Sodium carbonate (Na2C0 3 ) BDHA/WR Sodium chloride (NaCl) Fisher Sodium dodecylsulphate (SDS) Fisher Sodium fluoride (NaF) BDH/VWR/Fisher Sodium hydroxide Fisher Sodium orthovanadate (Na 3 V0 4 ) Fisher Sodium pyruvate Sigma Sulindac Sigma Sulindac sulfide Calbiochem Sulindac sulfone Calbiochem Tris (hydroxylmethyl) methylamine (Tris) Fisher Tris hydroxylmethyl aminomethane hydrochloride Fisher (Tris-HCI) Tween-20(polyoxyethylene-20-sorbitan monolaurate) Fisher Trypsin Gibco 2.1.2. Laboratory Supplies Molecular Weight Markers Biorad Nitrocellulose Biorad P81 filter paper V W R 3mm filter paper VWR X-Ray film Island Scientific 2.1.3. Plasmids pcDNA3-ILK WT, pcDNA3-ILK KD pcDNA3-PKB WT, pcDNA3-PKB KD pTopflash and pFopflash plasmid 2.1.4. Antibodies Anti-p-catenin (IHC) Anti-p-catenin (IB) Anti-Caspase-3 Anti-CK2 Anti-Erk Ant i -GAPDH Anti-GSK3p Anti-GSK3p Ser9 Anti-ILK (IB, IHC) Gift from S. Dedhar Gift from J . Woodgett Gift from H. Clevers Transduction Laboratories Santa Cruz Biotechnology Stressgen Biotechnology Corp Stressgen Biotechnology Corp Kinetek Pharmaceuticals Cayman Chemicals Stressgen Biotechnology Corp New England BioLabs Stressgen Biotechnology Corp 102 Anti-Parp Anti-PKB Anti-PKB Ser473 Goat anti-rabbit IgG linked to horse radish peroxidase conjugate Goat anti-mouse IgG linked to horse radish peroxidase conjugate Goat anti-rabbit IgG linked to FITC Goat anti-mouse IgG linked to rhodamine Secondary antibodies for immunohistochemistry Pharmigen Stressgen Biotechnology Corp New England BioLabs Calbiochem Calbiochem Jackson Labs Jackson Labs Ventana 2.1.5. Human Colonic Tissue For the experiments in sections 3.1.2. - 3.1.4., human polyp lesions (adenomas) and the corresponding normal control tissue from 13 patients with familial adenomatous polyposis were obtained from Dr. Steven Gallinger at Mt Sinai Hospital in Toronto, Ontario and immediately frozen in liquid nitrogen. For experiments in section 3.1.5., paraffin-embedded polypoid lesions from 10 patients were obtained through Anatomical Pathology at Vancouver Hospital and Health Sciences Centre (VH&HSC). For experiments in chapter 4, a total of 32 103 cases of human sporadic colon cancer with the normal adjacent control tissue were obtained through Dr. David Owen at VH&HSC. For experiments in chapter 4.2, paraffin-embedded colorectal cancers with positive lymph nodes from 10 patients were obtained through Anatomical Pathology at VH&HSC. 2.2. General Methods 2.2.1. Preparation of Human Tissue Samples The samples were serially sectioned (3-5 um in thickness) using a cryostat and approximately 20 slices were placed in 1 ml of homogenization buffer containing: 20 mM M O P S , 50 mM p-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP40, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM phenylmethanesulphonylfluoride and 10 ug/ml leupeptin. The lysates were then subjected to a shearing force with a pestle (3 X 20 s spins) and centrifuged @ 14, 000 rpm for 20 min. The supernatent was collected and stored at -70°C until analysis. Protein concentrations for all samples were quantitated by the Bradford method using bovine serum albumin (BSA) as the standard. 2.2.2. Evaluation of Protein Concentration The protein concentration for each sample was determined using the standard Bradford method (1976). Briefly, BSA protein standards ranging from from 0-30 \ig were prepared; 2.5 ml of Bradford reagent (100 mg Coomassie Blue G, 50 ml ethanol, 100 ml H 3 P 0 4 , 850 ml dH 2 0) was added to each of these 104 standards. The tubes were mixed gently by vortexing and allowed to stand for approximately 5 min before measuring the absorbance at 595 nm with a spectrophotometer. The absorbance values for the standards were used to develop a linear trace plot. For each of the samples to be quantitated, 2.5 ml of the Bradford reagent was added to 5 ul of the undiluted stock. The samples were mixed by vortexing and allowed to stand for 5 min. The absorbances were read and the protein concentrations were determined through linear regression. 2.2.3. Electrophoresis and Western Blotting 2.2.3.1. SDS-Polyacrylamide Gel Electrophoresis Protein samples for immunoblotting were resolved using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) . Aliquots of each sample were normalized to 1 mg/ml using standard homogenization buffer; each sample was diluted up to 800 ul. Two hundred ul of 5X Laemmli sample buffer (125 mM Tris-HCI (pH 6.8), 4% S D S (w/v), 20% glycerol (v/v), 10% p-mercaptoethanol, 0.01% bromophenol blue (w/v) was added to each sample to bring the final volume up to 1 ml. The samples were then boiled for 5 min and 30 uls of each of the samples was then loaded onto a S D S - P A G E gel. Proteins were resolved by electrophoresis on a 1.5 mm thick, 10% separating gel for 2.5 h at 100 V in running buffer (25 mM Tris, 192 mM glycine, 3.5 mM SDS). All immune complex kinase reactions were terminated by the addition of 10 jal of 5X Laemmli sample buffer and boiled for 5 min. Following this, the 105 samples were resolved as outlined above; however, using a 14% S D S - P A G E gel. The resulting gel was stained by immersion in Coomassie Blue Stain (0.5% Coomassie Brilliant Blue R, 45% methanol, 10% acetic acid solution) for 30 min and then destained in a solution containing (40% methanol, 10% acetic acid solution) overnight. The gel was then dried using a Bio-rad gel dryer and autoradiographed overnight. 2.2.3.2. Immunoblotting After resolving the proteins by S D S - P A G E , the gels were immersed for 5 min in transfer buffer (20 mM Tris, 120 mM glycine, 20% methanol (v/v), pH 8.6) to remove SDS. The nitrocellulose membrane was hydrated in transfer buffer for approximately 5 min prior to transfer. Subsequently, the gel and nitrocellulose membrane was assembled into a transfer sandwich between 2 pieces of 3MM filter paper. Proteins were transferred onto the nitrocellulose membrane in a Bio Rad transfer apparatus submersed in ice for 90 min at 300 mA. After the proteins were transferred to the nitrocellulose membrane, the efficiency of the transfer was assessed by staining with Ponceau S dye for approximately 1 min. Protein bands were visualized by removing the excess stain with water. The membranes were subsequently blocked in a 5% skim milk (w/v) solution diluted with TBS (50 mM Tris base, 150 mM NaCI, pH 7.5) overnight at 4°C. After blocking, the membranes were rinsed three consecutive times with TTBS (Tween-20, 0.05%) to remove excess blocking solution and then incubated 1 0 6 with the primary antibody for a period of 4 h. All primary antibodies were diluted according to the manufacturer's specifications in TTBS with 0.1 % azide. After the 4 h incubation period with the primary antibody, the blots were washed three times for 10 min each with TTBS to eliminate non-specific binding of the primary antibody. Following washing, the membranes were incubated with the appropriate secondary antibody conjugated to horse-radish peroxidase, diluted to the appropriate concentration (according to the manufacturer's specifications) in TTBS for 1 h at room temperature with gentle agitation. Excess secondary antibody was removed by thoroughly washing with TTBS for two 10 min intervals followed by one 10 min interval with TBS. The resulting membrane was exposed to enhanced chemiluminescence (ECL) for 1 min. The Western blot was then exposed to film to visualize the immunoreactive proteins. 2.2.3.3. Stripping Immunoblots for Reprobing Bound antibodies were removed from the membranes by incubating the membranes with a stripping solution containing 100 mM p-mercaptoethanol, 2% S D S (w/v), 62.5 mM Tris-HCI (pH 6.7) at 55°C for 30 min. Subsequently, the membranes were washed three consecutive times for 10 min with TTBS and then reblocked in 5% skim milk in TBS overnight and immunoblotted as outlined above. Membranes were stripped no more than two times. 107 2.2.4. Immunoprecipitation Four hundred ug of the protein lysate was subjected to a pre-clear step with a non-specific rabbit IgG antibody pre-absorbed to protein A Sepharose for a minimum of 1 hour at 4°C. The samples were then centrifuged at 6,000 rpm and equal volumes of the supernatent was taken and aliquoted into a new microfuge tube. The lysate was then incubated with 4 ug of the appropriate (anti-ILK or anti-PKB) antibody overnight at 4°C with gentle mixing. To each vial, 30 ul protein A Sepharose was added for an additional 1 h at 4°C. The lysates were then centrifuged and the supernatent was discarded. The protein A Sepharose beads conjugated to the antibody were washed twice with the standard lysis buffer and twice with protein kinase reaction buffer (100 mM Hepes pH 7.0, 20 mM MgCI 2 , 2 mM MnCI 2 , and 2 mM Na orthovanadate). 2.2.5. Immune Complex Kinase Assays The beads were pelleted and the reaction was started by the addition of 25 ul of the kinase reaction buffer (50 mM Hepes pH 7.0, 10 mM MgCI 2 , 1 mM MnCI 2 , 1 mM Na orthovanadate and 2 mM NaF), 5 ug of the appropriate substrate per tube (MBP for ILK assays; Histone 2B for PKB assays) and 0.5 ug of ATP (250 nM ATP, 1uCi[ r 3 2P] ATP) at 30°C for 20 min. The reaction was terminated with the addition of 10 ul of 5X Laemmli sample buffer and boiled for 5 min. The tubes were microfuged at maximum for 1 min and the proteins were electrophoresed on a 14% S D S - P A G E gel. The resulting gel was stained with 1 0 8 Coomassie Blue as outlined above. The phosphorylation status of the substrate was visualized by autoradiography and quantitated using densitometry. 2.2.6. Mono Q fractionation Extracts were fractionated by fast protein liquid chromatography (FPLC; Pharmacia) onto a Mono Q column using a standard procedure (Pelech et al., 1991). Approximately 1 mg of the appropriate sample was loaded onto Mono Q column (Pharmacia; 1 ml) equilibrated in column buffer (25 mM sodium-p-D-glycerophosphate, 10 mM M O P S , 5 mM EGTA, 2 mM MgCI 2 1 mM DTT and 2 mM Na 3 V0 4 ) and chromatographically resolved using a 10 ml linear 0-0.8 M NaCl gradient and 500 ul fractions were collected using an F P L C system and fraction collector. Aliqouts of these samples were prepared either for Immunoblotting or for protein kinase analysis as outlined in the proceeding sections. 2.2.7. Protein Kinase Assays Ten ug or ul of each either crude lysate or the fractionated sample was added to 10 ul of Assay dilution buffer (20 mM M O P S , pH 7.2, 25 mM p-glycerolphosphate, 20 mM MgCI 2 , 5 mM EGTA, 2 mM EDTA, 1 mM DTT and 1 mM sodium vanadate) containing 10 ug of the appropriate substrate (EGF receptor peptide for Mapk assays; casein for protein kinase CK2 assays) and 5 uM cAMP-dependent protein kinase inhibitor peptide. The reaction was initiated by the addition of 5 w l of ATP (250 uM ATP, 1uCi[ r 3 2P] ATP) at 30°C for 15 min. 109 Subsequently, 20 ul of the reaction mix was spotted onto P81 filter paper and washed extensively with 1% phosphoric acid, and then analyzed in a scintillation counter after the addition of 500 ul of scintillation fluid. 2.2.8. In vitro Kinase Assays Ten ul of the appropriate recombinant protein kinases (rILK, rPKB and rCK2) diluted in assay dilution buffer was incubated with varying dosages of either ASA, sulindac, sulindac sulfide, sulindac sulfone or NS-398. To each tube, 10 ul of the appropriate substrate was added (MBP for ILK; Histone 2B for P K B and casein for protein kinase CK2). The reaction was initiated with the addition of 5 ul of [ r 3 2 P] ATP and incubated at 30°C for 10 min. Subsequently, 10 ul of the reaction mixture was spotted onto a P81 Whatman filter paper and washed three consecutive times with 1% phosphoric acid for 10 min each. Enzymatic activity was assessed by scintillation counting as outlined above. 2.2.9. Immunohistochemistry Formalin-fixed paraffin-embedded tissue sections were obtained from the Division of Anatomical Pathology at Vancouver Hospital and Health Sciences Center (VH&HSC). Immunohistochemical staining for ILK (1:100) was carried out using a standard streptavidin-biotin technique. Antigen retrieval was performed by incubating the sections in a 0.01 M citrate buffer for 10 min at high power using a 925-W microwave oven. The slides were then incubated with the appropriate primary antibody at the pre-determined concentration overnight at 110 room temperature. Following incubation, the sections were rinsed three consecutive times with P B S and then incubated with the appropriate biotinylated secondary antibody for 1 h followed by incubation with a peroxidase-labelled streptavidin for an additional 1 h. A E C (3-amino-9-ethylcarbazole) substrate was used as the chromagen and the sections were counterstained with haematoxylin. The overall staining intensity (weak=1, intermediate=2 and strong=3) was scored by 3 independent individuals (Dr. David Owen, Dr. Baljinder Salh and Anthony Marotta) and the results were averaged for each case. These averaged values were used for statistical analysis. 2.2.10. Immunocytochemistry Cells were seeded on glass coverslips, ~ 70% confluency. Twenty-four hours later, the cells were treated accordingly. Experiments were terminated by fixing cells in 100% methanol overnight at -20 °C. After being blocked in P B S containing 4% bovine serum albumin, cells were stained with the mouse monoclonal anti-p-catenin (1:500) antibody and the rabbit polyclonal anti-ILK antibody (1:100) for 4 h. Cells were was three times with PBS and then incubated with the goat anti-mouse IgG conjugated to rhodamine (1:1000) and the goat anti-rabbit IgG conjugated to fluorescein isothiocyanate or FITC (1:1000). Subsequently, the cells were rinsed 3 times with PBS and then counterstained with 4', 6-diamidino-2-phenylindole or DAPI (0.1 ug/ml in PBS). Coverslips were mounted on glass slides with anti-fade and viewed with a fluorescent microscope. i l l 2.2.11. Cell Culture The human colon adenocarcinoma cell lines HT-29, Caco-2, and DLD-1 were obtained from the American Type Culture Collection (Rockville, MD). The human colon cancer cell line, HCT p53+/+ was a gift from B. Vogelstein. HT-29 and Caco-2 cells were cultured in M199 medium containing 1% penicillin/streptomycin supplemented with 10% heat inactivated fetal bovine serum. DLD-1 cells were cultured in RPMI1640 medium containing 2 mM L-glutamine, 10 mM H E P E S , 1 mM sodium pyruvate, 4.5 g/L glucose supplemented with 10% fetal bovine serum. The HCT-116 p53+/+ was cultured in McCoy's 5A media containing 1% penicillin/streptomycin supplemented with 10% fetal bovine serum. HEK-293T cells were a gift from Dr. Alice Mui. These cells were cultured in Dulbecco Minimum Essential Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin and L-glutamine (2 mM). Cells were plated at a density of 5x10 3 cells/well in a 96-well dish, 1x10 5 cells per well in a 6-well dish and 1x10 6 cells per 100 mm dish and grown in a humidified chamber at 37°C in 5% C 0 2 . 2.2.10.1 MTS Cell Viability Assay To assess the effects of non-steroidal anti-inflammatory agents on cell viability, the colorimetric MTS method from Promega was utilized. This assay is based upon the bioreduction of the MTS reagent to formazan; a water soluble compound. MTS is a tetrazolium salt (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) that when 112 mixed with the electron coupling agent P M S (phenazinemethosulfate) is bioreduced by the mitochondrial dehydrogenases to yield the soluble formazan product. The amount of formazan yielded is directly proportional to the number of metabolically active cells. Twenty p.1 of the MTS/PMS (333 iaM/25 uM) solution is added to 100 ul of the appropriate culture medium in a 96-well plate for 1-2 h in a humidified chamber at 5% C O 2 and 37°C. The experiments are completed by measuring the absorbance at 490 nm using an Elisa plate reader. The absorbance reading is directly proportional to the number of viable cells. 2.2.10.2. Transfections 2.2.10.2.1. Tcf-4 Reporter Assays To assess the effects of NSAIDs on Tcf-4-dependent gene transcription, Caco-2, DLD-1 and SW-480 cells were transiently transfected using lipofectamine. A total of 2 pg of cDNA was added to each 35 mm plate; 1 ug of the appropriate Tcf reporter (either the Topflash or the mutant Fopflash reporter) in the presence of 1 u.g of the control reporter, pRenilla. Cells were transfected overnight at 37° C in a 5% C 0 2 incubator. Following this, the cells were rinsed with sterile PBS and new medium containing 10% FBS was added for 24 h. 2.2.10.2.2. Over-expression Studies To assess the influence of the protein kinases ILK and PKB on cell viability in response to NSAIDs, HEK293 and HCT-116 cells were transiently transfected using Effectene. A total of 1 ug of plasmid cDNA was added to each 113 60 mm dish in 5 ml of the appropriate cell culture medium. The cells were left for 8 h at 37° C in a 5% C O 2 incubator. After this period, the medium containing the DNA-Effectene complexes was removed and 5 ml of new medium was added. The cells were incubated for an additional 24 h to allow for gene expression. 2.2.11. Bacterial Transformations One hundred uls of competent DH5aE. coli cells were combined with approximately 100 ng of the appropriate plasmid and gently mixed by agitation. The resulting mixture was incubated on ice for 15 min and then the bacterial cells were heat shocked for 1 min at 42°C. Following this the cells were transferred to ice for 1 min. Five hundred ul of LB broth (10 g bactotryptone, 5 g bactoyeast extract, 10 g NaCl, pH 7) was added to each tube and incubated at 37°C, 200 rpm for 30 min. Two hundred ul of this mixture was plated onto a LB/amplcillin agar plate (100 ug of ampicillin/per ml of LB) and then incubated for 18 h at 37°C. 2.2.11.1. Plasmid Preparation Large scale DNA preparations were performed using the Qiagen, QIAfilter Plasmid Maxi Kit. For each plasmid, a 3 ml starter culture inoculated with the appropriate transformed bacterial cell was incubated at 30°C and 225 rpm for 3 h. Following this period, the 3 ml started culture was added to 300 ml of LB broth. The culture was allowed to grow for 16 h at 30°C and 225 rpm. The cDNA pellets were resuspended in 100 pl of TE buffer and stored at -80°C. 114 2.2.11.2. DNA Yield The DNA concentration obtained from plasmid DNA purification was determined by UV spectrophotometry. Briefly, the plasmid DNA was diluted 1 in 10 with ddhbO and the optical density was measured at 260 nm to determine the DNA concentration obtained from the purification. The purity of the DNA obtained was assessed by measuring the relative ratio at O.D. 260 nm and at O.D. 280 2.2.12. Treatment Protocols 2.2.12.1. Stimulation Studies Time course studies were carried out for each protein kinase (ILK, PKB, and CK2) to delineate at which time points these kinases were activated in response to serum. These results were used to delineate a suitable time point to assess the effects NSAIDs on the activation of these kinases in response to serum. 2.2.12.2. Enzymatic Activation-Inhibitor Studies Caco-2 or DLD-1 cells were seeded at a density of 1 x 10 6 cells per 100 mm dish. Twenty-four h after seeding, the cells were serum starved for 16 h and pre-treated with the appropriate NSAID for an additional 2 h. Following this, the cells were stimulated with 10% F B S for 10 min in either the presence or the absence of the drug and rinsed once with ice-cold PBS . The P B S was aspirated off and the cells were harvested in 1 ml of homogenization buffer (20 mM M O P S , 50 mM p-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP40, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM phenylmethanesulphonylfluoride and 10 ^g/ml leupeptin). 115 2.2.12.3. Transcriptional Assays Following transfection, cells were placed in the appropriate cell culture medium containing 1% FBS in the presence or absence of the appropriate drug for a period of 24 h. The cells were harvested and assayed for transcriptional activity with a luminometer. To corroborate the effects of NSAIDs on Tcf-dependent gene transcription, the expression of the cyclin D1 protein, a down-stream target of Tcf-4, was analyzed. These studies were conducted in a similar fashion as outlined above. 2.2.12.4. Cell Viability Assays Cells were plated out at a density of 5 x 10 3 cells per well in a 96-well dish in the appropriate growth medium containing 10% FBS for 24 h. Following this, the cells were rinsed once with sterile P B S . One hundred ul of serum containing 1% FBS was added to each well in the presence or absence of the appropriate drug for 24 h in a humidified chamber at 5% C 0 2 and 37°C. These studies mirrored the transcriptional studies, to specifically demonstrate that the effects of NSAIDs on Tcf-4-dependent gene transcription were independent of the effects of these agents on apoptosis. 2.2.12.5. Preparation of inhibitors Aspirin and sulindac were purchased from Sigma. Sulindac sulfide, sulindac sulfone and NS-398 were purchased through Calbiochem. Aspirin, 116 sulindac sulfide and NS-398 were initially dissolved in 100% DMSO at 5 mM, 100 uM and 100 \xM, respectively. Sulindac and sulindac sulfone were dissolved in Tris buffer (pH 8) at 1 mM and 100 uM, respectively. All inhibitors were diluted to the desired concentration with the appropriate cell culture medium depending on the cell type. 2.3. Statistical analysis The relative amount of protein was measured by scanning the film using the BioRad gel-doc apparatus into a TIFF format file. The band densities were quantified in arbitrary units using the NIH Image Program (version 1.61). Results are expressed as mean S E M , with p<0.05 being considered significant using Student's T-test (unpaired, two-tail). To determine the significance of the immunohistochemal data, the scored values were analyzed using the Analysis of Variance (Anova):Single Factor Test. 117 Chapter 3. Characterization of Signaling Modules within Polypoid Lesions. 3.1. General Introduction It is well established that the single most important event involved in the initiation of colon cancer is the mutation of the adenomatous polyposis coli (APC) gene. Mutations in this gene are associated with the development of multiple adenomas within the large intestine of patients diagnosed with familial adenomatous polyposis (FAP). These lesions are precursors to colorectal cancer (CRC). Furthermore, 80-90% of all sporadic cases of C R C are believed to arise due to mutations in this gene. Mutation of the A P C gene results in the expression of a C-terminal truncated protein that is no longer able to form a complex with Axin and GSK3p. As a result, GSK3p is unable to phosphorylate p-catenin; this phosphorylation is required for the degradation p-catenin by the ubiquitin-mediated proteasomal complex. Consequently, stabilization of p-catenin is believed to parallel its translocation to the nucleus. However, whether additional signals are required to facilitate this translocation is currently unclear. A recent report suggests that other mechanisms could be involved in this process, based upon their data, which demonstrated a negative correlation between A P C immunoreactivity and p-catenin nuclear positivity (Iwamoto et al., 2000). Interestingly, stable over-expression of the integrin-linked kinase (ILK) in rat intestinal epithelial cells has been demonstrated to have similar consequences to the mutation of A P C on the sub-cellular distribution of p-catenin. Serum stimulation of cells stably over-118 expressing ILK resulted in a significant redistribution of (3-catenin from the cell membrane and cytosol into the nucleus. This translocation accompanied the activation of Tcf-4-dependent gene transcription (Novak et al., 1998). Moreover, another protein kinase, which has been reported to be over-expressed and exhibit increased casein phosphotransferase activity in both colonic adenomas and colorectal carcinomas, has been implicated in Wnt signaling. Specifically, CK2 has been shown to have a positive influence on the redistribution of p-catenin from the cytosol to the nucleus. 3.1.1. Introduction to ILK ILK is a recently identified protein serine/threonine kinase whose activity has been demonstrated to be dependent upon PI3-kinase (Delcommenne et al., 1998). More importantly, this protein kinase has been shown to have a positive influence on Wnt signaling. Particularly, ILK has been shown to favor the translocation of p-catenin from the cytosol into the nucleus (Novak et al., 1998), which is a common event in colon cancer. Additionally, ILK has been demonstrated to possess a number of oncogenic properties including the induction of anchorage-independent cell cycle progression and the enhancement of tumorigenicity in nude mice (Radeva et al., 1997). Furthermore, ILK has been demonstrated to directly regulate the activity of both PKB and GSK3p (Delcommenne et al., 1998; Persad et al., 2000; Persad et al., 2001). More importantly, administration of a specific inhibitor of ILK (KP-SD1), or transient transfection with the kinase-dead version of ILK has been recently reported to 119 attenuate Tcf-4-dependent gene transcription in colon cancer cell lines harboring mutations in either A P C or p-catenin (Tan et al., 2001). Based on these findings, it was postulated that dysregulation of ILK signaling might occur be involved in colorectal carcinogenesis. 3.1.2. Investigation of the ILK signaling nexus in FAP patients One of the main objectives of this thesis was to evaluate the ILK signaling nexus during disease progression from the earliest lesion, the colorectal adenoma to the metastatic deposit within adjacent lymph nodes. In the present study, the enzymatic activity and protein levels of ILK in polypoid lesions and normal colonic tissue from patients who were diagnosed with familial adenomatous polyposis were examined. Furthermore, the changes in ILK activity were correlated with effects on downstream targets involved in the ILK signaling nexus, specifically GSK3p. Colorectal adenomas and the corresponding normal control tissues were obtained from thirteen patients diagnosed with FAP and processed as outlined in the Materials and Methods section. The homogenates from these lesions were subjected to ILK immunoprecipitation and immune complex kinase assays, using myelin basic protein (MBP) as a substrate. The results presented in Figure 12 demonstrate that there was an increase in immunoprecipitated ILK M B P phosphotransferase activity in 10 of the 13 cases that were analyzed any where from 40-350% above the control sample for each case. Three of the cases did not display any measurable increase in activity when compared to the control. To 120 ensure that similar amounts of proteins were utilized for all experimental conditions, the expression of G A P D H was evaluated for each case (data not shown). Additionally, each of the lysates corresponding to either the polyp or patient matched control in each were normalized in each experiment by assaying for the specific protein concentration using the Bradford method as outlined in the Materials and Methods section. 121 Figure 12. Increased ILK/MBP Phosphotransferase Activity in Polyps from Patients Diagnosed with FAP. Lysates from either the polyp or the corresponding control samples for each case were subjected to an ILK immunoprecipitation-protein kinase assay using M B P as the substrate. The results for the 10 cases where a change was observed are expressed as a % change in ILK activity in the polyp over the control sample. Assays were performed at least in duplicate, in triplicate when sufficient lysates were present. 122 123 To delineate whether the changes in ILK activity in the 10 cases, which displayed increased M B P phospotransferase activity could be attributed to differences in the expression of the ILK protein, the levels of ILK in the polypoid lesions were assessed with respect to the control by Western blot analysis. The results presented in Figure 13A, which are representative of the 10 cases, demonstrate that elevated expression levels of ILK (lower panel) might account for the observed increase in ILK activity within these samples. To specifically elucidate whether there was a direct correlation between ILK activity and expression levels, the percentage change in ILK activity above the control was compared with the percent change in ILK protein expression above the control. The data indicated that although similar trends were observed between the two of these, there did not appear to be a direct correlation (see Figure 13B). Importantly, these increases in ILK expression and activity were determined to be independent of changes in the expression levels of the Map kinase, Erk1 and Erk2 (see Figure 15). Moreover analysis of all immunoreactive bands by Western blot analysis revealed that only one band appeared to undergo dramatic changes in expression. Based upon the molecular weight, this band was determined to be ILK (see Figure 13C). 124 Figure 13. Increased MBP Phosphotransferase Activity Correlates With Elevated Expression of the ILK Protein. A. Examination of ILK activity and expression. Upper panel, ILK/MBP phosphotransferase densitometry. Immunoprecipitated ILK M B P phosphotransferase performed in triplicate. Middle panel, corresponding autoradiograph demonstrating phosphorylation of MBP. Lower panel, anti-ILK immunoblot, examining the protein expression of ILK in the polyp and the corresponding control sample. B. Correlation between ILK activity and expression. Data represented as % change in ILK expression or activity above the control. C. Specificity of ILK antibody. Representative Western blot demonstrating specificity of the ILK antibody from Stressgen Biotechnologies. 125 3500 -4—I [= 3000 C5 P5 C6 P6 C7 P7 C9 P9 Case Number 126 5 6 7 9 Case Number 33 — 29 — 21 — C3 P3 C4 P4 Case Number 127 As outlined earlier, one of the downstream targets in the ILK signal transduction cascade is GSK3p. ILK has been reported to phosphorylate GSK3p on Ser9; phosphorylation at this site results in the inhibition of its GSK3p kinase activity. To evaluate whether changes in ILK activity correlated with effects on GSK3p, the phosphorylation status of GSK3p was evaluated by Western blot analysis. In addition to this the levels of p-catenin, a protein whose levels are controlled by GSK3p, and CK2a were analyzed. CK2 has been previously reported to be over-expressed in colorectal adenomas (Munstermann et al., 1990). 128 Figure 14. Effects of ILK on Downstream Targets in FAP. ILK activity is increased in the polyp compared with its respective control (representative M B P autoradiogram). Anti-ILK immunoblot; examining expression of ILK. Anti-p-catenin immunoblot; examining expression of p-catenin. Anti-P-GSK3p immunoblot; examining phosphorylation status of GSK3p. Anti-GSK3p immunoblot; examining expression of GSK3p. Anti-CK2a immunoblot; examining expression of the catalytic subunits of CK2. Ant i -GAPDH immunblot; used as an internal control for the experiments. The results are representative for the 10 cases, which displayed increases in ILK activity. 129 ILK p-catenin P-GSK3P < P-GSK3J3 < GSK3P < C K 2 a < CK2a ' G A P D H Control Polyp 130 The results for the representative patient (1 patient out of the 10 which displayed changes in ILK activity) that are presented in Figure 14 indicate that GSK3p was significantly phosphorylated at Ser9, which is indicative of its biochemical inhibition. These results are corroborated when examining the expression of GSK3p in the control and polyps samples; there appears to be a slower migrating band, which under most circumstances correlates with a post-translation modification like phosphorylation. Moreover, this increase in the inhibitory phosphorylation of GSK3p appeared to be independent of changes in the relative expression of the protein itself. GSK3p is the kinase that when complexed with Axin and APC, phosphorylates p-catenin promoting its degradation (Hinoi et al., 2000). Therefore within these patients, not only is complex formation impaired through the mutation of APC but as well the kinase that is integral in regulating the cytosolic levels of p-catenin is inhibited within these lesions. Perhaps, additional proteins or mechanisms other than APC can regulate the cytosolic levels of p-catenin and inhibition of GSK3p ensures that the former is over-expressed. As predicted, the protein levels of p-catenin were also elevated with respect to the control. To consolidate these findings, particularly ILK and GSK3p, the expression and activity levels of CK2 were evaluated. As outlined earlier, CK2 has been reported to be over-expressed and hyperactive in colorectal adenomas. The data clearly demonstrates that for this particular patient the levels of CK2a are increased in the polyp samples when compared to the control, as one would predict. The expression of GAPDH, a 'house-keeping' gene, was assessed in each case to ensure equal protein loading. 131 Based upon these novel findings, it was postulated that additional signals other than the mutation of the A P C gene might be required or involved in polyp formation. These findings are supported by a recent manuscript that reported a negative correlation between the loss of A P C expression and a nuclear p-catenin signal. Only 29% of the adenomatous polyps that were positive for a nuclear p-catenin signal were negative for A P C immunoreactivity. Iwamoto et al. postulated that perhaps mutation of either Axin or GSK3p might be involved in this process (Iwamoto et al., 2000). Based the work presented in this section, activation of the ILK signaling pathway combined with a loss of functional A P C might be an important mechanism in not only stabilizing the cytosolic levels of p-catenin but as well in facilitating its translocation to the nucleus in the case of FAP. This hypothesis is supported by a recent manuscript, which demonstrated that administration of a selective inhibitor to ILK (KP-SD1) or transfection with a dominant-negative version of ILK could block Tcf-4-dependent gene transcription in cells that are known to harbor mutations in the A P C gene (Tan et al., 2001). 3.1.3. Introduction to the Map kinase Signaling Nexus Map kinase, mainly Erk1 and Erk2, which lie downstream of the cellular proto-oncogene Ras, are known to play a positive role in a myriad of biological events including cell proliferation, differentiation and apoptosis. In the presence of a growth stimulant, activation of these protein kinases results in their translocation into the nucleus where they regulate transcription through transcription factors like Elk-1. 132 3.1.4. Investigation of Erk1 and Erk2 in FAP Patients To determine whether the protein levels of Erk1 and Erk2 are elevated and/or whether the phosphotransferase activity of these two kinases are increased in an analogous fashion to ILK in colorectal adenomas resected from patients diagnosed with FAP, a series of Western blots and protein kinase assays were performed. Briefly, crude protein kinase assays were performed to assess the activity of these isoforms using the E G F receptor peptide, which contains the Thr-669 site specifically phosphorylated by MAP kinase (Salh et al., 1999). The results presented in Figure 15A, which correspond to the mean activity levels of Erk1 and Erk2 in 11 cases of FAP, clearly indicates that there were no overall significant differences in the activity levels between the polyps and the patient matched control samples. To delineate whether there were any differences in the expression levels these two kinases, the relative expression of was evaluated by Western blotting. The data depicted in Figure 15B (represenative of the 11 cases analyzed) clearly demonstrate that there were no observable differences in the protein expression patterns of Erk1 and Erk2 in the polyp samples when compared to the control tissues. In addition to this, there did not appear to be any retardation in the electrophoretic mobility of the respective kinases in any of the samples analyzed which is indicative of a post-translation modification such as phosphorylation. A decrease in the electrophoretic mobility of Erk1 and Erk2 is believed to coincide with the activation of the former. 133 Figure 15. Examination of Erk1 and Erk2 expression/activity in FAP. A. Examination of Mean Erk activity in 11 patients with FAP. Mean crude E G F receptor phosphotransferase activity performed to specifically examine Erk1 and Erk2 activity. B. Examination of Erk1 and Erk2 expression in patients with FAP. Anti-Erk1-CT immunoblot; examining the expression levels of Erk1 and Erk2 in colorectal adenomas and the corresponding control samples. 134 6000 c o 4000 iS 5000 o Q. C o c ° - 3" 3000 o E o w rr UL o LU 2000 1000 Control Polyp IB: ERK1-CT Erk 1 Erk2 C5 P5 C6 P6 C7 P7 C9 P9 135 Based upon this data, it appears that the Map kinases, Erk1 and Erk2 are not dysregulated in colorectal adenomas and thus might not play a role in this disease. In addition to this, another report demonstrated that the activity of Erk1 and Erk2 was down-regulated in human colon cancers when compared to the normal control tissue (Attar et al., 1996). This is also supported by evidence demonstrating that mutations in Ras do not coincide with changes in these protein kinases in human pancreatic cancer (Yip-Schneider et al., 1999). Approximately, 90% of these cancers harbor mutations in the Ras oncogene. Thus, it is plausible to hypothesize that Ras might mediate its oncogenic effects through other signaling pathways, like the PI3-kinase pathway. Ras has not only been demonstrated to act upstream of PI3-kinase but as well PI3-kinase activity has been reported to be required for Ras-mediated transformation. In support of this, PI3-kinase has been recently reported to be hyperactive in colonic cancers (Benistant et al., 2000) as well as play a role in the intestinal cell differentiation (Tuhackova et al., 1999; Wang et al., 2001). 3.1.5. Introduction to the protein kinase CK2 The protein kinase CK2 has been reported to play a critical role in a number of biological processes. Since it's discovery, it has been demonstrated to phosphorylate well over 100 different substrates. More recently, transgenic over-expression of the catalytic subunit of protein kinase CK2 has been reported to result in the development of lymphomas as well as breast cancer in mice (Kelliher et al., 1996; Landesman-Bollag et al., 1998; Nusse et al., 1990; Seldin 136 and Leder, 1995). In addition to this, CK2 has been demonstrated to have a positive influence on the Wingless/Wnt signal transduction (Song et al., 2000a). However, whether this protein kinase functions as an oncogene or a tumor suppressor gene in vivo is currently unresolved. 3.1.6. Investigation of Protein Kinase CK2 in patients with FAP The protein kinase CK2 has been reported to be up-regulated in a number of transformed cell lines and rapidly proliferating tissues. Furthermore, this kinase has been previously reported to be over-expressed and exhibit increased casein phosphotransferase activity in colorectal adenomas (Munstermann et al., 1990). Thus, it was hypothesized that evaluating the enzymatic activity and protein expression of this kinase in polyp lesions might serve as an internal control to validate any findings that were potentially novel. The results presented in Figure 16A, which are representative of the 11 cases analyzed, demonstrated that not only is the expression of the CK2a and a' subunit elevated but as well its casein phosphotransferase activity is increased up to 2-fold. To delineate whether changes in CK2 activity directly correlated with increases in the protein expression, the % change in CK2 activity above the control was compared with the % change in CK2 expression above the control. 137 Figure 16. Increased C K 2 activity in F A P patients. A. Evaluation of CK2 activity and expression. Upper panel, crude casein phosphotransferase activity performed to specifically examine protein kinase CK2 activity. Lower panel; anti-CK2a, examining the expression levels of CK2a and CK2a' in colorectal adenomas and the corresponding control samples. B. Correlation between CK2 activity and expression. Data represented as % change in CK2 expression or activity above the control. 138 2000 % 1800 C3 P3 C5 P5 C6 P6 Case Number IB: CK2 C3 P3 C5 P5 C6 P6 Case Number 139 140 indicated that although changes in the expression and activity followed similar trends, there was not a direct correlation between the two of these. It is possible that CK2 plays a critical role in the development of human colon cancer. Recent evidence indicates that transgenic over-expression of the catalytic subunit of this protein kinase influences mammary carcinogenesis as well as the development of lymphomas (Song et al., 2000a). Conceivably, intervention with a selective inhibitor to this protein kinase might aid in the treatment of familial adenomatous polyposis based upon the data presented here and by others (Munstermann et al., 1990). 3.1.7. Investigation of the ILK signaling module in sporadic polyps To determine whether the dysregulation of ILK signaling observed in FAP patients also occurred in sporadic polyps, ten cases in which sporadic polyps were evident were selected for and the protein expression of ILK was analyzed using an immunohistochemical approach. The representative micrographs depicted in Figure 17A-F demonstrate that ILK expression is dramatically increased in the sporadic polyps when compared to the adjacent control tissue based upon the intensity of the red chromagen. To delineate whether these changes in ILK expression were statistically significant, the staining intensity was scored by 3 independent individuals as outlined in the Materials and Methods section. The data indicates that there was approximately a 1.7 fold difference in the mean expression of ILK between the control and polyp samples. These impressive changes were determined to highly significant (p<0.001). 141 Figure 17. Over -express ion of ILK in S p o r a d i c Po lyps . Examination of ILK expression in sporadic polyps by immunohistochemical analysis. Panels A - C ; One case examining ILK expression in the control crypts versus adenomatous crypts at a lower magnification (panel A, 100X). Panels B and C examining ILK expression in the same crypts at a higher magnification (200X). Panels D-F. 3 additional representative cases examining ILK expression. Panel G. Mean expression of ILK in polypoid lesions from 10 cases of sporadic colorectal cancer. 142 Control Polyp 3.2. Summary of the Results The results from these studies demonstrate that ILK, a protein kinase known to have similar consequences to the mutation of A P C on the sub-cellular distribution of p-catenin, is over-expressed and exhibits increased MBP phosphotransferase activity in polyp samples from patients diagnosed with FAP with respect to corresponding control tissue. Elevated ILK activity appeared to correlate with changes in the phosphorylation status of GSK3p. In this regard, ILK has been demonstrated to phosphorylate GSK3p; phosphorylation results in the inhibition of the latter. To corroborate this data, the expression of G A P D H , a 'house-keeping gene' and the protein expression/activity levels of protein kinase CK2 were examined. To add to this, the data in these studies demonstrated that changes in ILK expression were evident in sporadic polyps derived from patients with colorectal cancer. 3.3. General Conclusions and Future Directions ILK is a recently identified protein serine/threonine kinase that has been reported to regulate a myriad of biological functions. These include the induction of anchorage-independent cell cycle progression, the enhancement of tumorigenicity in nude mice and the activation of Tcf-4-dependent gene transcription by specifically promoting the translocation of p-catenin into the nucleus. Dysregulation of the APC-p-catenin signaling nexus is believed to initiate the development of not only adenomatous lesions in FAP patients but as well 145 sporadic cancers. Since ILK has been shown to have a positive influence on APC-p-catenin signaling, perhaps activation of the former is an important early event in the development of adenomatous lesions or even in the development of colon cancer in general. Furthermore, modulation of this kinase with a selective inhibitor may be equally efficacious as non-steroidal anti-inflammatory agents like sulindac in the treatment of the polyposis disorders. To assess the importance of this kinase in the development of these pre-neoplastic lesions it would be of interest to specifically over-express wild-type ILK in the colonic epithelium of mice to determine whether these mice develop adenomatous lesions in a similar fashion to the human disease using the Cre-lox system. Furthermore, it would be of therapeutic interest to delineate whether modulation of ILK activity with a selective inhibitor might cause regression of polypoid lesions using the Min mouse animal model that is believed to mimic the human FAP condition. In addition to this, it would be of scientific interest to delineate what regulates the expression of this protein kinase. Is it possible that A P C regulates ILK expression and activity? 146 Chapter 4. Investigation of Signaling Modules in Colorectal Cancer. 4.1. General Introduction Mutation of the adenomatous polyposis coli (APC) gene is an integral event in the genesis of colorectal cancer (Fearon et al., 1990; Kinzler et al., 1991). Mutation of this gene results in the expression of a protein that is no longer able to form a complex with Axin, p-catenin and GSK3p (Barker et al., 2000; Behrens et al., 1998; Rowan et al., 2000; Sparks et al., 1998). Consequently, there is an increase in the cytosolic levels of p-catenin. Stabilization of the latter is believed to result in its translocation to the nucleus where it binds to the Tcf-4 (T-cell factor) family of transcription factors resulting in the expression of a number of different genes that have been implicated in oncogenesis. These include cyclin D1, c-myc and the matrix metalloproteinase (MMP)-7 (Crawford et al., 1999; He et al., 1998; Shtutman et al., 1999). However, whether mutation of A P C alone is sufficient in dysregulating p-catenin signaling or whether additional signals are required for this disruption are currently unclear. In this regard, a prominent nuclear p-catenin signal was observed in cells that over-express the integrin-linked kinase (ILK), duplicating the events associated with the mutation of A P C . Translocation of p-catenin accompanied the activation of Tcf-4 dependent gene transcription (Novak et al., 1998). 147 4.1.1. ILK-C l in i ca l Perspectives ILK is a proto-oncogene that has not only been described as a putative immunohistochemical marker for the identification of Ewing's sarcoma (ES) and primitive neuroectodermal tumors (PNET) but as well, increased expression of the protein has been demonstrated to be inversely related to the 5 year survival rate in prostate cancer (Chung et al., 1998; Graff et al., 2001). In Chapter 3, the results demonstrated that ILK signaling is dysregulated in patients diagnosed with familial adenomatous polyposis (FAP) as well as sporadic polyposis (Marotta et al., 2001). To delineate whether this pathway was also disrupted in sporadic cases of colon cancer, this signaling nexus was investigated in both primary cancers as well as metastatic deposits within regional lymph nodes. 4.1.2. ILK expression is dysregulated in sporadic cases of colon cancer In chapter 3, ILK signaling was demonstrated to be dysregulated in polypoid lesions resected from patients diagnosed with the autosomal dominant condition, FAP. To delineate whether ILK signaling was also dysregulated in sporadic human colon cancer, the protein expression levels of ILK was initially investigated by immunohistochemical analysis. A total of 10 cases of sporadic C R C were analyzed. Intriguingly, all 10 cases showed dramatic increases in the expression of this protein kinase in the cancerous crypts when compared with the corresponding normal control. Representative micrographs are depicted in Figure 18. 148 Figure 18. Over -express ion of ILK in S p o r a d i c Co lorecta l C a n c e r s . Panel A; One case examining ILK expression in the control crypts versus cancerous crypts at a lower magnification (100X) as well at a higher magnification (200X). Panels B-l; 4 additional representative cases demonstrating enhanced ILK expression in the cancerous lesions (C, E, G, I) when compared with the normal control (B, D, F, H). 149 150 In Figure 18 A (upper micrograph), it is evident that the expression of ILK is increased in the region of the tumor with respect to the normal. Upon higher magnification, it is evident that ILK is predominately expression within the malignant acini based upon the intensity of the chromagen. This increase in ILK expression is further corroborated by the representative micrographs depicted in Figure 18 B-l, which represent 4 individual cases. These results further demonstrate that ILK expression is increased in the cancerous lesions (panels C, E, G, I) when compared with the adjacent control tissue (panels B, D, F, H). These changes in expression were evident in all the cases; however, it should be noted that approximately 5-10% of the neoplastic crypts within the same lesions displayed no change in the expression of ILK when compared with the normal controls. To determine whether the over-expression of ILK within these lesions was statistically significant, the relative staining intensity for each case was scored. Statisitical analysis of the data for the 10 cases revealed that the increase in ILK in the neoplastic crypts was highly significant (p<0.00005). There was approximately a 3-fold increase in the expression of ILK in the cancerous lesions (2.13) when compared to the normal adjacent tissue (0.75), see Figure 23. 4.1.3. ILK activity is increased in colonic cancers In Chapter 3, it was determined that over-expression of ILK coincided with an increase in the M B P phosphotransferase activity in polyps from patients with FAP. Based upon the immunohistochemical data obtained in the previous 151 section, the next objective in these studies was to determine whether ILK activity was also dysregulated in human colon cancer. In order to elucidate this, the activity and expression levels of ILK were examined in a total of 32 cases of sporadic human colon cancer. It is worth noting that in addition to quantifying the protein levels by Bradford analysis, the expression of G A P D H was evaluated for each patient match control and tumor sample to ensure for equal protein loading. The results from these studies, which are representative for all of the cases which demonstrated changes in ILK activity are presented in Figure 19A. 152 Figure 19. ILK Activity is Enhanced in Colorectal Cancers. A. Examination of ILK expression/activity in sporadic human colon cancer. Upper panel, ILK/MBP phosphotransferase densitometry. Immunoprecipitated ILK M B P phosphotransferase performed in triplicate. Middle panel, anti-ILK immunoblot, examining the protein expression of ILK in the tumor and the corresponding control sample. Lower panel; anti-Erk1-CT immunoblot, examining the expression of Erk1 and Erk2 in the control samples versus the corresponding tumor. B. Correlation between ILK activity and expression. Data represented as % change in ILK expression or activity above the control. 153 0.3 C1 T1 C2 T2 C3 T3 Case Number C1 T1 C2 T2 C3 T3 Case Number 154 The data indicated that there was anywhere from a 2-15 fold increase in immunoprecipated ILK/MBP phosphotransferase activity in the cancerous lesions when compared to the normal adjacent control tissue in general. In fact, these differences in ILK activity were apparent in 20 out of the 32 cases (63%) analyzed. To delineate whether changes in ILK expression could account for these observed changes in ILK activity, the relative protein expression of ILK was examined by Western blotting. The data indicated that changes in ILK activity were likely due to changes in ILK expression. To determine whether changes in ILK activity directly correlated with increases in the relative expression of ILK, the percent change in ILK activity above the control was compared with the percent change in ILK expression (see Figure 19B). Although, both the expression and activity followed similar trends, there did not appear to be a linear correlation between the two of these. It is possible that additional factors are involved in regulating the activity of ILK in this disease. As outlined above, 12 of the cases did not display any measurable differences in the activity levels of ILK. It is possible that this discrepancy between the expression and activity of ILK is attributable to such factors including tissue sampling or the extent of tumor vascularization. A more systematic analysis of protein/activity relationships within the tumor will be required to resolve this definitively. Despite these shortcomings, it is plausible to assume that over-expression of ILK might be an important event in the development of colon cancer in general. Importantly, increased expression of ILK appeared to be independent of differences in the expression of Erk1 and Erk2 (see Figure19A), in keeping with the findings in Chapter 3. 156 In Chapter 3, it was demonstrated that over-expression and increased ILK/MBP phosphotransferase activity correlated with changes on downstream targets involved in the ILK signaling nexus. To delineate whether these changes occurred universally in colon cancer, the effects of ILK on downstream targets was investigated. For the representative patient (similar results were obtained in the other 19 cases which displayed changes in ILK activity), changes in ILK expression appeared to account for the observed differences in ILK activity (see Figure 20). In addition to this, changes in ILK expression and activity appeared to coincide with differences in the expression of p-catenin. Furthermore, the results demonstrated that elevated expression of ILK appeared to be associated with a general increase in the expression of Lef-1. Analysis of ILK and Lef-1 expression in 9 separate cases where sufficient lysates were present revealed that there was a highly significant linear correlation between elevated expression of ILK and an increase in Lef-1 expression (p<0.005, see Figure 21). Over-expression of ILK has been reported to have a positive influence on Lef-1 expression (Novak et al., 1998). 157 Figure 20. Ef fects of ILK o n Downst ream Targets . ILK activity is increased in the polyp compared with its respective control (representative MBP autoradiogram). Anti-ILK immunoblot; examining expression of ILK. Anti-p-catenin immunoblot; examining expression of p-catenin. Anti-Lef-1 immunoblot; examining expression of Lef-1. Anti-P-GSK3p immunoblot; examining phosphorylation status of GSK3p. Anti-GSK3p immunoblot; examining expression of GSK3p. Ant i-GAPDH immunblot; used as an internal control for loading. The results are representative for the 20 cases, which displayed increases in ILK activity. 158 C1 T1 159 In support of the increased MBP phosphotransferase activity, we detected an impressive increase in the phosphorylation of GSK3p by immunoblotting with a phosphospecific GSK3p Ser9 antibody and by a gel-retardation in the GSK3p protein itself. Phosphorylation of GSK3p at this serine residue correlates with a decrease in its enzymatic activity. Since GSK3p is known to regulate the levels of p-catenin under normal circumstances, is it possible that inhibition of the enzymatic activity of this kinase is also necessary (in. addition to mutations in APC) for the dysregulation of Tcf4-dependent gene transcription as well as in the initiation of colorectal carcinogenesis? The expression of G A P D H was assessed to control for protein loading for each case analyzed. 160 Figure 21. Positive Correlation Between ILK Expression and the Levels of Lef-1 in Colorectal Cancers. The protein band intensities for both ILK and Lef-1 were quantitated by densitometry and the results were analyzed using linear regression. (* represents 2 or more data points). 161 0 1 2 3 4 5 ILK expression 162 4.1.4. ILK expression in regional lymph nodes Over-expression of ILK has been demonstrated to have a positive influence on the invasive phenotype of cancer cells. Specifically, ILK has been reported to alter the invasiveness of cells in culture by up-regulating the expression of MMP-9 (Troussard et al., 2000). To determine whether the expression levels of ILK were further increased in metastatic deposits within regional lymph nodes, ILK immunohistochemical analysis was performed. The representative results, which are presented in Figure 22, demonstrated that ILK is predominantly expressed in the malignant acini of these lymph nodes; however, a small proportion of the signal was detected in the inflammatory component of these glands. To determine whether the expression of ILK was significantly increased in these glands in comparison to the primary tumor, the expression of ILK was quantitated as outlined in the Materials and methods section. The data (see Figure 23) indicated that the expression of ILK is significantly (p<0. 00005) increased approximately 4-fold in the malignant acini of the regional lymph nodes (2.67) when compared to the normal colonic crypts (0.75). Additionally, statistical analysis indicated revealed that there was a significant difference in the expression of ILK between the primary cancer and the metastatic deposits within regional lymph nodes. Based upon the data obtained in Chapters 3 and 4, it appears that ILK expression is not only altered very early during the development of human colon cancer but as well during the metastatic process. It is possible that over-expression of and increased activity of ILK is a pivotal step in altering the 163 metastatic potential of cancer cells. In support of this, it has been demonstrated that over-expression of ILK leads to a down-regulation of E-cadherin; the latter is important in regulating cell-to-cell interactions (Wu, 1999). Furthermore, administration of a selective inhibitor of ILK has been reported to result in the induction of E-cadherin expression (Tan et al., 2001). Thus, activation of ILK might alter cellular architecture making cancer cells more prone to invade through the surrounding basement membrane. Certainly, ILK has been reported to promote epithelial to mesenchymal transition in mammary epithelium (Somasiri et al., 2001). 164 Figure 22. ILK Expression in Positive Lymph Nodes. Panel A, expression of ILK in a lymph node negative for tumor cell infiltration. Panels B-E, expression of ILK in 4 tumors with positive lymph nodes. Over-expression of the ILK protein has been demonstrated to have a positive. 165 166 Figure 23. ILK E x p r e s s i o n is S igni f icant ly Increased in Co lorecta l C a n c e r s . Mean expression of ILK in 10 cases of colon cancer with positive lymph nodes. 167 168 4.2. Summary of Results ILK (Integrin-linked kinase) is a protein serine/threonine kinase that has been reported to be involved in a number of biological events including anchorage-independent cell cycle progression (Radeva et al., 1997) and tumor cell invasion (Troussard et al., 2000). In Chapter 3, the ILK signaling nexus was demonstrated to be dysregulated in the hereditary condition FAP (familial adenomatous polyposis). To validate the relevance of this finding in human colon cancer, we examined the protein expression of ILK in ten cases by immunohistochemical analysis. The data indicated that ILK was significantly hyperexpressed in the malignant glands from the primary cancer (p<0. 00005) as well as in the malignant acini in the regional lymph nodes (p<0.00005) when compared to the adjacent normal control tissue for each case. Statistical differences were also observed between the primary and metastatic lesions. In addition to this, over-expression of ILK appeared to correlate with not only an increase in the MBP phosphotransferase activity of the immunoprecipitated ILK but as well appeared to correlate with changes on downstream targets like GSK3p. Furthermore, there was a direct correlation between the protein expression of ILK and the protein levels of Lef-1 (p=0.002). 4.3. General Conclusions and Future Directions The current dogma dictates that mutation or dysregulation of one of the components involved in a signaling pathway should be sufficient to promote its functional or biological effect. The fact that ILK, an enzyme that mimics the 169 effects of a mutation in A P C on the sub-cellular distribution of p-catenin, is over-expressed and exhibits an increased M B P phosphotransferase activity in colon cancer and these changes correlate with changes on downstream targets suggests that the dysregulation of this pathway is more than likely an important event in colorectal carcinogenesis. As in Chapter 3, these changes appeared to be independent of changes in the expression and activity of Erk1 and Erk2, two downstream targets of Ras. Ras transformation has been documented to be dependent upon PI3-kinase. Perhaps in the context of human colon cancer, mutation of Ras results in the activation of the PI3-kinase/ILK signaling nexus. Certainly, PI3-kinase has been demonstrated to be dysregulated in this disease. To assess the influence of this kinase in the development of colon cancer it would of interest to specifically over-express wild-type ILK in the colonic epithelium of wild-type A P C mice or in the Min mouse model to determine whether these mice develop colorectal cancer. In addition to this, it would be useful to determine whether ILK played a role in mediating drug resistance. Finally, it would be importance to determine whether administration of a selective inhibitor to ILK alone or in combination with other conventional chemotherapeutic drugs would be improve the 5 year survival rates associated with this disease. 170 Chapter 5. Modulation of Signaling Cascades by NSAIDs. 5.1. General Introduction It is well established that NSAIDs such as sulindac play a pivotal role in reducing the incidence of colorectal cancer and that this effect more than likely occurs at the stage of the aberrant crypt focus or in polyp development. Sulindac, the pro-form of the drug, has been reported to regress adenomas in FAP patients and suppress their growth in the Min mouse animal model (Beazer-Barclay et al., 1996; Chiu et al., 1997). However, the precise mechanism by which these agents elicit their anti-neoplastic properties is currently unresolved. NSAIDs are believed to exert their anti-tumor properties by specifically inhibiting the biochemical activity of the cyclooxygenase enzymes (DuBois and Smalley, 1996; Giardiello et al., 1995; Smalley and DuBois, 1997; Smith et al., 2000). Interestingly, Cox-2 has been demonstrated to be over-expressed in both pre-malignant adenomatous lesions as well as colorectal cancer; however, the precise role of this over-expression is currently unclear (Eberhart et al., 1994). Cox-2 has been implicated in not only regulating angiogenesis but as well preventing apoptosis. However, controversy exists over the latter. In this regard, one report demonstrated an inverse relationship between the expression of Cox-2 and the induction of apoptosis (Tsujii and DuBois, 1995). While another showed that exogenous expression of Cox-2 in a variety of cell lines resulted in a cell cycle arrest. This arrest occurred at the level of G 0 / G i ; the cells did not undergo apoptosis (Trifan et al., 1999). To add to this uncertainty, sulindac 171 sulfone, one of the metabolic metabolites of sulindac that lacks Cox-2 inhibitory properties, has also been reported to promote apoptosis (Piazza et al., 1997b). Based upon this information, it appears that NSAIDs may not exert their effects by specifically inhibiting Cox function alone. Recently, a number of studies have shown that NSAIDs are able to modulate a number of other biochemical pathways and that inhibition of these might be relevant to understanding the mechanism of action of these drugs. Specifically NS-398, a Cox-2 specific inhibitor has been reported to promote apoptosis by promoting the release of cytochrome C and the subsequent activation of caspase-9 and caspase-3 (Li et al., 2001b). In addition to this, these agents have been demonstrated to modulate N F - K B signaling. Sulindac has been shown to inhibit the phoshorylation of I K B by specifically inhibiting the I kappa kinases (IKK) a and p (Yamamoto et al., 1999b). Furthemore, Celecoxib, a Cox-2 specific inhibitor, has also been reported to modulate downstream events involved in the PI3-kinase signaling pathway. Specifically, this agent was shown to inhibit PKB activity (Hsu et al., 2000). This effect was not dependent upon PI3-kinase. In addition to this, these agents have also been shown to inhibit PPAR5 (He et al., 1999) as well as alter the Bax to Bcl -X L ratio (Zhang et al., 2000). Taken together, these studies indicate that alternative NSAID targets do exist, and understanding the mechanism of action of these drugs may aid in defining future therapy. In the present study, it was hypothesized that since ILK signaling was demonstrated to be dysregulated in polyposis and that controversy exists over 172 the precise mechanism(s) by these agents elicit their anti-neoplastic effects, that perhaps these drugs might influence the ILK signaling nexus by targeting this kinase directly. 5.2. ILK and NSAIDs A number of studies have demonstrated the importance of ILK in oncogenesis. To delineate whether NSAIDs might inhibit the enzymatic activity of ILK directly, in vitro kinase assays were performed using recombinant ILK in the presence of a number of different NSAIDs. The results presented in Table 1 clearly demonstrate that NSAIDs are able to modulate ILK activity directly in vitro at physiological relevant doses present within the colon (Duggan et al., 1980). At 500 y.M, sulindac sulfone, which was determined to be one of the most efficacious agents, inhibited in vitro ILK activity by 49% whereas the Cox-2 specific inhibitor, NS-398, at a similar dose, inhibited ILK by 41%. Sulindac, the pro-form of the drug, at 1 mM inhibited ILK by 33%. The sulfide derivative of this drug did not appear to have any effect on ILK activity in vitro (data not shown). These results suggest that although the sulfide derivative of sulindac is believed to be the major effector in tumor regression, perhaps the other metabolic metabolite of sulindac, sulindac sulfone, might also be involved in mediating the anti-tumor effects of these drugs. A S A also appeared to inhibit in vitro ILK activity. At 5 mM, A S A inhibited the activity of this kinase by a modest 19%. 173 Table 1. The effects of various NSAIDs on in vitro ILK activity Treatment: Effects of NS-398 on in vitro ILK activity NS-398 Mean Activity % Inhibition Control 25776 +645 25 uM 24656 + 2152 4 50 uM 24260 + 2255 6 100 uM 19864 + 1008 23* 500 LJM 15084 + 3627 41** Treatment: Effects of sulindac and sulindac sulfone on in vitro ILK activity Sulindac Mean Activity % Inhibition Control 23958 + 512 50 uM 21145 + 1145 12* 100 U M 19201 +2206 20* 500 laM 17652 + 569 20**** 1 mM 16165 + 2417 33** 5 mM 5908 + 750 Sulindac sulfone 50 uM 18874 + 1715 21** 100 uM 16020 + 1157 33**** 500 uM 12156 + 518 49**** 1 mM 11348 + 43 53**** Treatment: Effects of ASA on in vitro ILK activity ASA Mean Activity % Inhibition Control 32507 + 4165 500 uM 29390 + 2730 10 1 mM 30527 + 3590 6 5 mM 26397 + 4776 19 10 mM 19702 + 2417 39* Recombinant ILK was subjected to in vitro kinase assays in the presence of varying dosages of a number of NSAIDs for 15 min interval as outlined in the Materials and Methods section. Results were performed in triplicate on 3 separate occasions (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). 174 To further explore the possibility that NSAIDs might modulate ILK activity, the ability of these agents to block ILK in vivo was tested using physiologically relevant doses (Duggan et al., 1980). Briefly, Caco-2 cells were pre-treated with either A S A (5 mM) or sulindac (1 mM) for 2 h and then stimulated with serum for 10 minutes. The results presented in Figure 24 indicate that these agents significantly inhibit the serum-induced activation of ILK. Specifically, administration of sulindac resulted in an impressive 87% inhibition of the serum-induced activation of ILK while A S A resulted in a 68% reduction. Both of these effects were found to be statistically significant, p=0.02 and p=0.001, respectively. The inhibitory effects of these agents are further depicted in the M B P autoradiograph, which clearly demonstrates a difference in the phosphorylation of MBP in response to these drugs. The expression of ILK was assessed to demonstrate equal protein loading. Consequently, NSAIDs are able to modulate the specific activity of ILK since similar levels of the ILK protein were detected in all of the conditions. Based upon the in vitro and in vivo data obtained in these studies, it would appear that NSAIDs are able to modulate the ILK signaling nexus. However, whether or not NSAIDs effects on ILK activity are direct or indirect would require further delineation. Due to the differences in the efficacy of these agents in vitro and in vivo, it would appear that these drugs might influence other signaling molecules or pathways that ultimately regulate ILK activity, such as PI-3 kinase. However a recent report demonstrated that celecoxib, a Cox-2 specific inhibitor, could modulate PKB activity, a down-stream target of ILK, independently of 175 effects on PI-3 kinase (Hsu et al., 2000). Thus the effects of these agents on ILK might not be due to inhibition of PI-3 kinase. To address this issue, one would have to assess the effects of the NSAIDs used in these studies on PI-3 kinase. Furthermore, additional studies are required to delineate how the activity of ILK is regulated in vivo and by which signaling molecules. Once this information has been acquired, one could attempt to specifically evaluate the effects of these drugs on those signaling molecules. 176 F igure 24. Inhibition of ILK s igna l ing with NSAID administrat ion. Caco-2 cells were treated as outlined in the Materials and Methods section. Upper panel; ILK immunoprecipitation and immune complex kinase assays. Middle panel; representative MBP autoradiogram. Lower panel, ILK immunoblot. Similar findings were observed on at least three different occasions. (*p=0.02 and **p=0.001). 177 0 1.6 i E 10% Serum + + + A S A + Sulindac + 178 To substantiate the effects of NSAIDs on the inhibition of ILK in vivo, the phosphorylation status of GSK3p(a down-stream target of ILK), which reflects the biochemical activity of the latter, was assessed using a phospho-specific GSK3p Ser9 antibody. ILK has been reported to phosphorylate this site on GSK3p and directly inhibit its kinase activity. The results shown in Figure 25 indicate that inhibition of ILK by NSAIDs correlates with a decrease in the inhibitory phosphorylation of GSK3p. In fact, the phosphorylation levels, which were determined to be statistically significant (p=0.02), were almost reduced to basal levels. These results are further depicted in the GSK3p Ser9-immunoblot. It is possible that a reduction in Ser9 phosphorylation could coincide with an increase in the specific activity of GSK3p based upon the observation that there were similar amounts of the protein in all conditions. Perhaps, an increase in GSK3p activity in response to NSAIDs plays a critical role in the regulation of p-catenin. Expression of GSK3p and G A P D H were performed to demonstrate equal protein loading. 179 Figure 25. Modulat ion of ILK activity by NSAIDs is assoc ia ted with c h a n g e s in the phosphory la t ion status of G S K 3 p . Caco-2 cells were treated as outlined in the Materials and methods section. Upper panel, quantification of GSK3p Ser9 phosphorylation. Anti-P-GSK3p; analysis of Ser9 phosphorylation. Anti-GSK3p; analysis of GSK3p expression. Ant i -GAPDH; analysis of G A P D H expression. (#p=0.02). 180 E o V c Q D CO > * 4= CO J5 O o> 6* _ c QL (/) O - C Q_ 0.6 0.5 0.4 0.3 0.2 0.1 P-GSK3P GSK3P protein 10% Serum A S A Sulindac G A P D H + + 181 5.3. Effects of NSAIDs on Tcf-4-Mediated Gene Transcription Mutation of the A P C gene has been documented to result in the dysregulation of p-catenin signaling (Kinzler and Vogelstein, 1996). When this molecule translocates to the nucleus it binds with the Tcf-4 family of transcription factors, which results in the up-regulation of a number of different proteins that are believed to play a critical role in tumorigenesis. More importantly, over-expression of ILK has been demonstrated to have a similar effect on the sub-cellular localization of p-catenin, in that when ILK was over-expressed in rat intestinal epithelial cells the majority of p-catenin resided in the nucleus (Novak et al., 1998). This translocation accompanied the activation of Tcf-4-dependent gene transcription. Moreover, administration of a selective inhibitor to ILK or transfection with the dominant-negative version of ILK has been reported to inhibit Tcf-4-dependent gene transcription (Tan et al., 2001). The precise mechanism involved in this down-regulation is currently unclear. 5.3.1. Specific Effects of NSAIDs on Tcf-4-Reporter Activity Based upon the observation that ILK modulates Tcf-4-dependent gene transcription (Novak et al., 1998; Tan et al., 2001) and that NSAIDs are able to modulate ILK signaling, it was postulated that NSAIDs might also influence Tcf-4-dependent gene transcription. To address this, Caco-2 cells were transiently transfected with either the Topflash or the mutant Fopflash reporter in the presence of the pRenilla control reporter. The data represented in Figure 26 clearly indicates that adminstration of either A S A or sulindac for 24 h leads to a 1 8 2 down-regulation of Tcf-4-dependent gene transcription. Specifically, sulindac (1 mM) reduced transcription by 61% (p<0.01) whereas administration of A S A (5 mM) inhibited it by 40% (p<0.001), respectively. Similar findings were observed in DLD-1 and SW480 cells (data not shown). The accompanying pRenilla data indicates that the observed effects were specific for Tcf-4 since there were no observable differences between any of the control and treated groups. These results are in agreement with a recent manuscript showing that either A S A or indomethacin can modulate this pathway (Dihlmann et al., 2001). 183 Figure 26. NSAIDs Inhibit Tcf-4/l_ef-1 Mediated G e n e Transcr ip t ion . Caco-2 cells were treated and assayed for transcriptional activity with a luminometer as outlined in the Materials and methods section. Data are expressed as relative light units (RLU). All experiments were done in triplicate on at least three independent occasions. ( * p<0.01, ** p< 0.001). 184 8000 4^ 7000 6000 5000 £ 4000 3000 2000 1000 oJ-i Luc Ren Luc Ren Luc Ren Luc Ren T O P F O P A S A Su l i ndac 185 5.3.2. Effects of NSAIDs on the Expression of Cyclin D1 In the previous section, it was demonstrated that both A S A and sulindac, two non-steroidal anti-inflammatory agents, were able to modulate Tcf-4-dependent gene transcription. To confirm these effects on transcriptional activity, the protein expression of cyclin D1, a specific target of ILK, was examined in response to 24 h treatment with either of these agents in Caco-2 cells (D'Amico et al., 2000). Furthermore, the ability of other NSAID agents to down-regulate cyclin D1 was also assessed. To exclude the possibility that down-regulation of cyclin D1 was due to changes in cell viability, dose reponse curves were performed on each of the agents in question. 5.3.2.1. A S A Modulates Cyclin D1 Expression - Independent of Effects of A S A on Cell Viability As outlined above, administration of A S A led to a 40% reduction in Tcf-4-dependent gene transcription. To corroborate the effects of A S A on transcription, the protein expression of cyclin D1 was examined in response to administration of varying dosages of A S A for 24 h. The results presented in Figure 27A demonstrate that administration of A S A leads to a down-regulation in the protein expression of cyclin D1 with the most prominent effects being between 1 mM to 5 mM. Expression of G A P D H was examined to demonstrate that the effects of A S A were specific for cyclin D1 based upon comparisons of protein loading. To further validate that A S A blocked Tcf-4-dependent gene transcription, independently of effects on cell viability, the percentage of viable cells was measured using the 186 MTS assay, after 24 h of drug exposure to mirror the transcriptional studies in Caco-2 cells. The results depicted in Figure 27B demonstrate that administration of A S A (1 mM) has no appreciable effect on cell viability of Caco-2 cells, but at this dose there was a significant reduction in the expression of cyclin D1. Based upon the results from this study, it appears that administration of A S A can influence Tcf-4-mediated gene transcription independent of its effects on cell viability or perhaps down-regulation of transcription might be an important early event involved in the initiation of cell death. In this regard, down-regulation of p-catenin signaling has been a proposed to be an early event in the induction of apoptosis (Steinhusen et al., 2000). 187 Figure 27. Admin is t rat ion of A S A L e a d s to a Down-regulat ion of C y c l i n D1 -Independent of Ef fects of A S A o n Cel l Viabil ity. A A S A blocks cyclin D1 expression in a dose-dependent fashion. Caco-2 cells were incubated in the presence of varying dosages of A S A (50 iM to 5 mM) for 24 h as outlined in the Materials and methods section. B Transcriptional effects are independent of effects on cell viability. Cell viability was assessed in response to varying dosages of A S A as outline in the Materials and methods section. Results are represented as % change over the control. Similar results were obtained on at least three independent occasions. 188 189 5.3.2.2. Sulindac Modulates Cyclin D1 Expression - Independent of Effects of Sulindac on Cell Viability The chemopreventative effect of sulindac is widely recognized; however, the precise mechanism by which this agent elicits its effects is unclear presently. It has been hypothesized that inhibition of Cox function is responsible for its anti-neoplastic effects but the recent literature tends to suggest that alternative mechanism(s) might be involved. In the previous section, the data demonstrates that sulindac can modulate Tcf-4-dependent gene transcription. The results presented in section 5.2.2. indicate that 1 mM sulindac inhibited Tcf-4-dependent gene transcription by 61%. To substantiate the effects of sulindac on p-catenin mediated transcription, the expression levels of the cyclin D1 protein was assessed in response to 24 h administration of this agent. The results presented in Figure 28 demonstrate that there was a dramatic decrease in the levels of cyclin D1, between 500 ^M to 1 mM. To demonstrate that these changes in the protein levels of cyclin D1 were independent of sulindac's effects on cell viability, dose response MTS studies were performed on the Caco-2 cell line. The reduction in the levels of cyclin D1 were determined to be independent of the effects of sulindac on viability using the Caco-2 cell line. It is apparent that there was a significant reduction in the levels of cyclin D1 at 500 | aM , however there was little or no effect on the viability of the cells at this dose. 190 Figure 28. Admin is t rat ion of S u l i n d a c L e a d s to a Down-regulat ion of C y c l i n D1 - Independent of Effects of Su l indac o n Cel l Viabil ity. A Sulindac blocks cyclin D1 expression in a dose-dependent fashion. Caco-2 cells were incubated in the presence of varying dosages of sulindac (50 yM to 1 mM) for 24 h as outlined in the Materials and method section. B Transcriptional effects are independent of effects on cell viability. Cell viability was assessed in response to varying dosages of sulindac as outline in the Materials and methods section. Results are represented as % change over the control. Similar results were obtained on at least three independent occasions. 191 192 5.3.2.3. Metabolic Metabolites of Sulindac Block Cyclin D1 Expression -Independent of Effects of These Agents on Cell Viability Sulindac is the pro-form of the drug that lacks Cox-2 inhibitory properties. Intestinal bacteria and the liver metabolize sulindac to sulindac sulfide and sulindac sulfone (Thompson et al., 1995). Sulindac sulfide is believed to be the metabolite responsible for eliciting the effects of this drug by inhibiting Cox-2 function. Recently however, the sulfone derivative, which lacks Cox inhibitory properties, has been reported to mimic the anti-neoplastic effects of sulfide in that administration of the former resulted in the induction of apoptosis (Rahman et al., 2000; Reddy et al., 1999; Thompson et al., 1997). Based upon this information, it appears that NSAIDs might not exert their effects by specifically inhibiting Cox-2 function alone. To delineate whether the metabolites also affected cyclin D1 expression, the protein levels were assessed in dose-dependent manner in response to either sulindac sulfide or the sulfone derivative. The data presented in Figures 29 and 30, demonstrate that both the sulfide and sulfone derivatives are able to modulate cyclin D1 expression; however, the sulfide derivative appeared to be the better of the two agents. Specifically, 10 uM sulfide appeared to result in approximately a 2-fold decrease in the levels of cyclin D1, while the sulfone appeared to elicit its effects by 100 u.M. To determine if these effects were independent of their known effects on viability, dose response studies were performed on the Caco-2 cell line. Interestingly, the effects of these agents on the levels of cyclin D1 were also determined to be independent of their effects on cell viability in this cell system. 193 Specifically at 25 u M , there were very negligible effects on cell viability in response to the sulfide derivative. The absence of effects on viability was even more apparent at lower dosages of sulfide (data not shown). Similarily at a 100 nM, the sulfone derivative had very little effect on cell viability. Thus both agents are able to modulate the expression of cyclin D1 and these effects appear to be independent of effects on cell viability. 194 Figure 29. Admin is t rat ion of Su l indac Sulf ide results in a Down-regulat ion of Cyc l i n D1 - Independent of Ef fects of Sul f ide on Cel l Viabil ity. A Sulindac sulfide blocks cyclin D1 expression in a dose-dependent fashion. Caco-2 cells were incubated in the presence of varying dosages of sulindac sulfide (500 nM to 100 uM) for 24 h as outlined in the Materials and methods section. B Transcriptional effects are independent of effects on cell viability. Cell viability was assessed in response to varying dosages of sulindac as outline in the Materials and methods section. Results are represented as % change over the control. Similar results were obtained on at least three independent occasions. 195 Cyclin D1 G A P D H 500 1 10 100 nM pJvl JLIM u.M Sulindac Sulfide 120% 100% 80% 60% 4 0 % 20% 0% Sulindac Sulfide 196 Figure 30. Admin is t rat ion of Su l indac Su l fone results in a Down-regulat ion of Cyc l i n D1 - Independent of Effects of Su l fone o n Cel l Viabil ity. A Sulindac sulfone blocks cyclin D1 expression in a dose-dependent fashion. Caco-2 cells were incubated in the presence of varying dosages of sulindac sulfone (500 nM to 500 uM) for 24 h as outlined in the Materials and methods section. B Transcriptional effects are independent of effects on cell viability. Cell viability was assessed in response to varying dosages of sulindac as outline in the Materials and methods section. Results are represented as % change over the control. Similar results were obtained on at least three independent occasions. 197 Cyclin D1 G A P D H 500 uM Sulindac Sulfone 120% i C 100 50 25 LiM u.M uM Sulindac Sulfone 198 5.3.2.4. Effects of NSAIDs on the Sub-cellular Distribution of p-catenin. Activation of Tcf-4 dependent gene transcription is a tightly regulated process. The stabilization of p-catenin expression and its translocation from the cytosol into the nucleus is a pivotal step in the activation of Tcf-4 mediated gene transcription (Behrens et al., 1996; Huber et al., 1996; Korinek et al., 1997; Morin et al., 1997; Porfiri et al., 1997). In the previous sections, NSAIDs were shown to not only inhibit this transcriptional apparatus but as well were shown to result in a decrease in the levels of the cell cycle regulated protein cyclin D1. Cyclin D1 is a putative downstream target of the former. To gain a better understanding for the underlying mechanism(s) involved in this inhibition of Tcf-4 mediated gene transcription, the sub-cellular distribution of p-catenin was evaluated in response to NSAID agents, like asprin and sulindac. It was postulated that perhaps NSAIDs inhibit transcription by altering the cellular distribution patterns of p-catenin. 199 Figure 31. T h e effects of NSAIDs o n p-catenin. A. The effects of NSAID agents on the sub-cellular distribution of p-catenin. Caco-2 cells were treated (5 mM A S A and 1 mM sulindac) and stained with the appropriate antibodies as outlined in the Materials and methods section. (Red staining represents p-catenin while green staining represents ILK.) B. Effects of NSAIDs on the protein expression of p-catenin. Anti-p-catenin immunoblot; examining expression p-catenin in response to these drugs. 2 0 0 p-catenin Dapi The results presented in Figure 31A indicate that administration of either aspirin (5 mM) or sulindac (1 mM) does not have any prominent effect on the sub-cellular distribution of p-catenin. p-catenin was present at the level of the cell membrane, within the cytosol as well as in the nucleus. These results are in agreement with a recent manuscript that investigated the effects of either aspirin or indomethacin on the cellular distribution of p-catenin (Dihlmann et al., 2001). Furthermore, there did not appear to be any effect on the distribution patterns of ILK in any of the conditions. Another mechanism involved in the regulation of this transcriptional machinery is believed to be the stabilization of p-catenin. Under normal circumstances, the levels of the former are regulated through the Axin complex. Complex formation results in the phosphorylation of p-catenin, which is mediated by GSK3p (Maniatis, 1999). The data presented in section 5.2.1. indicated that there was a decrease in the phosphorylation at the Ser-9 site on GSK3p in response to NSAID treatment. Phosphorylation at this site is indicative of its inhibition. Perhaps, dephosphorylation at this site might coincide with the activation of GSK3p. Based upon this hypothesis, it was postulated that perhaps that the reduction in transcription was due to a decrease in the protein levels of p-catenin. The data presented in Figure 31B indicate that the levels of p-catenin are unchanged in response to NSAID treatment. Thus, the inhibition of transcription does not appear to be directly due to changes in either the distribution or expression levels of p-catenin. It is plausible, that this inhibition is due to the inhibition of the upstream signaling molecules that ultimately control this 202 pathway. These may include protein kinase CK2, ILK or quite possibly PKB; all of which have been demonstrated to have a positive influence on Tcf-4-dependent gene transcription [Song, 2000 #525; Novak, 1998 #151; Fukumoto, 2001 #627]. 5.4. PKB and NSAIDs The PKB family, which has been demonstrated to be dependent upon PI3-kinase activity (Burgering and Coffer, 1995; Cross et al., 1995; Franke et al., 1995), is known to mediate a plethora of biological responses like gluconeogenesis. More recently, this kinase has received a great deal of attention for its role in apoptosis. In addition to this, a number of studies have demonstrated that this protein kinase might play a role in oncogenesis (Bellacosa et al., 1995; Cheng et al., 1996; Davies et al., 1999; Nakatani et al., 1999b) 5.4.1. Sulindac Inhibits PKB activity in vitro ILK, a protein kinase whose activity is modulated by NSAIDs, is known to regulate PKB activity. ILK has been reported to directly phosphorylate PKB on Ser473, which results in the activation of the latter. Interestingly, celecoxib, a Cox-2 specific inhibitor, has been reported to inhibit PKB activity. Inhbition of PKB activity was determined to be independent of effects on PI-3 kinase activity (Hsu et al., 2000). Based upon this, it was postulated that the inhibition of PKB activity might be attributable to the effects of these agents on ILK. To rule out the possibility that these drugs targeted PKB directly, in vitro protein kinase assays were performed in the presence of sulindac using recombinant PKB. Surprisingly, 203 the results presented in Figure 32 demonstrate that sulindac significantly inhibited PKB activity in vitro. The IC 5 0 value was determined to be approximately 180 uM. 204 Figure 32. Su l indac inhibits P K B in vitro. Recombinant PKB was assayed in the presence of varying doses of sulindac (50 nM to 1 mM) using HH2B as a substrate for 10 min. (*p<0.002, **p<0.005). 205 Sulindac 206 To validate the in vitro inhibitory effects of sulindac on P K B activity, immunoprecipitated PKB protein kinase assays were performed on lysates perpared from the human colon adenocarcinoma cells, DLD-1, that were treated with either A S A (5mM) or sulindac (1mM). The results presented in Figure 33 indicate that both aspirin and sulindac significantly inhibited the serum-induced activation of PKB with sulindac being the more effective of the two. Specifically, A S A inhibited PKB activity by 40% (p<0.01) whereas sulindac inhibited PKB activity by 70% (p<0.003). 2 0 7 Figure 33. Inhibition of PKB with NSAID administration. A. NSAIDs inhibit P K B in vivo. DLD-1 cells were treated with A S A (5 mM) or sulindac (1 mM) as outlined in the Materials and Methods section. Upper panel; PKB immunoprecipitation and immune complex kinase assays. Lower panel; representative HH2B autoradiogram. B. Effects of NSAIDs on Ser473 phosphorylation. Upper panel; quantification of PKB Ser473 phosphorylation. Anti-PKB Ser473 immunoblot, assessment of PKB Ser473 phosphorylation status. Anti-PKB immunoblot; analysis of PKB expression. (*p<0.01, **p<0.003, #p<0.001, ##p=0.001). 208 w •= (D c ro ro 5z CD o x 5 Q 14000 12000 10000 8000 6000 4000 2000 0 HH2B B O + i c o £ £ ^ si CO 2 CQ £ £ $ CL Q P-PKB Ser473 PKB G A P D H 1 0 % Serum ASA Sulindac + + 209 5.4.2. The Effects of NSAIDs on PKB Ser473 Phosphorylation In section 5.2.1. NSAIDs were demonstrated to attenuate the serum-induced activation of ILK. To determine whether the effects of NSAIDs on PKB activity in vivo, were direct or attributable to changes in the phosphorylation status of Ser473, lysates from the untreated and treated cells were electrophoresed and blotted with the phospho-specific Ser473 PKB antibody. The data presented in Figure 33B indicate that these agents appear to inhibit PKB activity by inhibiting the phosphorylation at the Ser473 site. Specifically, A S A resulted in almost a 55% decrease (p<0.001) in the serum-induced phosphorylation of PKB at Ser473, whereas sulindac resulted in approximately an 85% reduction (p=0.001), respectively. The levels of PKB and G A P D H were assessed to control for protein loading. From these studies, it is possible to assume that the inhibitory effects of these agents might occur primarily at the level of ILK; however, one cannot rule out the possibility that these agents might also inhibit PKB directly based upon the in vitro kinase data. Perhaps, inhibition of these signaling proteins is an important event in NSAID-mediated apoptosis. In this regard, both ILK and PKB have been documented to inhibit apoptosis (Attwell et al., 2000; Brunet et al., 1999; Datta et al., 1997; De Vita et al., 2000). 5.5. Introduction to protein kinase CK2 Protein kinase CK2 formerly referred to, as casein kinase 2, is a protein serine/threonine kinase that has been shown to phosphorylate well over 100 210 different substrates like p-catenin and Dsh, components of the Wnt signaling nexus (Song et al., 2000a). Furthermore, elevated expression of protein kinase CK2 has been documented in transformed cell lines, in rapidly proliferating tissues and in a number of tumors of diverse etiology including colorectal adenomas and cancer (Daya-Makin et al., 1994; Faust et al., 1996; Gapany et al., 1995; Guerra and Issinger, 1999; Issinger, 1993; Munstermann et al., 1990; Prowald et al., 1984; Schneider et al., 1986; Stalter et al., 1994). 5.5.1. The Effects of NSAIDs on protein kinase CK2 As outlined above, protein kinase CK2 not only has been reported to influence on Wnt signaling but as well has been demonstrated to be elevated in both colorectal adenomas and colorectal cancers (Munstermann et al., 1990; Song et al., 2000a). In section 5.2.2 NSAID agents were shown to inhibit Wnt signaling by inhibiting Tcf-4-dependent transcription. Taken together, it was hypothesized that NSAIDs might also inhibit the biochemical activity of protein kinase CK2. To address whether these agents might inhibit this protein kinase in vitro, recombinant protein kinase CK2 assays were performed in the presence of sulindac and its metabolites, sulindac sulfide and sulfone. The results presented in Figure 34 demonstrated that both sulindac and the sulfone metabolite significantly inhibited CK2 activity in vitro. Specifically, sulindac inhibited CK2 activity by 95% (p<0.05) whereas the sulfone derivative inhibited CK2 by almost 98% (p<0.0005). No effect was observed with the sulfide derivative. 211 Figure 34. Su l indac inhibits protein k inase C K 2 in vitro. Recombinant CK2 was assayed in the presence of sulindac (500 yM), sulindac sulfide (500 nM) and sulindac sulfone (500 ^M) using casein as a substrate for 10 min. (*p<0.05 and **p<0.0005). 212 500 uM To delineate whether sulindac was able to modulate CK2 activity in vivo, the human adenocarcinoma cell line DLD-1, was serum starved and then pre-treated with the drug for a 2 h interval. The results presented in Figure 35 demonstrate that sulindac significantly inhibited the serum-induced activation of CK2. Particularly, at 500 uM, sulindac inhibited the serum-stimulated CK2 activity by 82% (p<0.0005) while at 1 mM, CK2 activity was reduced to basal levels (p<0.0005). Furthermore, there were no apparent differences in the elution profile for CK2 between the control, stimulated or treated groups (data not shown). Thus, not only do NSAIDs inhibit the biochemical activity of ILK and PKB, but as well the activity of protein kinase CK2. Whether or not these represent likely in vivo targets or whether it is due to some non-specific effects of these drugs would require further delineation in a clinical setting or using animal models. Perhaps, inhibiting these upstream activators of Wnt signaling plays an important role in the induction of apoptosis. It has been proposed by others that down-regulation of Tcf-4 dependent gene transcription is a critical step in the induction of apoptosis (Steinhusen et al., 2000). 214 Figure 35. NSAIDs inhibit the b iochemica l activation of protein k inase CK2 in vivo. DLD-1 cells were treated with either A S A (5 mM) or sulindac (1 mM), stimulated with serum (St) for 10 mins and subsequently fractionated over a Mono Q column as outlined in the Materials and methods section. The fractionated samples (15-19) were assayed using casein as a substrate. Anti-CK2a immunoblot; analysis of CK2 elution profile. (*p<0.0005). 215 12000 0 J 15 16 17 18 19 Fraction no# IB: CK2 <~ CK2a < - CK2a' 15 16 17 18 19 Fraction no# 216 5.6. NSAID Mediated Cell Death - Cox-2 Dependency NSAIDs have been reported to influence cell viability (Akashi et al., 2000); however, whether inhibition of Cox-2 is involved in this process is still unclear. A number of reports appear to indicate that Cox-2 is not required for this process. To gain a better understanding of the effects of NSAID on cell viability and the role Cox-2 plays in the process, the effects of these agents on cell viability were evaluated in three colonic cell lines which have been reported to expression differential amounts of Cox-2: DLD-1; Caco-2, and HCT-116. 5.6.1. Differential Effects of NSAIDs on Cell Viability 5.6.1.1. Effects of A S A on Cell Viability Epidemiological studies indicate that ingestion of aspirin not only reduces the overall incidence of colon cancer but as well reduces the mortality associated with this particular malignancy (DuBois, 1995; Gann et al., 1993; Kune et al., 1988; Peleg et al., 1994; Rosenberg et al., 1991; Suh et al., 1993; Thun et al., 1991; Tonelli et al., 1994; Waddell and Loughry, 1983). However, the precise mechanism of action is currently unclear and thought to involve both Cox-1 and Cox-2. To determine whether A S A might elicit its anti-neoplastic effects independently of Cox-2 function, 3 cell lines with varying amounts of Cox-2 were selected for this study and were exposed to varying concentrations of this drug. The results presented in Figure 36 demonstrate that both the Caco-2 and DLD-1 cell lines were fairly insensitive to this agent at the selected concentrations. On 217 the otherhand, the HCT-116 cells appeared to be extremely sensitive to this drug. Specifically, 1 mM A S A appeared to have very little if any effect on the viability of either the Caco-2 cells or the DLD-1 cells whereas there was a 50% reduction in viability at this dose in the HCT-116 cells. The HCT-116 cell line has been reported to lack detectable expression of Cox-2 indicating that the effects of the drugs might be independent of Cox-2 function. More recently, A S A has been reported to mediate its effects on apoptosis through the release of cytochrome C from the mitochrondria. Release of cytochrome C is associated with the activation of caspase-9, which in turn activates caspase-3 (Li et al., 2001a; Li et al., 2001b). 218 Figure 36. Effects of A S A on Colon Cancer Cell Viability: Influence of Cox-2 Expression. The effects of A S A on cell viability were tested in three different cell lines (Caco-2, DLD-1 and HCT-116) as outlined in the Materials and methods section. Results are represented as % change over the control. Similar results were obtained on at least three independent occasions. 219 A S A 5.6.1.2. Effects of Sulindac on Cell Viability Administration of sulindac, the pro-drug which lacks Cox-2 inhibitory properties, has been demonstrated to promote regression of polyp lesions from not only patients diagnosed with FAP but as well in the Min mouse animal model (Beazer-Barclay et al., 1996; Oshima et al., 1996; Stoner et al., 1999). However, the precise mechanism by which this agent exerts its effects is currently unresolved. To determine whether sulindac, the pro-form of the drug, which lacks anti-inflammatory properties, is capable of reducing cell viability in the colon independently of Cox-2, three different colorectal adenocarcinoma cell lines were exposed to varying doses of the drug. The results presented in Figure 37 indicate that both the Caco-2 and DLD-1 cells are extremely insensitive to the parent compound at the selected doses. However, the HCT-116 cell line appeared to be extremely sensitive to sulindac. Specifically, there was approximately a 58% reduction in cell viability at 1 mM and 46% reduction in the viability at 500 ^M in this cell line. Since the HCT-116 cell line does not express detectable levels of the Cox-2 protein, this data lends further support to the notion that there are Cox-independent mechanisms that do exist and are likely involved NSAID-mediated cell death. 221 Figure 37. Ef fects of Su l indac o n C o l o n C a n c e r Cel l Viabil ity: Influence of Cox -2 E x p r e s s i o n . The effect of sulindac on cell viability was tested in three different cell lines (Caco-2, DLD-1 and HCT-116) as outlined in the Materials and methods section. Results are represented as % change over the control. Similar results were obtained on at least three independent occasions. 222 1 2 0 % i o -•—» c 100% o o 8 0 % o >> ' 6 0 % (0 4 0 % > 2 0 % 0% 120% o c 100% o o <4— 8 0 % o >* 6 0 % !5 CO 4 0 % > 2 0 % 0% "o 120% c o 100% o o 8 0 % 6 0 % CO 4 0 % > 2 0 % 0% Caco-2 DLD-1 HCT-116 1 500 100 mM (iM |iM Sulindac 223 5.6.1.3. Effects of Sulindac-Metabolites on Cell Viability Both sulindac sulfide and sulindac sulfone have been documented to promote decrease cell viability; however, the sulfone derivative is known to lack Cox-2 inhibitory properties suggesting that inhibition of Cox-2 function is not required for this process. To determine whether sulindac sulfone, an agent that has no Cox-2 inhibitory properties, could mimic the effects of sulindac sulfide on cell viability in colon cancer cell lines (Piazza et al., 1995), the effects of these two drugs were evaluated at equimolar concentrations in the Caco-2, DLD-1 and HCT-116 cell lines. The results presented in Figures 38 and 39 demonstrate that sulindac sulfide was a potent promoter of cell death in all three cells lines tested in comparison to the sulindac sulfone. Specifically at 100 nM, there appeared to be no appreciable effects on viability in DLD-1 and Caco-2 with sulindac sulfone whereas significant effects on cell viability were observed with sulindac sulfide in all three cells lines. There was a 42%, a 34% and a 62% reduction in cell viability in the DLD1, the Caco-2 and the HCT-116 cells with the sulfide derivative respectively. Since sulindac sulfide is believed to specifically inhibit Cox-2, one cannot negate the importance this molecule in mediating cell survival. However, the HCT-116 cell line has been reported to lack detectable expression of Cox-2 suggesting that there are Cox-independent events involved in the induction of NSAID-mediated cell death. In support of this, one group has reported that exougenous administration of PGH2 cannot attenuate the effects of these agents on cell viabililty (Chiu et al., 1997). However, one cannot dispute the reports that have shown that over-expression of Cox-2 confers a cyto-protective advantage 224 (Ishiko et al., 2001; Lin et al., 2001). Perhaps, elevated expression of Cox-2 might play a role in mediating drug-insensitivity by reducing the availability of the drug, specifically sulindac sulfide. This metabolite would bind to Cox-2 with a higher affinity and decrease the amount of available drug that is required to elicit its anti-neoplastic effects via additional mechanisms. Perhaps this is why certain patients develop colorectal cancers while on sulindac. 225 Figure 38. Ef fects of Su l indac Sul f ide o n C o l o n C a n c e r Cel l Viabil ity: Influence of Cox-2 E x p r e s s i o n . The effect of sulindac sulfide on cell viability was tested in three different cell lines (Caco-2, DLD-1 and HCT-116) as outlined in the Materials and methods section. Results are represented as % change over the control. Similar results were obtained on at least three independent occasions. 226 Sulindac Sulfide Figure 39. Effects of Sulindac Sulfone on Colon Cancer Cell Viability: Influence of Cox-2 Expression. The effect of sulindac sulfone on cell viability was tested in three different cell lines (Caco-2, DLD-1 and HCT-116) as outlined in the Materials and methods section. Results are represented as % change over the control. Similar results were obtained on at least three independent occasions. 228 Caco-2 DLD-1 HCT-116 100 50 25 (iM JIM LIM Sulindac Sulfone 5.6.2. Sulindac Sulfide-Mediated Apoptosis Recently, agents like A S A and NS-398 have been reported to promote cytochrome C release, which would coincide with not only the activation of capase-9 and caspase-3 but as well with the induction of apoptosis (Li et al., 2001a; Li et al., 2001b). From the studies in the previous section, it appears that NSAIDs might elicit their effects on apoptosis through mechanisms independent of Cox-2. To specifically delineate whether these agents promote apoptosis through a caspase-dependent process, DLD-1 cells were exposed to various NSAIDs for a 24 h period. The results presented in Figure 40 demonstrate that at 24 h, only the sulindac sulfide appeared to activate caspase-3. These findings are in agreement with the results in section 5.5.1.3, which demonstrated that the sulindac sulfide derivative is a relatively more potent promoter of apoptosis. No effects were observed with ASA, sulindac, sulindac sulfone and NS-398 at this time point. To corroborate the effects of sulindac sulfide on caspase-3 activation, the expression of P A R P , a caspase-3 sensitive protein was evaluated in a dose-dependent manner. The data indicates that caspase-3 is preferentially activated with 100 wVI sulfide and that activation at this dose coincides with the cleavage of P A R P . More recently, PKB has been demonstrated to be susceptible to caspase-3 dependent cleavage (Bachelder et al., 1999). To determine whether the sulfide-mediated activation of caspase-3 results in the cleavage of this protein, the expression of PKB was evaluated. The data indicates that in response to sulindac sulfide, PKB is cleaved between 50 to 100 yM. Importantly, PKB has 2 3 0 been demonstrated to inhibit the release of cytochrome C from the mitochondria (Kennedy et al., 1999). Perhaps, PKB is cleaved in a sulfide-dependent manner to inhibit the inhibitory effects of PKB on this pathway. Based upon these results, it appears that sulindac sulfide might exert its effects by altering events, which lead to the activation of cellular caspases, rather than by inhibiting Cox-2 function. However, one cannot dispute the importance of Cox-2, there clearly exist Cox-dependent and -independent mechanims involved in the regulation of apoptosis. 231 Figure 40. Ef fects of NSAIDs o n C a s p a s e Pathway. A Differential Effects of NSAIDs on the activation of Caspase-3. DLD-1 cells were treated as described in the Materials and methods section. Anti-caspase-3 immunoblot; evaluation of caspase-3 cleavage. B Effects of sulindac sulfide on downstream targets of caspase-3. DLD-1 cells were incubated with varying doses of sulindac sulfide (1 uM to 100 uM) as outlined in the Materials and methods section. Ant i -PARP immunoblot; analysis of P A R P cleavage. Anti-Caspase-3 immunoblot; assessment of caspase-3 cleavage. Anti-PKB immunblot, analysis of PKB expression. Ant i-GAPDH immunblot; control for equal protein loading. Similar results were obtained on at least three independent occasions. 232 Caspase-3 5mM 1mM 100um A S A Sulindac Sulfide 100um 100um Sulfone NS-398 B Uncleaved P A R P Cleaved P A R P Caspase-3 j^ i^ l^ P K B G A P D H 1 10 25 50 100 J J M fxM u.M | iM \iM Sulindac Sulfide 233 5.7. Effects of Protein Kinases on NSAID mediated apoptosis 5.7.1. Over-expression of ILK modulates effects of sulindac on cell survival Over-expression of ILK has been reported to influence a number of different biological properties including cell growth and apoptosis (Attwell et al., 2000; Radeva et al., 1997). Specifically, stable over-expression of ILK in a mouse mammary epithelial cell line has been shown to inhibit anchorage-dependent cell death or anoikis. Furthermore, inhibition of ILK in a PTEN mutant prostate cancer cell line has been reported to result in the induction of apoptosis. To determine whether over-expression of ILK is involved in mediating drug resistance, wild-type ILK and the 'kinase-dead' version of ILK were transiently transfected into either colorectal cancer cell line, HCT-116 or the human embryonic kidney (Hek293T) cells. The Hek293T cell line was chosen in addition to the colorectal cancer cell line because of the ease and efficiency of transfection (Korinek et al., 1997). The results that are presented in Figure 41 indicate that over-expression of wild-type ILK in Hek293T cells, which was detected by immunoblotting with an anti-V5 antibody (lower panel), attenuates the effects of sulindac on cell viability. Specifically, administration of sulindac (500 uM) in Hek293T cells transfected with the empty vector resulted in a 32% decrease in cell viability whereas there was a 6% decrease in the Hek293T cells that were transfected with wild-type ILK (p<0.05). Overexpression of ILK however did not suppress the effects of 5-Fu or sulindac sulfide. Interestingly, similar effects on cell viability were observed in 234 those cells transfected with the 'kinase-dead' version of ILK (data not shown). Unfortunatley, no effects on cell viability were observed in the HCT-116 cell line due to problems with transfection. It is possible that the effects of ILK on cell viability might in part be achieved by inhibiting the activation of caspase 3 in a caspase 9-dependent manner. In this regard, NSAIDs have been reported to promote the release of cytochrome C from the mitochondria (Li et al., 2001a; Li et al., 2001b). Certainly, over-expression of ILK has been demonstrated to inhibit anchorage-dependent cell death in a capase-3 dependent manner (Attwell et al., 2000). However, based upon the data presented in Figure 40 one could potentially refute this hypothesis. Specifically, sulindac sulfide was the only agent that appeared to have an effect on caspase-3 at 24 h. Thus, if ILK inhibits the activation of caspase-3 one would assume over-expression of ILK should attenuate the effects of this drug on cell viability. Perhaps, the suppressive effects of ILK might be attributable to the observation that sulindac is able to inhibit the biochemically activity of ILK whereas the sulfide derivative did not. Is it possible that sulindac interacts directly with ILK, and as a result reduces drug availability? The data obtained in the in vitro kinase experiments would appear to suggest this; however, further investigation is required to determine whether sulindac binds directly with ILK. In any regards, is it possible that when ILK is over-expressed it reduces the efficacy of sulindac by binding with ILK and as a result decreasing the concentration of available drug, which is to be converted to its active metabolite by the intestinal flora in humans? In support of this, it was determined 235 that the effects of ILK on survival in the case if sulindac are not dependent upon the activity of the kinase as the 'kinase-dead' mutant of ILK was also determined to attenuate sulindac-induced apoptosis (data not shown). This would require further delineation. On the basis of the data one could assume that over-expression of ILK in colorectal tumors might represent not only a pivotal step in terms of growth but as well in mediating drug resistance. Perhaps, over-expression of ILK in the context of FAP, as suggested above, reduces the amount of sulindac sulfide, the active metabolite of sulindac. Thus by disrupting the interaction between sulindac and ILK by way of a specific inhibitor, one could postulate that this could improve the responsiveness to sulindac. Studies in cell culture and in animal models would help to confirm this. 236 Figure 41. ILK suppresses effects of NSAIDS on cell viability. Hek293T cells were transiently transfected with either the wild-type ILK or the empty vector and incubated with either 500 ^.M sulindac or 500 uM 5-Fu or 100 ^M Sulindac sulfide as outlined in the Materials and methods section. The data is represented as a % change in the absorbance of the treated cells versus the untreated group. Similar results on 3 different occasions. (*p<0.05). 237 A V5-tagged ILK pcDNA3 ILK WT 238 5.7.2. Over-expression of PKB modulates effects of sulindac on cell survival A number of studies have underscored the importance of PKB in mediating cell survival. One of the first studies to demonstrate that P K B was involved in mediating this process demonstrated that IGF-I mediated cell survival was attenuated by transfection with a dominant-negative version of PKB. Furthermore, this study demonstrated IGF-1 independence in cells that were transfected with either the wild-type PKB or a constitutive-active mutant (Dudek et al., 1997). These initial observations were corroborated by a number of other studies, which reported that PKB could promote survival in a variety of cell lines. More importantly, PKB has been shown to inhibit cell death in response to a number of different apoptotic stimuli including growth factor withdrawal, UV irradiation, DNA damage, matrix detachment, cell cycle discordance and treatment of cells with either anti-Fas or TGFp antibodies (Page et al., 2000). To determine whether PKB is involved in mediating NSAID tumor cell resistance, HCT-116 and HEK293T cells were transfected with either the wild-type PKB or the 'kinase-dead' mutant as outlined above. The results that are presented in Figure 42 demonstrate that over-expression of wild-type PKB, which was detected by immunoblotting with an anti-HA antibody (lower panel), attenuates the effects of sulindac on cell viability. Specifically, administration of sulindac (500 uM) in cells transfected with the empty vector resulted in a 36% decrease in cell viability whereas there was only a 16% decrease in the cells that were transfected with wild-type PKB (p<0.005). Once again no effects were observed with sulindac sulfide or 5-Fu. No effects 239 were observed in the HCT-116 cells. This effect was attributed to inherent problems involved in transfection of these cells. The results obtained with P K B in this section, specifically in response to sulindac sulfide, the major activator of caspase-3 in human colon cancer cell lines, are quite surprising since over-expression of PKB , a caspase-3 sensitive protein, has been previously reported to inhibit caspase-3 activity by blocking the efflux of cytochrome C from the mitochondria (Kennedy et al., 1999). Furthermore, the protein levels of PKB were shown to decrease with increasing doses of sulindac sulfide (see Figure 40), which could be attributed to the activation of caspase-3. Thus, one could postulate that over-expressing PKB should block sulfide-mediated apoptosis. However, this was not the case in these studies. On this basis and in agreement with the findings in section 5.7.1, one could postulate that perhaps PKB's anti-apoptotic effects are due to the expression of the PKB protein itself and that the effects of PKB in reponse to sulindac are due to the fact that sulindac might bind to PKB based upon the in vitro data. However, one would have to specifically address this matter. To further support this notion, similar effects to the catalytically inactive mutant of ILK on cell viability were observed with the 'kinase-dead' mutant of PKB (data not shown). This data suggests that PKB's effects might be due to elevated expression of the protein rather than the enzymatic activity. To add to this, sulindac sulfide was determined to be ineffective at inhibiting PKB activity in vitro, suggesting that perhaps the differential effects of these agents is attributed to the notion that the sulfide derivative is not able to associate with PKB. 240 In terms of colon cancer and more specifically the treatment of this disease, one would not only have to determine whether PKB is over-expressed and/or overactive as well as to delineate if P K B is targeted by NSAIDs in vivo. Based upon a number of reports, which highlight the potential importance of P K B in oncogenesis, it is reasonable to propose that intervention with an inhibitor to PKB in combination with either one of the NSAID or chemotherapeutic agents might be a more effective means of treatment than the current protocols. This would obviously require pre-clinical validation using cell culture and animal models. 241 Figure 42. P K B s u p p r e s s e s effects of N S A I D S o n cel l viability. Hek293T cells were transiently transfected with either the wild-type PKB or the empty vector and incubated with either 500 i a M sulindac or 500 (J.M 5-Fu or 100 i a M Sulindac sulfide as outlined in the Materials and methods section. The data is represented as a % change in the absorbance of the treated cells versus the untreated group. Similar results on 3 different occasions. (*p<0.005). 242 • Sulindac n Sulfide pCDNA3 PKB < HA-tagged PKB pcDNA3 PKB WT 243 5.7.3. Over-expression of CK2 modulates effects of sulindac on cell survival Protein kinase CK2 is a ubiquitously expressed protein serine/threonine kinase that has been reported to phosphorylate well over 100 different substrates. This protein kinase has been demonstrated to be up-regulated in transformed cell lines, rapidly proliferating tissues and a number of tumors of diverse etiology (Daya-Makin et al., 1994; Faust et al., 1996; Gapany et al., 1995; Guerra and Issinger, 1999; Issinger, 1993; Munstermann et al., 1990; Prowald et al., 1984; Schneider et al., 1986; Stalter et al., 1994). Furthermore, dysregulated expression of the CK2a subunit is oncogenic. (Kelliher et al., 1996; Landesman-Bollag et al., 1998; Nusse et al., 1990; Seldin and Leder, 1995). More recently, CK2 has also been demonstrated to play a role in preventing apoptosis. Since CK2 was also demonstrated to be inhibited by sulindac both in vitro and in vivo, the effects of CK2 on cell viability in response to drug treatment was assessed. The results that are presented in Figure 43 demonstrate that over-expression of the catalytic subunit of CK2, which was detected by immunoblotting with an anti-myc antibody (lower panel), modulates the effects of sulindac on cell viability. Specifically, administration of sulindac (500 uM) in cells transfected with the empty vector resulted in a 29% decrease in cell viability whereas there was a 7% decrease in the cells that were transfected with CK2a (p<0.05). No effects were observed in response to sulindac sulfide or 5-Fu. The effects of CK2 on cell viability might be achieved through its shuttling to the nucleus or through the phosphorylation of the apoptotic protein Bid. Phosphorylation of Bid has been shown to inhibit apoptosis by inhibiting its 244 cleavage in a caspase-8 dependent manner. Cleavage of Bid results in the release of cytochrome C from the mitochondria; this release results in the subsequent activation of the caspase-9 pathway (Desagher et al., 2001). However, it is plausible that the effects of CK2 on sulindac mediated cell death could be attributed to sequestration of sulindac by CK2 when it is over-expressed. Since protein kinase CK2 is over-expressed in colon cancer, perhaps elevated expression of this protein not only confers a positive growth influence but as well confers a cytoprotective effect on chemical induced cell death by decreasing the levels of sulindac, which is to be converted to its active metabolites by bacteria within the colon. Thus, it is possible that intervention with a selective inhibitor to this protein kinase in combination with agents like sulindac could be a more effective approach in the treatment of colon cancer. In order to determine the effectiveness of this treatment protocol, one could utilize both cell culture and animal models. 245 Figure 43. CK2 suppresses effects of NSAIDS on cell viability. Hek293T cells were transiently transfected with either the CK2aor the empty vector and incubated with either 500 fiM sulindac or 500 uM 5-Fu or 100 uM Sulindac sulfide as outlined in the Materials and methods section. The data is represented as a % change in the absorbance of the treated cells versus the untreated group. Similar results on 3 different occasions. (*p<0.05). 2 4 6 247 5.8. Summary of Results The chemopreventative effects of NSAIDs are widely recognized; however, the precise mechanism by which these agents elicit their anti-neoplastic effects is unclear. The data presented in this chapter, indicates that NSAIDs might partly elicit their growth suppressive effects by inhibiting components of the Wnt signaling nexus including ILK, PKB and protein kinase CK2. Inhibition of these protein kinases by NSAIDs, all of which have been demonstrated to have a postive influence of the Wnt signaling nexus, not only coincided with a decrease in Tcf-4-dependent gene transcription but also the effects of these agents on transcription were determined to be independent of apoptosis. Furthermore, the results indicated that administration of these agents coincided with a decrease in the phosphorylation of GSK3p on Ser9. Phosphorylation of this site is indicative of its inhibition; thus a decrease in this phosphorylation might represent the activation of this protein kinase. Additionally, the results demonstrated that over-expression of either ILK, PKB or CK2a modulated sulindac but not 5-Fu or sulindac sulfide mediated apoptosis. Morevover, the data presented here, suggested that the effects of these drugs on cell viability might in fact be independent of Cox-2 function. It is plausible to assume that these agents might promote elicit their effects on viability through the release of cytochrome C and the subsequent activation of caspases - 9 and -3. This is not unlikely based upon a number of recent reports that have shown 248 that these agents can promote the release of cytochrome C from the mitochondria. 5.9. General Conclusion and Future Directions It is estimated that approximately 50% of the patients diagnosed with C R C respond poorly to treatment; however, very little is known what mediates chemoresistance. 5-Fluorouracil (5-Fu) is a DNA damaging agent, which is believed to mutate the DNA. This would theoretically result in the induction of p53-mediated apoptosis. However, the precise role of p53 in this process is not well established based upon the data from human clinical studies. Based upon the results from the studies in this chapter, it would be interesting to delineate whether administration of sulindac sulfide, which is a potent promoter of apoptosis, by itself or in combination with 5-Fu would be more effective at treating this disease than with 5-Fu alone. Furthermore, it would be extremely interesting to determine whether or not inhibitors to ILK, PKB or CK2 alone or in combination with each other, with NSAIDs or conventional chemotherapeutic agents could improve the 5-year survival rates for this disease. Certainly, the BCR-Abl tyrosine kinase inhibitor, STI-571, has proven to be effective in the treatment of CML (Maura et al., 2001). 2 4 9 Chapter 6 . Conclusion 6.1. Summary of Results One of the most common mutations involved in the initiation of human colon cancer involves the adenomatous polyposis coli (APC) gene . Mutation of A P C is believed to be associated with the dysregulation of p-catenin signaling in that this molecule is predominantly translocated to the nucleus. Within the nucleus, this molecule up-regulates the transcription of a number of genes that are important not only in mediating cell growth but as well tissue remodeling (Kinzler and Vogelstein, 1996). Interestingly, stable over-expression of the integrin-linked kinase (ILK) in rat intestinal epithelial cells has been demonstrated to have a similar consequence to the mutation of A P C on the sub-cellular distribution of p-catenin (Novak et al., 1998). Specifically, over-expression of ILK resulted in a dramatic increase in the nuclear levels of p-catenin; this coincided with the activation of Tcf-4-dependent gene transcription. In addition to this both P K B and protein kinase CK2 have been shown to have a postive influence on p-catenin mediated gene transcription (Fukumoto et al., 2001; Song et al., 2000a). Protein kinase CK2 has previously been shown to be aberrantly expressed in both colorectal adenomas and colorectal cancers (Munstermann et al., 1990). To delineate whether ILK signaling might also be disrupted in an analogous fashion to protein kinase CK2 during the progression of human colon cancer, this signaling nexus was investigated in the precursor adenomatous 250 lesions, primary colorectal cancers and metastatic deposits within the regional lymph nodes. The data from these studies demonstrated that changes in the ILK protein occur very early in the development of colon cancer and that these changes in ILK expression reflect changes in ILK activity. Furthermore, these changes appeared to correlate with effects on downstream targets involved in this pathway, including GSK3p and Lef-1. Since sulindac and aspirin are the two most important therapeutic/ chemopreventative agents demonstrated in colorectal carcinogenesis in both animals and humans, the possibility that these agents might partly exert their anti-neoplastic properties by inhibiting ILK signaling was explored. The data demonstrated that these agents not only inhibited ILK but as well directly inhibited both PKB and CK2. It is possible that the effects of these agents on in vivo PKB activity might be attributable to both direct and indirect effects. ILK, which is sensitive to these agents both in vitro and in vivo, has been reported to phosphorylate PKB on Ser473. Therefore, it is possible that changes in PKB might reflect changes in the phosphorylation of PKB at Ser473. In support of this, administration of these agents appeared to result in a decrease in the phosphorylation at this site. However, one cannot rule out the possibility that these agents directly target PKB based upon the in vitro data. Furthermore, these studies showed that administration of these drugs resulted in a decrease in the Ser-9 phosphorylation of GSK3p. Phosphorylation at this site is indicative of its inhibition. Perhaps, a decrease in this phosphorylation might represent an increase in the activity of GSK3p. Moreover, administration of these drugs 2 5 1 resulted in a decrease in Tcf-4-dependent gene transcription. These effects were determined to be independent of their effects on apoptosis. Additionally, the results demonstrated that over-expression of ILK, PKB or CK2 modulated sulindac but not sulindac sulfide or 5-Fu mediated apoptosis. Moreover, the results suggested that the apoptotic effects of these agents might be independent of Cox-2. It is possible that these agents might mediate their effects through cellular caspases. 6.2. Future Directions The results from these studies indicate that ILK signaling is disrupted in human colon cancer; however, whether this dysregulation is intimately involved in the development of colon cancer would require further delineation. It will be of importance to show that selective over-expression of ILK within the intestine favors tumorigenesis in the colon. To add to these studies, it would be of interest to show that administration of a selective ILK inhibitor could cause regression of adenomatous lesions using the Min mouse animal model. In addition to this, it would be of interest to delineate how the expression of ILK is dysregulated in colon cancer. Perhaps A P C or p-catenin is involved in this process. Furthermore, I believe it would be of extreme value to address whether administration of sulindac sulfide by itself or in combination with 5-Fu might be more beneficial than 5-Fu alone in the treatment of human colon cancer. In addition to this, targeting ILK directly might be of extreme importance in the treatment of colon cancer in general. In support of this, a number of recent studies have 252 demonstrated the importance of inhibiting signal transduction pathways in the treatment of human cancers. Specifically, antibodies which inhibit signaling via the epidermal growth factor receptor pathway (EGF), HER-2/Neu as well as the tyrosine kinase inhibitor STI (signal transduction inhibitor number)-571 have been demonstrated to be extremely effective in the treatment of human breast cancer and chronic myeloid leukemia (CML), respectively (Ishiko et al., 2001; Joensuu et al., 2001). In addition to this, a novel irreversible inhibitor of the E G F receptor kinase EKI-569, has been demonstrated to inhibit tumorigenesis in the Min mouse model (Torrance et al., 2000). The authors also demonstrate that in combination, both sulindac and EKI-569 have profound effects on inhibiting intestinal neoplasia. They speculate that this could serve as a novel means of chemoprevention in humans in the future. Perhaps, specific inhibition of ILK alone or in combination with other drugs could provide a new means of treatment for human colon cancer. 253 Chapter 7. Re ferences 7.1. List of Re ferences Aberle, H., Bauer, A., Stappert, J . , Kispert, A., and Kemler, R. (1997). beta-catenin is a target for the ubiquitin-proteasome pathway. Embo J 16, 3797-3804. Adjei, A. A. (2001). Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 93, 1062-1074. Ahmed, N. N., Grimes, H. L , Bellacosa, A., Chan, T. O., and Tsichlis, P. N. (1997). Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc Natl Acad Sci U S A 94, 3627-3632. Akashi, H., Han, H. J . , lizaka, M., and Nakamura, Y. (2000). Growth-suppressive effect of non-steroidal anti-inflammatory drugs on 11 colon-cancer cell lines and fluorescence differential display of genes whose expression is influenced by sulindac. Int J Cancer 88, 873-880. Akiyama, Y., Nagasaki, H., Yagi, K. O., Nomizu, T., and Yuasa, Y. (2000). Beta-catenin and adenomatous polyposis coli (APC) mutations in adenomas from hereditary non-polyposis colorectal cancer patients. Cancer Lett 157, 185-191. Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., Norman, D. G., Gaffney, P., Reese, C. B., MacDougall, C. N., Harbison, D., et al. (1997a). 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol 7, 776-789. 254 Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997b). Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. CurrBiol 7, 261-269. Alessi, D. R., Saito, Y., Campbell, D. G., Cohen, P., Sithanandam, G., Rapp, U., Ashworth, A., Marshall, C. J . , and Cowley, S. (1994). Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. Embo J 13, 1610-1619. Altomare, D. A., Guo, K., Cheng, J . Q., Sonoda, G., Walsh, K., and Testa, J . R. (1995). Cloning, chromosomal localization and expression analysis of the mouse Akt2 oncogene. Oncogene 11, 1055-1060. Anderson, K. E., Coadwell, J . , Stephens, L. R., and Hawkins, P. T. (1998). Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B. Curr Biol 8, 684-691. Anderson, N. G. , Mailer, J . L , Tonks, N. K., and Sturgill, T. W. (1990). Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343, 651-653. Andjelkovic, M., Jones, P. F., Grossniklaus, U., Cron, P., Schier, A. F., Dick, M., Bilbe, G. , and Hemmings, B. A. (1995). Developmental regulation of expression and activity of multiple forms of the Drosophila R A C protein kinase. J Biol Chem 270, 4066-4075. Arcaro, A., Volinia, S., Zvelebil, M. J . , Stein, R., Watton, S. J . , Layton, M. J . , Gout, I., Ahmadi, K., Downward, J . , and Waterfield, M. D. (1998). Human 255 phosphoinositide 3-kinase C2beta, the role of calcium and the C2 domain in enzyme activity. J Biol Chem 273, 33082-33090. Askham, J . M., Moncur, P., Markham, A. F., and Morrison, E. E. (2000). Regulation and function of the interaction between the A P C tumour suppressor protein and EB1. Oncogene 19, 1950-1958. Attar, B. M., Atten, M. J . , and Holian, O. (1996). MAPK activity is down-regulated in human colon adenocarcinoma: correlation with P K C activity. Anticancer Res 16, 395-399. Attwell, S., Roskelley, C , and Dedhar, S. (2000). The integrin-linked kinase (ILK) suppresses anoikis. Oncogene 19, 3811-3815. Avruch, J . (1998). Insulin signal transduction through protein kinase cascades. Mol Cell Biochem 782, 31-48. Axelrod, J . D., Miller, J . R., Shulman, J . M., Moon, R. T., and Perrimon, N. (1998). Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev 12, 2610-2622. Bachelder, R. E., Ribick, M. J . , Marchetti, A., Falcioni, R., Soddu, S., Davis, K. R., and Mercurio, A. M. (1999). p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB. J Cell Biol 747, 1063-1072. Bailey, A., Norris, A. L., Leek, J . P., Clissold, P. M., Carr, I. M., Ogilvie, D. J . , Morrison, J . F., Meredith, D. M., and Markham, A. F. (1995). Yeast artificial chromosome cloning of the beta-catenin locus on human chromosome 3p21-22. Chromosome Res 3, 201-203. 256 Bakin, A. V., Tomlinson, A. K., Bhowmick, N. A., Moses, H. L , and Arteaga, C. L. (2000). Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration. J Biol Chem 275, 36803-36810. Balendran, A., Biondi, R. M., Cheung, P. C , Casamayor, A., Deak, M., and Alessi, D. R. (2000). A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1. J Biol Chem 275, 20806-20813. Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney, P., Currie, R., Downes, C. P., and Alessi, D. R. (1999a). PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr Biol 9, 393-404. Balendran, A., Currie, R., Armstrong, C. G., Avruch, J . , and Alessi, D. R. (1999b). Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252. J Biol Chem 274, 37400-37406. Bamba, H., Ota, S., Kato, A., Adachi, A., Itoyama, S., and Matsuzaki, F. (1999). High expression of cyclooxygenase-2 in macrophages of human colonic adenoma. Int J Cancer 83, 470-475. Banin, S., Moyal, L , Shieh, S., Taya, Y., Anderson, C. W., Chessa, L , Smorodinsky, N. I., Prives, C , Reiss, Y., Shiloh, Y., and Ziv, Y. (1998). Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674-1677. 257 Barak, Y., Juven, T., Haffner, R., and Oren, M. (1993). mdm2 expression is induced by wild type p53 activity. Embo J 12, 461-468. Barbieri, M. A., Hoffenberg, S., Roberts, R., Mukhopadhyay, A., Pomrehn, A., Dickey, B. F., and Stahl, P. D. (1998). Evidence for a symmetrical requirement for Rab5-GTP in in vitro endosome-endosome fusion. J Biol Chem 273, 25850-25855. Barker, N., Morin, P. J . , and Clevers, H. (2000). The Yin-Yang of TCF/beta-catenin signaling. Adv Cancer Res 77, 1-24. Barth, A. I., Nathke, I. S., and Nelson, W. J . (1997). Cadherins, catenins and A P C protein: interplay between cytoskeletal complexes and signaling pathways. Curr Opin Cell Biol 9, 683-690. Beazer-Barclay, Y., Levy, D. B., Moser, A. R., Dove, W. F., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. (1996). Sulindac suppresses tumorigenesis in the Min mouse. Carcinogenesis 17, 1757-1760. Behrens, J . (1999). Cadherins and catenins: role in signal transduction and tumor progression. Cancer Metastasis Rev 18, 15-30. Behrens, J . , Jerchow, B. A., Wurtele, M., Grimm, J . , Asbrand, C , Wirtz, R., Kuhl, M., Wedlich, D., and Birchmeier, W. (1998). Functional interaction of an axin homolog, conductin, with beta-catenin, A P C , and GSK3beta. Science 280, 596-599. Behrens, J . , von Kries, J . P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R., and Birchmeier, W. (1996). Functional interaction of beta-catenin with the transcription factor LEF-1 . Nature 382, 638-642. 258 Bellacosa, A., Chan, T. O., Ahmed, N. N., Datta, K., Malstrom, S., Stokoe, D., McCormick, F., Feng, J . , and Tsichlis, P. (1998). Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene 17, 313-325. Bellacosa, A., de Feo, D., Godwin, A. K., Bell, D. W., Cheng, J . Q., Altomare, D. A., Wan, M., Dubeau, L , Scambia, G., Masciullo, V., and et al. (1995). Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer 64, 280-285. Bellacosa, A., Testa, J . R., Staal, S. P., and Tsichlis, P. N. (1991). A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 254, 274-277. Benistant, C , Chapuis, H., and Roche, S. (2000). A specific function for phosphatidylinositol 3-kinase alpha (p85alpha-p110alpha) in cell survival and for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in de novo DNA synthesis of human colon carcinoma cells. Oncogene 19, 5083-5090. Ben-Ze'ev, A., and Geiger, B. (1998). Differential molecular interactions of beta-catenin and plakoglobin in adhesion, signaling and cancer. Curr Opin Cell Biol 10, 629-639. Besson, A., Robbins, S. M., and Yong, V. W. (1999). PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis. Eur J Biochem 263, 605-611. Bhattacharjee, R. N., Hamada, F., Toyoshima, K., and Akiyama, T. (1996). The tumor suppressor gene product A P C is hyperphosphorylated during the M phase. Biochem Biophys Res Commun 220, 192-195. 259 Bhattacharya, G., and Boman, B. M. (1995). Phosphorylation of the adenomatous polyposis coli protein and its possible regulatory effects in cells. Biochem Biophys Res Commun 208, 103-110. Bienz, M. (1999). A P C : the plot thickens. Curr Opin Genet Dev 9, 595-603. Bienz, M., and Clevers, H. (2000). Linking colorectal cancer to Wnt signaling. Cell 103, 311-320. Biggs, W. H., 3rd, Meisenhelder, J . , Hunter, T., Cavenee, W. K., and Arden, K. C. (1999). Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci U S A 96, 7421-7426. Biondi, R. M., Kieloch, A., Currie, R. A., Deak, M., and Alessi, D. R. (2001). The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. Embo J 20, 4380-4390. Blume-Jensen, P., Janknecht, R., and Hunter, T. (1998). The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136. Curr Biol 8, 779-782. Boland, C. R., Sinicrope, F. A., Brenner, D. E., and Carethers, J . M. (2000). Colorectal cancer prevention and treatment. Gastroenterology 118, S115-128. Boolbol, S. K., Dannenberg, A. J . , Chadburn, A., Martucci, C , Guo, X. J . , Ramonetti, J . T., Abreu-Goris, M., Newmark, H. L., Lipkin, M. L., DeCosse, J . J . , and Bertagnolli, M. M. (1996). Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res 56, 2556-2560. 260 Bos, J . L. (1989). ras oncogenes in human cancer: a review. Cancer Res 49, 4682-4689. Boulton, T. G., Gregory, J . S., and Cobb, M. H. (1991). Purification and properties of extracellular signal-regulated kinase 1, an insulin-stimulated microtubule-associated protein 2 kinase. Biochemistry 30, 278-286. Boutros, M., Mihaly, J . , Bouwmeester, T., and Mlodzik, M. (2000). Signaling specificity by Frizzled receptors in Drosophila. Science 288, 1825-1828. Bowtell, D., Fu, P., Simon, M., and Senior, P. (1992). Identification of murine homologues of the Drosophila son of sevenless gene: potential activators of ras. Proc Natl Acad Sci U S A 89, 6511-6515. Brabletz, T., Jung, A., Dag, S., Hlubek, F., and Kirchner, T. (1999). beta-catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am J Pathol 155, 1033-1038. Brennan, P., Babbage, J . W., Burgering, B. M., Groner, B., Reif, K., and Cantrell, D. A. (1997). Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell cycle regulator E2F. Immunity 7, 679-689. Brodbeck, D., Cron, P., and Hemmings, B. A. (1999). A human protein kinase Bgamma with regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain. J Biol Chem 274, 9133-9136. Brown, R. A., Domin, J . , Arcaro, A., Waterfield, M. D., and Shepherd, P. R. (1999). Insulin activates the alpha isoform of class II phosphoinositide 3-kinase. J Biol Chem 274, 14529-14532. 261 Browne, S. J . , MacFarlane, M., Cohen, G. M., and Paraskeva, C. (1998). The adenomatous polyposis coli protein and retinoblastoma protein are cleaved early in apoptosis and are potential substrates for caspases. Cell Death Differ 5, 206-213. Brunet, A., Bonni, A., Zigmond, M. J . , Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J . , Arden, K. C , Blenis, J . , and Greenberg, M. E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857-868. Burgering, B. M., and Coffer, P. J . (1995). Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376, 599-602. Buschmann, T., Minamoto, T., Wagle, N., Fuchs, S. Y., Adler, V., Mai, M., and Ronai, Z. (2000). Analysis of JNK, Mdm2 and p14(ARF) contribution to the regulation of mutant p53 stability. J Mol Biol 295, 1009-1021. Caca, K., Kolligs, F. T., Ji , X., Hayes, M., Qian, J . , Yahanda, A., Rimm, D. L., Costa, J . , and Fearon, E. R. (1999). Beta- and gamma-catenin mutations, but not E-cadherin inactivation, underlie T-cell factor/lymphoid enhancer factor transcriptional deregulation in gastric and pancreatic cancer. Cell Growth Differ 10, 369-376. Cadigan, K. M., and Nusse, R. (1997). Wnt signaling: a common theme in animal development. Genes Dev 11, 3286-3305. Cao, X., Eu, K. W., Seow-Choen, F., and Cheah, P. Y. (1999). Germline mutations are frequent in the A P C gene but absent in the beta-catenin gene in 262 familial adenomatous polyposis patients. Genes Chromosomes Cancer 25, 396-398. Cardone, M. H., Roy, N., Stennicke, H. R., Salvesen, G. S., Franke, T. F., Stanbridge, E., Frisch, S., and Reed, J . C. (1998). Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318-1321. Cavallo, R. A., Cox, R. T., Moline, M. M., Roose, J . , Polevoy, G. A., Clevers, H., Peifer, M., and Bejsovec, A. (1998). Drosophila Tcf and Groucho interact to repress Wingless signalling activity. Nature 395, 604-608. Chan, T. A., Morin, P. J . , Vogelstein, B., and Kinzler, K. W. (1998). Mechanisms underlying nonsteroidal antiinflammatory drug-mediated apoptosis. Proc Natl Acad Sci U S A 95, 681-686. Chang, H. W., Aoki, M., Fruman, D., Auger, K. R., Bellacosa, A., Tsichlis, P. N., Cantley, L. C., Roberts, T. M., and Vogt, P. K. (1997). Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase. Science 276, 1848-1850. Chardin, P., Camonis, J . H., Gale, N. W., van Aelst, L , Schlessinger, J . , Wigler, M. H., and Bar-Sagi, D. (1993). Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science 260, 1338-1343. Chaudhry, P. S., and Casillas, E. R. (1989). Casein kinase II activity and polyamine-stimulated protein phosphorylation of cytosolic and plasma membrane proteins in bovine sperm. Arch Biochem Biophys 271, 98-106. 263 Chen, P., Lee, K. S., and Levin, D. E. (1993). A pair of putative protein kinase genes (YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae. Mol Gen Genet 236, 443-447. Chen, R. H., and McCormick, F. (2001). Selective targeting to the hyperactive beta-catenin/T-cell factor pathway in colon cancer cells. Cancer Res 61, 4445-4449. Cheney, I. W., Johnson, D. E., Vaillancourt, M. T., Avanzini, J . , Morimoto, A., Demers, G. W., Wills, K. N., Shabram, P. W., Bolen, J . B., Tavtigian, S. V., and Bookstein, R. (1998). Suppression of tumorigenicity of glioblastoma cells by adenovirus-mediated MMAC 1/PTEN gene transfer. Cancer Res 58, 2331-2334. Cheng, J . Q., Ruggeri, B., Klein, W. M., Sonoda, G., Altomare, D. A., Watson, D. K., and Testa, J . R. (1996). Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci U S A 93, 3636-3641. Chiariello, E., Roz, L., Albarosa, R., Magnani, I., and Finocchiaro, G. (1998). PTEN/MMAC1 mutations in primary glioblastomas and short-term cultures of malignant gliomas. Oncogene 16, 541-545. Chiu, C. H., McEntee, M. F., and Whelan, J . (1997). Sulindac causes rapid regression of preexisting tumors in Min/+ mice independent of prostaglandin biosynthesis. Cancer Res 57, 4267-4273. Chung, D. H., Lee, J . I., Kook, M. C , Kim, J . R., Kim, S. H., Choi, E. Y., Park, S. H., and Song, H. G. (1998). ILK (betal-integrin-linked protein kinase): a novel 264 immunohistochemical marker for Ewing's sarcoma and primitive neuroectodermal tumour. Virchows Arch 433, 113-117. Cobb, M. H., Boulton, T. G., and Robbins, D. J . (1991). Extracellular signal-regulated kinases: ERKs in progress. Cell Regul 2, 965-978. Cochet, C , Feige, J . J . , Pirollet, F., Keramidas, M., and Chambaz, E. M. (1982). Selective inhibition of a cyclic nucleotide independent protein kinase (G type casein kinase) by quercetin and related polyphenols. Biochem Pharmacol 31, 1357-1361. Coffer, P. J . , and Woodgett, J . R. (1991). Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem 201, 475-481. Cohen, P., Alessi, D. R., and Cross, D. A. (1997). PDK1, one of the missing links in insulin signal transduction? F E B S Lett 410, 3-10. Cong, L. N., Chen, H., Li, Y., Zhou, L., McGibbon, M. A., Taylor, S. I., and Quon, M. J . (1997). Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11, 1881-1890. Cook, D., Fry, M. J . , Hughes, K., Sumathipala, R., Woodgett, J . R., and Dale, T. C. (1996). Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C. Embo J 15, 4526-4536. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J . (1994). Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77, 841-852. 265 Cranley, J . P., Petras, R. E., Carey, W. D., Paradis, K., and Sivak, M. V. (1986). When is endoscopic polypectomy adequate therapy for colonic polyps containing invasive carcinoma? Gastroenterology 91, 419-427. Crawford, H. C , Fingleton, B. M., Rudolph-Owen, L. A., Goss, K. J . , Rubinfeld, B., Polakis, P., and Matrisian, L. M. (1999). The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors. Oncogene 18, 2883-2891. Critchfield, J . W., Coligan, J . E., Folks, T. M., and Butera, S. T. (1997). Casein kinase II is a selective target of HIV-1 transcriptional inhibitors. Proc Natl Acad Sci U S A 94, 6110-6115. Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785-789. Cross, D. A., Watt, P. W., Shaw, M., van der Kaay, J . , Downes, C. P., Holder, J . C , and Cohen, P. (1997). Insulin activates protein kinase B, inhibits glycogen synthase kinase-3 and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue. F E B S Lett 406, 211-215. Crowder, R. J . , and Freeman, R. S. (1999). The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic A M P , requires phosphatidylinositol 3-kinase and Akt. J Neurochem 73, 466-475. Cui, H., Meng, Y., and Bulleit, R. F. (1998). Inhibition of glycogen synthase kinase 3beta activity regulates proliferation of cultured cerebellar granule cells. Brain Res Dev Brain Res 111, 177-188. 266 Dahia, P. L , Aguiar, R. C , Alberta, J . , Kum, J . B., Caron, S., Sill, H., Marsh, D. J . , Ritz, J . , Freedman, A., Stiles, C , and Eng, C. (1999). PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanismsin haematological malignancies. Hum Mol Genet 8, 185-193. D'Amico, M., Hulit, J . , Amanatullah, D. F., Zafonte, B. T., Albanese, C , Bouzahzah, B., Fu, M., Augenlicht, L. H., Donehower, L. A., Takemaru, K., et al. (2000). The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways. J Biol Chem 275, 32649-32657. Datta, K., Franke, T. F., Chan, T. O., Makris, A., Yang, S. I., Kaplan, D. R., Morrison, D. K., Golemis, E. A., and Tsichlis, P. N. (1995). AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation. Mol Cell Biol 15, 2304-2310. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997). Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231-241. Davies, M. A., Koul, D., Dhesi, H., Berman, R., McDonnell, T. J . , McConkey, D., Yung, W. K., and Steck, P. A. (1999). Regulation of Akt/PKB activity, cellular growth, and apoptosis in prostate carcinoma cells by M M A C / P T E N . Cancer Res 59, 2551-2556. Daya-Makin, M., Sanghera, J . S., Mogentale, T. L , Lipp, M., Parchomchuk, J . , Hogg, J . C , and Pelech, S. L. (1994). Activation of a tumor-associated protein 267 kinase (p40TAK) and casein kinase 2 in human squamous cell carcinomas and adenocarcinomas of the lung. Cancer Res 54, 2262-2268. de Groot, R. P., Auwerx, J . , Bourouis, M., and Sassone-Corsi, P. (1993). Negative regulation of Jun/AP-1: conserved function of glycogen synthase kinase 3 and the Drosophila kinase shaggy. Oncogene 8, 841-847. De Vita, G., Berlingieri, M. T., Visconti, R., Castellone, M. D., Viglietto, G. , Baldassarre, G. , Zannini, M., Bellacosa, A., Tsichlis, P. N., Fusco, A., and Santoro, M. (2000). Akt/protein kinase B promotes survival and hormone-independent proliferation of thyroid cells in the absence of dedifferentiating and transforming effects. Cancer Res 60, 3916-3920. Dedhar, S., Williams, B., and Hanhigan, G. (1999). Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling. Trends Cell Biol 9, 319-323. del Peso, L., Gonzalez-Garcia, M., Page, O , Herrera, R., and Nunez, G. (1997) . lnterleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278, 687-689. Delcommenne, M., Tan, C , Gray, V., Rue, L., Woodgett, J . , and Dedhar, S. (1998) . Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci U S A 95, 11211-11216. Desagher, S., Osen-Sand, A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot, L., Antonsson, B., and Martinou, J . C. (2001). 268 Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8. Mol Cell 8, 601-611. Di Cristofano, A., Pesce, B., Cordon-Cardo, C , and Pandolfi, P. P. (1998). Pten is essential for embryonic development and tumour suppression. Nat Genet 19, 348-355. Diehl, J . A., Cheng, M., Roussel, M. F., and Sherr, C. J . (1998). Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12, 3499-3511. Dihlmann, S., Siermann, A., and von Knebel Doeberitz, M. (2001). The nonsteroidal anti-inflammatory drugs aspirin and indomethacin attenuate beta-catenin/TCF-4 signaling. Oncogene 20, 645-653. Dimberg, J . , Hugander, A., Sirsjo, A., and Soderkvist, P. (2001). Enhanced expression of cyclooxygenase-2 and nuclear beta-catenin are related to mutations in the A P C gene in human colorectal cancer. Anticancer Res 21, 911-915. Dolcet, X., Egea, J . , Soler, R. M., Martin-Zanca, D., and Cornelia, J . X. (1999). Activation of phosphatidylinositol 3-kinase, but not extracellular-regulated kinases, is necessary to mediate brain-derived neurotrophic factor-induced motoneuron survival. J Neurochem 73, 521-531. Domin, J . , Pages, F., Volinia, S., Rittenhouse, S. E., Zvelebil, M. J . , Stein, R. O , and Waterfield, M. D. (1997). Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin. Biochem J 326, 139-147. 269 DuBois, R. N. (1995). Nonsteroidal anti-inflammatory drug use and sporadic colorectal adenomas. Gastroenterology 108, 1310-1314. DuBois, R. N., and Smalley, W. E. (1996). Cyclooxygenase, NSAIDs, and colorectal cancer. J Gastroenterol 31, 898-906. Dudek, H., Datta, S. R., Franke, T. F., Birnbaum, M. J . , Yao, R., Cooper, G. M., Segal, R. A., Kaplan, D. R., and Greenberg, M. E. (1997). Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275, 661-665. Dudley, D. T., Pang, L , Decker, S. J . , Bridges, A. J . , and Saltiel, A. R. (1995). A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A 92, 7686-7689. Dugan, D.E., Hooke, K.F. and Hwang, S.S. (1980). Kinetics of the tissue distributions of sulindac and metabolites. Relevance to sites and rates of bioactivation. Drug Metab. Dispos. 8, 241-246. Eberhart, C. E., Coffey, R. J . , Radhika, A., Giardiello, F. M., Ferrenbach, S., and DuBois, R. N. (1994). Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 107, 1183-1188. Egyhazi, E., Ossoinak, A., Tayip, U., Kazimierczuk, Z., and Shugar, D. (1982). Specific inhibition of hnRNA synthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Requirement of a free 3'-hydroxyl group, but not 2'-or 5'-hydroxyls. Biochim Biophys Acta 697, 213-220. 270 Ekbom, A., Helmick, C , Zack, M., and Adami, H. O. (1990). Ulcerative colitis and colorectal cancer. A population-based study. N Engl J Med 323, 1228-1233. el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J . M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993). WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817-825. Embi, N., Rylatt, D. B., and Cohen, P. (1980). Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem 107, 519-527. Esteller, M., Corn, P. G., Baylin, S. B., and Herman, J . G. (2001). A gene hypermethylation profile of human cancer. Cancer Res 61, 3225-3229. Eves, E. M., Xiong, W., Bellacosa, A., Kennedy, S. G. , Tsichlis, P. N., Rosner, M. R., and Hay, N. (1998). Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line. Mol Cell Biol 18, 2143-2152. Fagotto, F., Jho, E., Zeng, L., Kurth, T., Joos, T., Kaufmann, C , and Costantini, F. (1999). Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization. J Cell Biol 145, 741-756. Faust, R. A., Gapany, M., Tristani, P., Davis, A., Adams, G. L., and Ahmed, K. (1996). Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation. Cancer Lett 101, 31-35. Fearon, E. R., Cho, K. R., Nigra, J . M., Kern, S. E., Simons, J . W., Ruppert, J . M., Hamilton, S. R., Preisinger, A. C , Thomas, G., Kinzler, K. W., and et al. 271 (1990). Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247, 49-56. Fosslien, E. (2000). Biochemistry of cyclooxygenase (COX)-2 inhibitors and molecular pathology of COX-2 in neoplasia. Crit Rev Clin Lab Sci 37, 431-502. Franke, T. F., Kaplan, D. R., Cantley, L. C , and Toker, A. (1997). Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275, 665-668. Franke, T. F., Tartof, K. D., and Tsichlis, P. N. (1994). The SH2-like Akt homology (AH) domain of c-akt is present in multiple copies in the genome of vertebrate and invertebrate eucaryotes. Cloning and characterization of the Drosophila melanogaster c-akt homolog Dakt l . Oncogene 9, 141-148. Franke, T. F., Yang, S. I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995). The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81, 727-736. Fry, M. J . (1994). Structure, regulation and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1226, 237-268. Fujita, E., Jinbo, A., Matuzaki, H., Konishi, H., Kikkawa, U., and Momoi, T. (1999). Akt phosphorylation site found in human caspase-9 is absent in mouse caspase-9. Biochem Biophys Res Commun 264, 550-555. Fukazawa, T., Fujiwara, T., Morimoto, Y., Shao, J . , Nishizaki, M., Kadowaki, Y., Hizuta, A., Owen-Schaub, L. B., Roth, J . A., and Tanaka, N. (1999). Differential involvement of the CD95 (Fas/APO-1) receptor/ligand system on 272 apoptosis induced by the wild-type p53 gene transfer in human cancer cells. Oncogene 18, 2189-2199. Fukuda, M., Gotoh, Y., and Nishida, E. (1997). Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase. Embo J 16, 1901-1908. Fukui, Y., and Hanafusa, H. (1989). Phosphatidylinositol kinase activity associates with viral p60src protein. Mol Cell Biol 9, 1651-1658. Fukumoto, S., Hsieh, C. M., Maemura, K., Layne, M. D., Yet, S. F., Lee, K. H., Matsui, T., Rosenzweig, A., Taylor, W. G., Rubin, J . S., et al. (2001). Akt participation in the Wnt signaling pathway through Dishevelled. J Biol Chem 276, 17479-17483. Furnari, F. B., Lin, H., Huang, H. S., and Cavenee, W. K. (1997). Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain. Proc Natl Acad Sci U S A 94, 12479-12484. Gann, P. H., Manson, J . E., Glynn, R. J . , Buring, J . E., and Hennekens, C. H. (1993). Low-dose aspirin and incidence of colorectal tumors in a randomized trial. J Natl Cancer Inst 85, 1220-1224. Gapany, M., Faust, R. A., Tawfic, S., Davis, A., Adams, G. L., and Ahmed, K. (1995). Association of elevated protein kinase CK2 activity with aggressive behavior of squamous cell carcinoma of the head and neck. Mol Med 1, 659-666. Gautreau, A., Poullet, P., Louvard, D., and Arpin, M. (1999). Ezrin, a plasma membrane-microfilament linker, signals cell survival through the 273 phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci U S A 96, 7300-7305. Gerber, H. P., McMurtrey, A., Kowalski, J . , Yan, M., Keyt, B. A., Dixit, V., and Ferrara, N. (1998). Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 273, 30336-30343. Ghosh, A. K., Grigorieva, I., Steele, R., Hoover, R. G., and Ray, R. B. (1999). PTEN transcriptionally modulates c-myc gene expression in human breast carcinoma cells and is involved in cell growth regulation. Gene 235, 85-91. Giardiello, F. M., Offerhaus, G. J . , and DuBois, R. N. (1995). The role of nonsteroidal anti-inflammatory drugs in colorectal cancer prevention. Eur J Cancer 31 A, 1071-1076. Gibson, S., Tu, S., Oyer, R., Anderson, S. M., and Johnson, G. L. (1999). Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation. J Biol Chem 274, 17612-17618. Giovannucci, E., Ascherio, A., Rimm, E. B., Colditz, G. A., Stampfer, M. J . , and Willett, W. C. (1995). Physical activity, obesity, and risk for colon cancer and adenoma in men. Ann Intern Med 122, 327-334. Giovannucci, E., and Willett, W. C. (1994). Dietary factors and risk of colon cancer. Ann Med 26, 443-452. Gold, M. R., Scheid, M. P., Santos, L , Dang-Lawson, M., Roth, R. A., Matsuuchi, L , Duronio, V., and Krebs, D. L. (1999). The B cell antigen receptor 274 activates the Akt (protein kinase B)/glycogen synthase kinase-3 signaling pathway via phosphatidylinositol 3-kinase. J Immunol 163, 1894-1905. Gomez, N., and Cohen, P. (1991). Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature 353, 170-173. Goss, K. H., and Groden, J . (2000). Biology of the adenomatous polyposis coli tumor suppressor. J Clin Oncol 18, 1967-1979. Gotoh, Y., Matsuda, S., Takenaka, K., Hattori, S., Iwamatsu, A., Ishikawa, M., Kosako, H., and Nishida, E. (1994). Characterization of recombinant Xenopus MAP kinase kinases mutated at potential phosphorylation sites. Oncogene 9, 1891-1898. Graff, J . R., Deddens, J . A., Konicek, B. W., Colligan, B. M., Hurst, B. M., Carter, H. W., and Carter, J . H. (2001). Integrin-linked kinase expression increases with prostate tumor grade. Clin Cancer Res 7, 1987-1991. Graff, J . R., Konicek, B. W., McNulty, A. M., Wang, Z., Houck, K., Allen, S., Paul, J . D., Hbaiu, A., Goode, R. G. , Sandusky, G. E., et al. (2000). Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression. J Biol Chem 275, 24500-24505. Grimes, C. A., and Jope, R. S. (2001). The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol 65, 391-426. Guerra, B., and Issinger, O. G. (1999). Protein kinase CK2 and its role in cellular proliferation, development and pathology. Electrophoresis 20, 391-408. 275 Guo, S., Rena, G., Cichy, S., He, X., Cohen, P., and Unterman, T. (1999). Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J Biol Chem 274, 17184-17192. Haggitt, R. C , Glotzbach, R. E., Soffer, E. E., and Wruble, L. D. (1985). Prognostic factors in colorectal carcinomas arising in adenomas: implications for lesions removed by endoscopic polypectomy. Gastroenterology 89, 328-336. Hall, A. (1994). A biochemical function for ras-at last. Science 264, 1413-1414. Hall, P. A., Meek, D., and Lane, D. P. (1996). p53-integrating the complexity. J Pathol 180, 1-5. Hanger, D. P., Hughes, K., Woodgett, J . R., Brion, J . P., and Anderton, B. H. (1992). Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase. Neurosci Lett 147, 58-62. Hanif, R., Pittas, A., Feng, Y., Koutsos, M. .I., Qiao, L., Staiano-Coico, L., Shiff, S. I., and Rigas, B. (1996). Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway. Biochem Pharmacol 52, 237-245. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 247, 42-52. 276 Hannigan, G. E., Leung-Hagesteijn, C., Fitz-Gibbon, L., Coppolino, M. G., Radeva, G., Filmus, J . , Bell, J . C , and Dedhar, S. (1996). Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase. Nature 379, 91-96. Hart, M., Concordet, J . P., Lassot, I., Albert, I., del los Santos, R., Durand, H., Perret, C , Rubinfeld, B., Margottin, F., Benarous, R., and Polakis, P. (1999): The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell. Curr Biol 9, 207-210. Hart, M. J . , de los Santos, R., Albert, I. N., Rubinfeld, B., and Polakis, P. (1998). Downregulation of beta-catenin by human Axin and its association with the A P C tumor suppressor, beta-catenin and G S K 3 beta. Curr Biol 8, 573-581. Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997). Mdm2 promotes the rapid degradation of p53. Nature 387, 296-299. Haystead, T. A., Dent, P., Wu, J . , Haystead, C. M., and Sturgill, T. W. (1992). Ordered phosphorylation of p42mapk by MAP kinase kinase. F E B S Lett 306, 17-22. He, T. C , Chan, T. A., Vogelstein, B., and Kinzler, K. W. (1999). PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99, 335-345. He, T. O , Sparks, A. B., Rago, C , Hermeking, H., Zawel, L., da Costa, L. T., Morin, P. J . , Vogelstein, B., and Kinzler, K. W. (1998). Identification of c -MYC as a target of the A P C pathway. Science 281, 1509-1512. 277 Heldin, C. H. (1995). Dimerization of cell surface receptors in signal transduction. Cell 80, 213-223. Hinoi, T., Yamamoto, H., Kishida, M., Takada, S., Kishida, S., and Kikuchi, A. (2000). Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3 beta-dependent phosphorylation of beta-catenin and down-regulates beta-catenin. J Biol Chem 275, 34399-34406. Hoeflich, K. P., Luo, J . , Rubie, E. A., Tsao, M. S., Jin, O., and Woodgett, J . R. (2000). Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 406, 86-90. Hrubey, T. W., and Roach, P. J . (1990). Phosphoserine in peptide substrates can specify casein kinase II action. Biochem Biophys Res Commun 172, 190-196. Hsu, A. L., Ching, T. T., Wang, D. S., Song, X., Rangnekar, V. M., and Chen, C. S. (2000). The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 275, 11397-11403. Hsu, W., Zeng, L., and Costantini, F. (1999). Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain. J Biol Chem 274, 3439-3445. Huber, O., Korn, R., McLaughlin, J . , Ohsugi, M., Herrmann, B. G., and Kemler, R. (1996). Nuclear localization of beta-catenin by interaction with transcription factor LEF-1 . Mech Dev 59, 3-10. 278 Ikeda, S., Kishida, M., Matsuura, Y., Usui, H., and Kikuchi, A. (2000). G S K -3beta-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by beta-catenin and protein phosphatase 2A complexed with Axin. Oncogene 19, 537-545. Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S., and Kikuchi, A. (1998). Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. Embo J 17, 1371-1384. Ishiko, O., Sumi, T., Yoshida, H., Matsumoto, Y., Honda, K., Deguchi, M., Yamada, R., and Ogita, S. (2001). Association between overexpression of cyclooxygenase-2 and suppression of apoptosis in advanced cancer of the uterine cervix after cyclic balloon-occluded arterial infusion. Oncol Rep 8, 1259-1263. Issinger, O. G. (1993). Casein kinases: pleiotropic mediators of cellular regulation. Pharmacol Ther 59, 1-30. Iwamoto, M., Ahnen, D. J . , Franklin, W. A., and Maltzman, T. H. (2000). Expression of beta-catenin and full-length A P C protein in normal and neoplastic colonic tissues. Carcinogenesis 21, 1935-1940. Janji, B., Melchior, C , Vallar, L , and Kieffer N. (2000). Cloning of an isoform of integrin-linked kinase (ILK) that is upregulated in HT-144 melanoma cells following TGF-beta1 stimulation. Oncogene 22, 3069-3077. Jayaraman, L., and Prives, C. (1999). Covalent and noncovalent modifiers of the p53 protein. Cell Mol Life Sci 55, 76-87. 279 Jefferies, H. B., Fumagalli, S., Dennis, P. B., Reinhard, C , Pearson, R. B., and Thomas, G. (1997). Rapamycin suppresses 5 T O P mRNA translation through inhibition of p70s6k. Embo J 16, 3693-3704. Jeffrey, P. D., Gorina, S., and Pavletich, N. P. (1995). Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science 267, 1498-1502. Jen, J . , Powell, S. M., Papadopoulos, N., Smith, K. J . , Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. (1994). Molecular determinants of dysplasia in colorectal lesions. Cancer Res 54, 5523-5526. Jimenez, C , Jones, D. R., Rodriguez-Viciana, P., Gonzalez-Garcia, A., Leonardo, E., Wennstrom, S., von Kobbe, C , Toran, J . L., L, R. B., Calvo, V., et al. (1998). Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase. Embo J 17, 743-753. Joensuu, H., Roberts, P. J . , Sarlomo-Rikala, M., Andersson, L. C , Tervahartiala, P., Tuveson, D., Silberman, S., Capdeville, R., Dimitrijevic, S., Druker, B., and Demetri, G. D. (2001). Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 344, 1052-1056. Jones, P. F., Jakubowicz, T., Pitossi, F. J . , Maurer, F., and Hemmings, B. A. (1991). Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A 88, 4171-4175. Kane, L. P., Shapiro, V. S., Stokoe, D., and Weiss, A. (1999). Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol 9, 601-604. 280 Kapeller, R., and Cantley, L. C. (1994). Phosphatidylinositol 3-kinase. Bioessays 16, 565-576. Kaplan, D. R., Whitman, M., Schaffhausen, B., Raptis, L , Garcea, R. L., Pallas, D., Roberts, T. M., and Cantley, L. (1986). Phosphatidylinositol metabolism and polyoma-mediated transformation. Proc Natl Acad Sci U S A 83, 3624-3628. Kastan, M. B., Zhan, Q., el-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J . , Jr. (1992). A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, 587-597. Kauffmann-Zeh, A., Rodriguez-Viciana, P., Ulrich, E., Gilbert, C , Coffer, P., Downward, J . , and Evan, G. (1997). Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 385, 544-548. Kawahara, K., Morishita, T., Nakamura, T., Hamada, F., Toyoshima, K., and Akiyama, T. (2000). Down-regulation of beta-catenin by the colorectal tumor suppressor A P C requires association with Axin and beta-catenin. J Biol Chem 275, 8369-8374. Kawasaki, Y., Senda, T., Ishidate, T., Koyama, R., Morishita, T., Iwayama, Y., Higuchi, O., and Akiyama, T. (2000). Asef, a link between the tumor suppressor A P C and G-protein signaling. Science 289, 1194-1197. Kelley, T. W., Graham, M. M., Doseff, A. I., Pomerantz, R. W., Lau, S. M., Ostrowski, M. C , Franke, T. F., and Marsh, C. B. (1999). Macrophage colony-281 stimulating factor promotes cell survival through Akt/protein kinase B. J Biol Chem 274, 26393-26398. Kelliher, M. A., Seldin, D. C , and Leder, P. (1996). Tal-1 induces T cell acute lymphoblastic leukemia accelerated by casein kinase llalpha. Embo J 15, 5160-5166. Kennedy, S. G., Kandel, E. S., Cross, T. K., and Hay, N. (1999). Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol Cell Biol 19, 5800-5810. Kennedy, S. G., Wagner, A. J . , Conzen, S. D., Jordan, J . , Bellacosa, A., Tsichlis, P. N., and Hay, N. (1997). The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev 11, 701-713. Kim, L , and Kimmel, A. R. (2000). GSK3 , a master switch regulating cell-fate specification and tumorigenesis. Curr Opin Genet Dev 10, 508-514. King, C. C , Gardiner, E. M., Zenke, F. T., Bohl, B. P., Newton, A. C , Hemmings, B. A., and Bokoch, G. M. (2000). p21-activated kinase (PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1 (PDK1). J Biol Chem 275, 41201-41209. Kinzler, K. W., Nilbert, M. C , Su, L. K., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J . , Preisinger, A. C., Hedge, P., McKechnie, D., and et al. (1991). Identification of FAP locus genes from chromosome 5q21. Science 253, 661-665. Kinzler, K. W., and Vogelstein, B. (1996). Lessons from hereditary colorectal cancer. Cell 87, 159-170. 282 Kinzler, K. W., and Vogelstein, B. (1997). Cancer-susceptibility genes. Gatekeepers and caretakers. Nature 386, 761, 763. Kishida, S., Yamamoto, H., Hino, S., Ikeda, S., Kishida, M., and Kikuchi, A. (1999). DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability. Mol Cell Biol 19, 4414-4422. Kishida, S., Yamamoto, H., Ikeda, S., Kishida, M., Sakamoto, I., Koyama, S., and Kikuchi, A. (1998). Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J Biol Chem 273, 10823-10826. Kitagawa, M., Hatakeyama, S., Shirane, M., Matsumoto, M., Ishida, N., Hattori, K., Nakamichi, I., Kikuchi, A., and Nakayama, K. (1999). An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin. Embo J 18, 2401-2410. Ko, L. J . , and Prives, C. (1996). p53: puzzle and paradigm. Genes Dev 10, 1054-1072. Kobayashi, M., Nagata, S., Iwasaki, T., Yanagihara, K., Saitoh, I., Karouji, Y., Ihara, S., and Fukui, Y. (1999). Dedifferentiation of adenocarcinomas by activation of phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A 96, 4874-4879. Koh, T. J . , Bulitta, C. J . , Fleming, J . V., Dockray, G. J . , Varro, A., and Wang, T. C. (2000). Gastrin is a target of the beta-catenin/TCF-4 growth-signaling pathway in a model of intestinal polyposis. J Clin Invest 106, 533-539. 283 Kohn, A. D., Summers, S. A., Birnbaum, M. J . , and Roth, R. A. (1996). Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271, 31372-31378. Kontos, C. D., Stauffer, T. P., Yang, W. P., York, J . D., Huang, L , Blanar, M. A., Meyer, T., and Peters, K. G. (1998). Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt. Mol Cell Biol 18, 4131-4140. Korinek, V., Barker, N., Morin, P. J . , van Wichen, D., de Weger, R., Kinzler, K. W., Vogelstein, ,, B., and Clevers, H. (1997). Constitutive transcriptional activation by a beta-catenin-Tcf complex in A P C - / - colon carcinoma. Science 275, 1784-1787. Kouhara, H., Hadari, Y. R., Spivak-Kroizman, T., Schilling, J . , Bar-Sagi, D., Lax, I., and Schlessinger, J . (1997). A lipid-anchored Grb2-binding protein that links FGF-receptor activation to the Ras /MAPK signaling pathway. Cell 89, 693-702. Kraus, C , Liehr, T., Hulsken, J . , Behrens, J . , Birchmeier, W., Grzeschik, K. H., and Ballhausen, W. G. (1994). Localization of the human beta-catenin gene (CTNNB1) to 3p21: a region implicated in tumor development. Genomics 23, 272-274. Kulik, G., Klippel, A., and Weber, M. J . (1997). Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 77, 1595-1606. 284 Kune, G. A., Kune, S., and Watson, L. F. (1988). Colorectal cancer risk, chronic illnesses, operations, and medications: case control results from the Melbourne Colorectal Cancer Study. Cancer Res 48, 4399-4404. Kurose, K., Zhou, X. P., Araki, T., Cannistra, S. A., Maher, E. R., and Eng, C. (2001). Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas. Am J Pathol 158, 2097-2106. Landesman-Bollag, E., Channavajhala, P. L , Cardiff, R. D., and Seldin, D. C. (1998). p53 deficiency and misexpression of protein kinase CK2alpha collaborate in the development of thymic lymphomas in mice. Oncogene 16, 2965-2974. Landesman-Bollag, E., Romieu-Mourez, R., Song, D. H., Sonenshein, G. E., Cardiff, R. D., and Seldin, D. C. (2001). Protein kinase CK2 in mammary gland tumorigenesis. Oncogene 20, 3247-3257. Landis, S. H., Murray, T., Bolden, S., and Wingo, P. A. (1998). Cancer statistics, 1998. C A Cancer J Clin 48, 6-29. Lashner, B. A., Kane, S. V., and Hanauer, S. B. (1990). Colon cancer surveillance in chronic ulcerative colitis: historical cohort study. Am J Gastroenterol 85, 1083-1087. Lau, K. F., Miller, C. C , Anderton, B. H., and Shaw, P. C. (1999). Molecular cloning and characterization of the human glycogen synthase kinase-3beta promoter. Genomics 60, 121-128. 285 Lavoie, L , Band, C. J . , Kong, M., Bergeron, J . J . , and Posner, B. I. (1999). Regulation of glycogen synthase in rat hepatocytes. Evidence for multiple signaling pathways. J Biol Chem 274, 28279-28285. Le Marchand, L., Wilkens, L. R., Kolonel, L. N., Hankin, J . H., and Lyu, L. C. (1997). Associations of sedentary lifestyle, obesity, smoking, alcohol use, and diabetes with the risk of colorectal cancer. Cancer Res 57, 4787-4794. Leroy, D., Heriche, J . K., Filhol, O., Chambaz, E. M., and Cochet, C. (1997). Binding of polyamines to an autonomous domain of the regulatory subunit of protein kinase CK2 induces a conformational change in the holoenzyme. A proposed role for the kinase stimulation. J Biol Chem 272, 20820-20827. Leroy, D., Valero, E., Filhol, O., Heriche, J . K., Goldberg, Y., Chambaz, E. M., and Cochet, C. (1994). Modulation of the molecular organization and activity of casein kinase 2 by naturally occurring polyamines. Cell Mol Biol Res 40, 441-453. Leverrier, Y., Thomas, J . , Mathieu, A. L., Low, W., Blanquier, B., and Marvel, J . (1999). Role of PI3-kinase in Bcl-X induction and apoptosis inhibition mediated by IL-3 or IGF-1 in Baf-3 cells. Cell Death Differ 6, 290-296. Li, D. M., and Sun, H. (1997). TEP1 , encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res 57, 2124-2129. Li, D. M., and Sun, H. (1998). PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G l cell cycle arrest in human glioblastoma cells. Proc Natl Acad Sci U S A 95, 15406-15411. 286 Li, J . , Yen, C , Liaw, D., Podsypanina, K., Bose, S., Wang, S. I., Puc, J . , Miliaresis, C , Rodgers, L., McCombie, R., et al. (1997). P T E N , a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943-1947. Li, M., Wu, X., and Xu, X. C. (2001a). Induction of apoptosis by cyclo-oxygenase-2 inhibitor NS398 through a cytochrome C-dependent pathway in esophageal cancer cells. Int J Cancer 93, 218-223. Li, M., Wu, X., and Xu, X. C. (2001b). Induction of apoptosis in colon cancer cells by cyclooxygenase-2 inhibitor NS398 through a cytochrome c-dependent pathway. Clin Cancer Res 7, 1010-1016. Liaw, D., Marsh, D. J . , Li, J . , Dahia, P. L., Wang, S. I., Zheng, Z., Bose, S., Call, K. M., Tsou, H. C , Peacocke, M., e ra / . (1997). Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat Genet 16, 64-67. Lickert, H., Bauer, A., Kemler, R., and Stappert, J . (2000). Casein kinase II phosphorylation of E-cadherin increases E-cadherin/beta-catenin interaction and strengthens cell-cell adhesion. J Biol Chem 275, 5090-5095. Lim, H. Y., Joo, H. J . , Choi, J . H., Yi , J . W., Yang, M. S., Cho, D. Y., Kim, H. S., Nam, D. K., Lee, K. B., and Kim, H. C. (2000). Increased expression of cyclooxygenase-2 protein in human gastric carcinoma. Clin Cancer Res 6, 519-525. Lin, M. T., Lee, R. C , Yang, P. C , Ho, F. M., and Kuo, M. L. (2001). Cyclooxygenase-2 inducing Mcl-1-dependent survival mechanism in human lung 2 8 7 adenocarcinoma CL1.0 cells. Involvement of phosphatidylinositol 3-kinase/Akt pathway. J Biol Chem 276, 48997-49002. Litchfield, D. W., Arendt, A., Lozeman, F. J . , Krebs, E. G., Hargrave, P. A., and Palczewski, K. (1990a). Synthetic phosphopeptides are substrates for casein kinase II. F E B S Lett 267, 117-120. Litchfield, D. W., Lozeman, F. J . , Piening, C , Sommercorn, J . , Takio, K., Walsh, K. A., and Krebs, E. G. (1990b). Subunit structure of casein kinase II from bovine testis. Demonstration that the alpha and alpha' subunits are distinct polypeptides. J Biol Chem 265, 7638-7644. Liu, Q., Sasaki, T., Kozieradzki, I., Wakeham, A., Itie, A., Dumont, D. J . , and Penninger, J . M. (1999). SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. Genes Dev 13, 786-791. Liu, W., Dong, X., Mai, M., Seelan, R. S., Taniguchi, K., Krishnadath, K. K., Hailing, K. C , Cunningham, J . M., Boardman, L. A., Qian, C , et al. (2000). Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/TCF signalling. Nat Genet 26, 146-147. Lloyd, A. C , Paterson, H. F., Morris, J . D., Hall, A., and Marshall, C. J . (1989). p21H-ras-induced morphological transformation and increases in c-myc expression are independent of functional protein kinase C. Embo J 8, 1099-1104. Lo, S. H., Weisberg, E., and Chen, L. B. (1994). Tensin: a potential link between the cytoskeleton and signal transduction. Bioessays 76, 817-823. 288 Lynch, D. K., Ellis, C. A., Edwards, P. A., and Hiles, I. D. (1999). Integrin-linked kinase regulates phosphorylation of serine 473 of protein kinase B by an indirect mechanism. Oncogene 18, 8024-8032. Lynch, H. T., Smyrk, T., McGinn, T., Lanspa, S., Cavalieri, J . , Lynch, J . , Slominski-Castor, S., Cayouette, M. O , Priluck, I., and Luce, M. C. (1995). Attenuated familial adenomatous polyposis (AFAP). A phenotypically and genotypically distinctive variant of FAP. Cancer 76, 2427-2433. Madrid, L. V., Mayo, M. W., Reuther, J . Y., and Baldwin, A. S., Jr. (2001). Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 276, 18934-18940. Maehama, T., and Dixon, J . E. (1998). The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 273, 13375-13378. Maekawa, M., Sugano, K., Sano, H., Miyazaki, S., Ushiama, M., Fujita, S., Gotoda, T., Yokota, T., Ohkura, H., Kakizoe, T., and Sekiya, T. (1998). Increased expression of cyclooxygenase-2 to -1 in human colorectal cancers and adenomas, but not in hyperplastic polyps. Jpn J Clin Oncol 28, 421-426. Mai, M., Qian, C , Yokomizo, A., Smith, D. I., and Liu, W. (1999). Cloning of the human homolog of conductin (AXIN2), a gene mapping to chromosome 17q23-q24. Genomics 55, 341-344. Maniatis, T. (1999). A ubiquitin ligase complex essential for the NF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways. Genes Dev 13, 505-510. 289 Mao, J . , Wang, J . , Liu, B., Pan, W., Farr, G. H., 3rd, Flynn, C , Yuan, H., Takada, S., Kimelman, D., Li, L., and Wu, D. (2001). Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol Cell 7, 801-809. Marin, O., Meggio, F., Draetta, G., and Pinna, L. A. (1992). The consensus sequences for cdc2 kinase and for casein kinase-2 are mutually incompatible. A study with peptides derived from the beta-subunit of casein kinase-2. F E B S Lett 301, 111-114. Marotta, A., Tan, C , Gray, V., Malik, S., Gallinger, S., Sanghera, J . , Dupuis, B., Owen, D., Dedhar, S., and Salh, B. (2001). Dysregulation of integrin-linked kinase (ILK) signaling in colonic polyposis. Oncogene 20, 6250-6257. Marsh, D. J . , Dahia, P. L., Coulon, V., Zheng, Z., Dorion-Bonnet, F., Call, K. M., Little, R., Lin, A. Y., Eeles, R. A., Goldstein, A. M., et al. (1998). Allelic imbalance, including deletion of PTEN/MMACI, at the Cowden disease locus on 10q22-23, in hamartomas from patients with Cowden syndrome and germline PTEN mutation. Genes Chromosomes Cancer 21, 61-69. Martegani, E., Vanoni, M., Zippel, R., Coccetti, P., Brambilla, R., Ferrari, C , Sturani, E., and Alberghina, L. (1992). Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator. Embo J 11, 2151-2157. Masure, S., Haefner, B., Wesselink, J . J . , Hoefnagel, E., Mortier, E., Verhasselt, P., Tuytelaars, A., Gordon, R., and Richardson, A. (1999). Molecular 290 cloning, expression and characterization of the human serine/threonine kinase Akt-3. Eur J Biochem 265, 353-360. Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992). Xenopus MAP kinase activator: identification and function as a key intermediate in the phosphorylation cascade. Embo J 77, 973-982. Mauro, M. J. , ' O'Dwyer, M. E., and Druker, B. J . (2001). ST1571, a tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia: validating the promise of molecularly targeted therapy. Cancer Chemother Pharmacol 48, S77-78. Mayer, B. J . , Ren, R., Clark, K. L , and Baltimore, D. (1993). A putative modular domain present in diverse signaling proteins. Cell 73, 629-630. Meek, D. W. (1994). Post-translational modification of p53. Semin Cancer Biol 5, 203-210. Meili, R., Ellsworth, C , Lee, S., Reddy, T. B., Ma, H., and Firtel, R. A. (1999). Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. Embo J 78,2092-2105. Midgley, C. A., and Lane, D. P. (1997). p53 protein stability in tumour cells is not determined by mutation but is dependent on Mdm2 binding. Oncogene 75, 1179-1189. Miwa, W., Yasuda, J . , Murakami, Y., Yashima, K., Sugano, K., Sekine, T., Kono, A., Egawa, S., Yamaguchi, K., Hayashizaki, Y., and Sekiya, T. (1996). 291 Isolation of DNA sequences amplified at chromosome 19q13.1-q13.2 including the AKT2 locus in human pancreatic cancer. Biochem Biophys Res Commun 225,968-974. Miyashita, T., and Reed, J . C. (1995). Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293-299. Mohri, T., Adachi, Y., Ikehara, S., Hioki, K., Tokunaga, R., and Taketani, S. (1999). Activated Rac1 selectively up-regulates the expression of integrin alpha6beta4 and induces cell adhesion and membrane ruffles of nonadherent colon cancer Colo201 cells. Exp Cell Res 253, 533-540. Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J . , Godsave, S., Korinek, V., Roose, J . , Destree, O., and Clevers, H. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391-399. Moodie, S. A., Willumsen, B. M., Weber, M. J . , and Wolfman, A. (1993). Complexes of Ras .GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 260, 1658-1661. Moreno, F. J . , Medina, M., Perez, M., Montejo de Garcini, E., and Avila, J . (1995). Glycogen synthase kinase 3 phosphorylates recombinant human tau protein at serine-262 in the presence of heparin (or tubulin). F E B S Lett 372, 65-68. Morimoto, A. M., Berson, A. E., Fujii, G. H., Teng, D. H., Tavtigian, S. V., Bookstein, R., Steck, P. A., and Bolen, J . B. (1999). Phenotypic analysis of 292 human glioma cells expressing the MMAC1 tumor suppressor phosphatase. Oncogene 18, 1261-1266. Morin, P. J . , Sparks, A. B., Korinek, V., Barker, N., Clevers, H., Vogelstein, B., and Kinzler, K. W. (1997). Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or A P C . Science 275, 1787-1790. Morisco, C , Seta, K., Hardt, S. E., Lee, Y., Vatner, S. F., and Sadoshima, J . (2001). Glycogen synthase kinase 3beta regulates GATA4 in cardiac myocytes. J Biol Chem 276, 28586-28597. Morris, G. F., Bischoff, J . R., and Mathews, M. B. (1996). Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53. Proc Natl Acad Sci U S A 93, 895-899. Morrison, D. K., Heidecker, G. , Rapp, U. R., and Copeland, T. D. (1993). Identification of the major phosphorylation sites of the Raf-1 kinase. J Biol Chem 268, 17309-17316. Moule, S. K., Welsh, G. I., Edgell, N. J . , Foulstone, E. J . , Proud, C. G. , and Denton, R. M. (1997). Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and beta-adrenergic agonists in rat epididymal fat cells. Activation of protein kinase B by wortmannin-sensitive and -insensitive mechanisms. J Biol Chem 272, 7713-7719. Munemitsu, S., Souza, B., Muller, O., Albert, I., Rubinfeld, B., and Polakis, P. (1994). The A P C gene product associates with microtubules in vivo and promotes their assembly in vitro. Cancer Res 54, 3676-3681. 293 Munstermann, U., Fritz, G., Seitz, G. , Lu, Y. P., Schneider, H. R., and Issinger, O. G. (1990). Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue. Eur J Biochem 189, 251-257. Murata, H., Kawano, S., Tsuji, S., Tsuji, M., Sawaoka, H., Kimura, Y., Shiozaki, H., and Hori, M. (1999). Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma. Am J Gastroenterol 94, 451-455. Murthy, S. S., Tosolini, A., Taguchi, T., and Testa, J . R. (2000). Mapping of AKT3, encoding a member of the Akt/protein kinase B family, to human and rodent chromosomes by fluorescence in situ hybridization. Cytogenet Cell Genet 88, 38-40. Musacchio, A., Gibson, T., Rice, P., Thompson, J . , and Saraste, M. (1993). The PH domain: a common piece in the structural patchwork of signalling proteins. Trends Biochem Sci 18, 343-348. Myers, M. P., Pass, I., Batty, I. H., Van der Kaay, J . , Stolarov, J . P., Hemmings, B. A., Wigler, M. H., Downes, C. P., and Tonks, N. K. (1998). The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proc Natl Acad Sci U S A 95, 13513-13518. Nagase, H., Miyoshi, Y., Horii, A., Aoki, T., Ogawa, M., Utsunomiya, J . , Baba, S., Sasazuki, T., and Nakamura, Y. (1992). Correlation between the location of germ-line mutations in the A P C gene and the number of colorectal polyps in familial adenomatous polyposis patients. Cancer Res 52, 4055-4057. 294 Nakatani, K., Sakaue, H., Thompson, D. A., Weigel, R. J . , and Roth, R. A. (1999a). Identification of a human Akt3 (protein kinase B gamma) which contains the regulatory serine phosphorylation site. Biochem Biophys Res Commun 257, 906-910. Nakatani, K., Thompson, D. A., Barthel, A., Sakaue, H., Liu, W., Weigel, R. J . , and Roth, R. A. (1999b). Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines. J Biol Chem 274, 21528-21532. Nathke, I. S. (1999). The adenomatous polyposis coli protein. Mol Pathol 52, 169-173. Nathke, I. S., Adams, C. L., Polakis, P., Sellin, J . H., and Nelson, W. J . (1996) . The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration. J Cell Biol 134, 165-179. Nelen, M. R., van Staveren, W. C , Peeters, E. A., Hassel, M. B., Gorlin, R. J . , Hamm, H., Lindboe, C. F., Fryns, J . P., Sijmons, R. H., Woods, D. G., et al. (1997) . Germline mutations in the PTEN/MMAC1 gene in patients with Cowden disease. Hum Mol Genet 6, 1383-1387. Novak, A., and Dedhar, S. (1999). Signaling through beta-catenin and Lef/Tcf. Cell Mol Life Sci 56, 523-537. Novak, A., Hsu, S. C , Leung-Hagesteijn, C , Radeva, G., Papkoff, J . , Montesano, R., Roskelley, C , Grosschedl, R., and Dedhar, S. (1998). Cell 295 adhesion and the integrin-linked kinase regulate the LEF-1 and beta-catenin signaling pathways. Proc Natl Acad Sci U S A 95, 4374-4379. Nugent, F. W., Haggitt, R. C , and Gilpin, P. A. (1991). Cancer surveillance in ulcerative colitis. Gastroenterology 100, 1241-1248. Nusse, R., Theunissen, H., Wagenaar, E., Rijsewijk, F., Gennissen, A., Otte, A., Schuuring, E., and van Ooyen, A. (1990). The Wnt-1 (int-1) oncogene promoter and its mechanism of activation by insertion of proviral DNA of the mouse mammary tumor virus. Mol Cell Biol 10, 4170-4179. Oshima, M., Dinchuk, J . E., Kargman, S. L., Oshima, H., Hancock, B., Kwong, E., Trzaskos, J . M., Evans, J . F., and Taketo, M. M. (1996). Suppression of intestinal polyposis in Ape delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 87, 803-809. Page, O , Lin, H. J . , Jin, Y., Castle, V. P., Nunez, G. , Huang, M., and Lin, J . (2000). Overexpression of Akt/AKT can modulate chemotherapy-induced apoptosis. Anticancer Res 20, 407-416. Pages, G., Brunet, A., L'Allemain, G., and Pouyssegur, J . (1994). Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK1). Embo J 13, 3003-3010. Paradis, S., and Ruvkun, G. (1998). Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev 12, 2488-2498. 296 Paramio, J . M., Navarro, M., Segrelles, C , Gomez-Casero, E., and Jorcano, J . L. (1999). PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. Oncogene 18, 7462-7468. Parry, R. V., Reif, K., Smith, G., Sansom, D. M., Hemmings, B. A., and Ward, S. G. (1997). Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B. Eur J Immunol 27, 2495-2501. Payne, D. M., Rossomando, A. J . , Martino, P., Erickson, A. K., Her, J . H., Shabanowitz, J . , Hunt, D. F., Weber, M. J . , and Sturgill, T. W. (1991). Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). Embo J 10, 885-892. Peifer, M., and Polakis, P. (2000). Wnt signaling in oncogenesis and embryogenesis-a look outside the nucleus. Science 287, 1606-1609. Peifer, M., Rauskolb, C , Williams, M., Riggleman, B., and Wieschaus, E. (1991). The segment polarity gene armadillo interacts with the wingless signaling pathway in both embryonic and adult pattern formation. Development 111, 1029-1043. Peifer, M., and Wieschaus, E. (1990). The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin. Cell 63, 1167-1176. Pelech, S. L., and Charest, D. L. (1995). MAP kinase-dependent pathways in cell cycle control. Prog Cell Cycle Res 7, 33-52. 297 Pelech, S. L , Sanghera, J . S., and Daya-Makin, M. (1990). Protein kinase cascades in meiotic and mitotic cell cycle control. Biochem Cell Biol 68, 1297-1330. Peleg, II, Maibach, H. T., Brown, S. H., and Wilcox, C. M. (1994). Aspirin and nonsteroidal anti-inflammatory drug use and the risk of subsequent colorectal cancer. Arch Intern Med 154, 394-399. Persad, S., Attwell, S., Gray, V., Delcommenne, M., Troussard, A., Sanghera, J . , and Dedhar, S. (2000). Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells. Proc Natl Acad Sci U S A 97, 3207-3212. Persad, S., Attwell, S., Gray, V., Mawji, N., Deng, J . T., Leung, D., Yan, J . , Sanghera, J . , Walsh, M. P., and Dedhar, S. (2001). Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem 276, 27462-27469. Pianetti, S., Arsura, M., Romieu-Mourez, R., Coffey, R. J . , and Sonenshein, G. E. (2001). Her-2/neu overexpression induces NF-kappaB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IkappaB-alpha that can be inhibited by the tumor suppressor PTEN. Oncogene 20, 1287-1299. Piazza, G. A., Alberts, D. S., Hixson, L. J . , Paranka, N. S., Li, H., Finn, T., Bogert, C , Guillen, J . M., Brendel, K., Gross, P. H., et al. (1997a). Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels. Cancer Res 57, 2909-2915. 298 Piazza, G. A., Rahm, A. K., Finn, T. S., Fryer, B. H., Li, H., Stoumen, A. L., Pamukcu, R., and Ahhen, D. J . (1997b). Apoptosis primarily accounts for the growth-inhibitory properties of sulindac metabolites and involves a mechanism that is independent of cyclooxygenase inhibition, cell cycle arrest, and p53 induction. Cancer Res 57, 2452-2459. Piazza, G. A., Rahm, A. L., Krutzsch, M., Sperl, G., Paranka, N. S., Gross, P. H., Brendel, K., Burt, R. W., Alberts, D. S., Pamukcu, R., and et al. (1995). Antineoplastic drugs sulindac sulfide and sulfone inhibit cell growth by inducing apoptosis. Cancer Res 55, 3110-3116. Pinna, L. A. (1990). Casein kinase 2: an 'eminence grise' in cellular regulation? Biochim Biophys Acta 7054, 267-284. Pinna, L. A. (1997). Protein kinase CK2. Int J Biochem Cell Biol 29, 551-554. Plyte, S. E., Hughes, K., Nikolakaki, E., Pulverer, B. J . , and Woodgett, J . R. (1992). Glycogen synthase kinase-3: functions in oncogenesis and development. Biochim Biophys Acta 7774, 147-162. Polakis, P. (1999). The oncogenic activation of beta-catenin. Curr Opin Genet Dev 9, 15-21. Porfiri, E., Rubinfeld, B., Albert, I., Hovanes, K., Waterman, M., and Polakis, P. (1997). Induction of a beta-catenin-LEF-1 complex by wnt-1 and transforming mutants of beta-catenin. Oncogene 75, 2833-2839. Prescott, S. M., and White, R. L. (1996). Self-promotion? Intimate connections between A P C and prostaglandin H synthase-2. Cell 87, 783-786. Prives, C , and Hall, P. A. (1999). The p53 pathway. J Pathol 787, 112-126. 2 9 9 Prowald, K., Fischer, H., and Issinger, O. G. (1984). Enhanced casein kinase II activity in human tumour cell cultures. F E B S Lett 176, 479-483. Radeva, G., Petrocelli, T., Behrend, E., Leung-Hagesteijn, C , Filmus, J . , Slingerland, J . , and Dedhar, S. (1997). Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression. J Biol Chem 272, 13937-13944. Rahman, M. A., Dhar, D. K., Masunaga, R., Yamanoi, A., Kohno, H., and Nagasue, N. (2000). Sulindac and exisulind exhibit a significant antiproliferative effect and induce apoptosis in human hepatocellular carcinoma cell lines. Cancer Res 60, 2085-2089. Ramakrishna, S., D'Angelo, G., and Benjamin, W. B. (1990). Sequence of sites on ATP-citrate lyase and phosphatase inhibitor 2 phosphorylated by multifunctional protein kinase (a glycogen synthase kinase 3 like kinase). Biochemistry 29, 7617-7624. Ramaswamy, S., Nakamura, N., Vazquez, F., Batt, D. B., Perera, S., Roberts, T. M., and Sellers, W. R. (1999). Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci U S A 96, 2110-2115. Ratcliffe, M. J . , Itoh, K., and Sokol, S. Y. (2000). A positive role for the PP2A catalytic subunit in Wnt signal transduction. J Biol Chem 275, 35680-35683. Reddy, B. S., Kawamori, T., Lubet, R. A., Steele, V. E., Kelloff, G. J . , and Rao, C. V. (1999). Chemopreventive efficacy of sulindac sulfone against colon 300 cancer depends on time of administration during carcinogenic process. Cancer Res 59, 3387-3391. Redpath, N. T., Foulstone, E. J . , and Proud, C. G. (1996). Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway. Embo J 15, 2291-2297. Robbins, D. J . , and Cobb, M. H. (1992). Extracellular signal-regulated kinases 2 autophosphorylates on a subset of peptides phosphorylated in intact cells in response to insulin and nerve growth factor: analysis by peptide mapping. Mol Biol Cell 3, 299-308. Robbins. Pathological Basis of Disease, 6 t h Ed. Sauders. Cotran, Kumar, Collins. 1999. Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J . , Waterfield, M. D., and Downward, J . (1994). Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370, 527-532. Rodriguez-Viciana, P., Warne, P. H., Khwaja, A., Marte, B. M., Pappin, D., Das, P., Waterfield, M. D., Ridley, A., and Downward, J . (1997). Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89, 457-467. Rodriguez-Viciana, P., Warne, P. H., Vanhaesebroeck, B., Waterfield, M. D., and Downward, J . (1996). Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation. Embo J 75, 2442-2451. Romashkova, J . A., and Makarov, S. S. (1999). NF-kappaB is a target of AKT in anti-apoptotic P D G F signalling. Nature 407, 86-90. 301 Roose, J . , Huls, G., van Beest, M., Moerer, P., van der Horn, K., Goldschmeding, R., Logtenberg, T., and Clevers, H. (1999). Synergy between tumor suppressor A P C and the beta-catenin-Tcf4 target Tcf1. Science 285, 1923-1926. Roose, J . , Molenaar, M., Peterson, J . , Hurenkamp, J . , Brantjes, H., Moerer, P., van de Wetering, M., Destree, O., and Clevers, H. (1998). The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. Nature 395, 608-612. Rosenberg, L , Palmer, J . R., Zauber, A. G., Warshauer, M. E., Stolley, P. D., and Shapiro, S. (1991). A hypothesis: nonsteroidal anti-inflammatory drugs reduce the incidence of large-bowel cancer. J Natl Cancer Inst 83, 355-358. Rossomando, A. J . , Payne, D. M., Weber, M. J . , and Sturgill, T. W. (1989). Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. Proc Natl Acad Sci U S A 86, 6940-6943. Rossomando, A. J . , Sanghera, J . S., Marsden, L. A., Weber, M. J . , Pelech, S. L , and Sturgill, T. W. (1991). Biochemical characterization of a family of serine/threonine protein kinases regulated by tyrosine and serine/threonine phosphorylations. J Biol Chem 266, 20270-20275. Rowan, A. J . , Lamlum, H., Ilyas, M., Wheeler, J . , Straub, J . , Papadopoulou, A., Bicknell, D., Bodmer, W. F., and Tomlinson, I. P. (2000). A P C mutations in sporadic colorectal tumors: A mutational "hotspot" and interdependence of the "two hits". Proc Natl Acad Sci U S A 97, 3352-3357. 302 Rubinfeld, B., Albert, I., Porfiri, E., Fiol, C , Munemitsu, S., and Polakis, P. (1996). Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science 272, 1023-1026. Ruggeri, B. A., Huang, L , Wood, M., Cheng, J . Q., and Testa, J . R. (1998). Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas. Mol Carcinog 27, 81-86. Sakanaka, C , and Williams, L. T. (1999). Functional domains of axin. Importance of the C terminus as an oligomerization domain. J Biol Chem 274, 14090-14093. Salh, B., Marotta, A., Matthewson, C , Ahluwalia, M., Flint, J . , Owen, D., and Pelech, S. (1999). Investigation of the Mek-MAP kinase-Rsk pathway in human breast cancer. Anticancer Res 79, 731-740. Sano, H., Kawahito, Y., Wilder, R. L , Hashiramoto, A., Mukai, S., Asai, K., Kimura, S., Kato, H., Kondo, M., and Hla, T. (1995). Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res 55, 3785-3789. Satoh, S., Daigo, Y., Furukawa, Y., Kato, T., Miwa, N., Nishiwaki, T., Kawasoe, T., Ishiguro, H., Fujita, M., Tokino, T., et al. (2000). AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat Genet 24, 245-250. Schneider, H. R., Reichert, G. H., and Issinger, O. G. (1986). Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa 303 phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells. Eur J Biochem 161, 733-738. Schubert, K. M., Scheid, M. P., and Duronio, V. (2000). Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473. J Biol Chem 275, 13330-13335. Scott, P. H., Brunn, G. J . , Kohn, A. D., Roth, R. A., and Lawrence, J . C , Jr. (1998). Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc Natl Acad Sci U S A 95, 7772-7777. Sebolt-Leopold, J . S. (2000). Development of anticancer drugs targeting the M A P kinase pathway. Oncogene 19, 6594-6599. Seed, M. P., Freemantle, C. N., Alam, C. A., Colville-Nash, P. R., Brown, J . R., Papworth, J . L., Somerville, K. W., and Willoughby, D. A. (1997). Apoptosis induction and inhibition of colon-26 tumour growth and angiogenesis: findings on COX-1 and COX-2 inhibitors in vitro & in vivo and topical diclofenac in hyaluronan. Adv Exp Med Biol 433, 339-342. Seger, R., Seger, D., Reszka, A. A., Munar, E. S., Eldar-Finkelman, H., Dobrowolska, G. , Jensen, A. M., Campbell, J . S., Fischer, E. H., and Krebs, E. G. (1994). Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells. Evidence that MAPKK involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and Vl l l . J Biol Chem 269, 25699-25709. 304 Sehgal, P. B., and Tamm, I. (1978). Halogenated benzimidazole ribosides, Novel inhibitors of RNA synthesis. Biochem Pharmacol 27, 2475-2485. Seldin, D. C , and Leder, P. (1995). Casein kinase II alpha transgene-induced murine lymphoma: relation to theileriosis in cattle. Science 267, 894-897. Shattuck-Brandt, R. L , Lamps, L. W., Heppner Goss, K. J . , DuBois, R. N., and Matrisian, L. M. (1999). Differential expression of matrilysin and cyclooxygenase-2 in intestinal and colorectal neoplasms. Mol Carcinog 24, 177-187. Shayesteh, L., Lu, Y., Kuo, W. L., Baldocchi, R., Godfrey, T., Collins, C , Pinkel, D., Powell, B., Mills, G. B., and Gray, J . W. (1999). PIK3CA is implicated as an oncogene in ovarian cancer. Nat Genet 27, 99-102. Sheikh, M. S., and Fornace, A. J . , Jr. (2000). Death and decoy receptors and p53-mediated apoptosis. Leukemia 14, 1509-1513. Shiff, S. J . , and Rigas, B. (1997). Nonsteroidal anti-inflammatory drugs and colorectal cancer: evolving concepts of their chemopreventive actions. Gastroenterology 773, 1992-1998. Shiff, S. J . , and Rigas, B. (1999). The role of cyclooxygenase inhibition in the antineoplastic effects of nonsteroidal antiinflammatory drugs (NSAIDs). J Exp Med 790, 445-450. Shivapurkar, N., Huang, L., Ruggeri, B., Swalsky, P. A., Bakker, A., Finkelstein, S., Frost, A., and Silverberg, S. (1997). K-ras and p53 mutations in aberrant crypt foci and colonic tumors from colon cancer patients. Cancer Lett 775, 39-46. 305 Shou, C , Farnsworth, C. L , Neel, B. G., and Feig, L. A. (1992). Molecular cloning of cDNAs encoding a guanine-nucleotide-releasing factor for Ras p21. Nature 358, 351-354. Shtutman, M., Zhurinsky, J . , Simcha, I., Albanese, C., D'Amico, M., Pestell, R., and Ben-Ze'ev, A. (1999). The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A 96, 5522-5527. Sinicrope, F. A., Lemoine, M., Xi , L , Lynch, P. M., Cleary, K. R., Shen, Y., and Frazier, M. L. (1999). Reduced expression of cyclooxygenase 2 proteins in hereditary nonpolyposis colorectal cancers relative to sporadic cancers. Gastroenterology 117, 350-358. Sivaraman, V. S., Wang, H., Nuovo, G. J . , and Malbon, C. C. (1997). Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest 99, 1478-1483. Skolnik, E. Y., Batzer, A., Li, N., Lee, C. H., Lowenstein, E., Mohammadi, M., Margolis, B., and Schlessinger, J . (1993). The function of G R B 2 in linking the insulin receptor to Ras signaling pathways. Science 260, 1953-1955. Skurat, A. V., and Roach, P. J . (1995). Phosphorylation of sites 3a and 3b (Ser640 and Ser644) in the control of rabbit muscle glycogen synthase. J Biol Chem 270, 12491-12497. Smalley, M. J . , Sara, E., Paterson, H., Naylor, S., Cook, D., Jayatilake, H., Fryer, L. G., Hutchinson, L., Fry, M. J . , and Dale, T. C. (1999). Interaction of axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription. Embo J 18, 2823-2835. 306 Smalley, W. E., and DuBois, R. N. (1997). Colorectal cancer and nonsteroidal anti-inflammatory drugs. Adv Pharmacol 39, 1-20. Smith, K. J . , Levy, D. B., Maupin, P., Pollard, T. D., Vogelstein, B., and Kinzler, K. W. (1994). Wild-type but not mutant A P C associates with the microtubule cytoskeleton. Cancer Res 54, 3672-3675. Smith, W. L., DeWitt, D. L., and Garavito, R. M. (2000). Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 69, 145-182. Smith, W. L , Garavito, R. M., and DeWitt, D. L. (1996). Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 277, 33157-33160. Somasiri, A., Howarth, A., Goswami, D., Dedhar, S., and Roskelley, C. D. (2001). Overexpression of the integrin-linked kinase mesenchymally transforms mammary epithelial cells. J Cell Sci 774, 1125-1136. Song, D. H., Sussman, D. J . , and Seldin, D. C. (2000a). Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells. J Biol Chem 275, 23790-23797. Song, J . , Medline, A., Mason, J . B., Gallinger, S., and Kim, Y. I. (2000b). Effects of dietary folate on intestinal tumorigenesis in the apcMin mouse. Cancer Res 60, 5434-5440. Songyang, Z., Baltimore, D., Cantley, L. C , Kaplan, D. R., and Franke, T. F. (1997). Interleukin 3-dependent survival by the Akt protein kinase. Proc Natl Acad Sci U S A 94, 11345-11350. 307 Sparks, A. B., Morin, P. J . , Vogelstein, B., and Kinzler, K. W. (1998). Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer. Cancer Res 58, 1130-1134. Sperber, B. R., Leight, S., Goedert, M., and Lee, V. M. (1995). Glycogen synthase kinase-3 beta phosphorylates tau protein at multiple sites in intact cells. Neurosci Lett 197, 149-153. Spirio, L., Olschwang, S., Groden, J . , Robertson, M., Samowitz, W., Joslyn, G., Gelbert, L., Thliveris, A., Carlson, M., Otterud, B., and et al. (1993). Alleles of the A P C gene: an attenuated form of familial polyposis. Cell 75, 951-957. Staal, S. P. (1987). Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci U S A 84, 5034-5037. Staal, S. P., Hartley, J . W., and Rowe, W. P. (1977). Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma. Proc Natl Acad Sci U S A 74, 3065-3067. Stalter, G., Siemer, S., Becht, E., Ziegler, M., Remberger, K., and Issinger, O. G. (1994). Asymmetric expression of protein kinase CK2 subunits in human kidney tumors. Biochem Biophys Res Commun 202, 141-147. Stambolic, V., Suzuki, A., de la Pompa, J . L., Brothers, G. M., Mirtsos, C , Sasaki, T., Ruland, J . , Penninger, J . M., Siderovski, D. P., and Mak, T. W. (1998). Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29-39. 308 Stambolic, V., Tsao, M. S., Macpherson, D., Suzuki, A., Chapman, W. B., and Mak, T. W. (2000). High incidence of breast and endometrial neoplasia resembling human Cowden syndrome in pten+/- mice. Cancer Res 60, 3605-3611. Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K., Lin, H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T., Davis, T., et al. (1997). Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 15, 356-362. Steinhusen, U., Badock, V., Bauer, A., Behrens, J . , Wittman-Liebold, B., Dorken, B., and Bommert, K. (2000). Apoptosis-induced cleavage of beta-catenin by caspase-3 results in proteolytic fragments with reduced transactivation potential. J Biol Chem 275, 16345-16353. Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G. F., Holmes, A. B., Gaffney, P. R., Reese, C. B., McCormick, F., Tempst, P., era/ . (1998). Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 279, 710-714. Stigare, J . , Buddelmeijer, N., Pigon, A., and Egyhazi, E. (1993). A majority of casein kinase II alpha subunit is tightly bound to intranuclear components but not to the beta subunit. Mol Cell Biochem 729, 77-85. Stoner, G. D., Budd, G. T., Ganapathi, R., DeYoung, B., Kresty, L. A., Nitert, M., Fryer, B., Church, J . M., Provencher, K., Pamukcu, R., era/ . (1999). Sulindac sulfone induced regression of rectal polyps in patients with familial adenomatous polyposis. Adv Exp Med Biol 470, 45-53. 3 0 9 Storm, S. M., Cleveland, J . L , and Rapp, U. R. (1990). Expression of raf family proto-oncogenes in normal mouse tissues. Oncogene 5, 345-351. Strovel, E. T., Wu, D., and Sussman, D. J . (2000). Protein phosphatase 2Calpha dephosphorylates axin and activates LEF-1-dependent transcription. J Biol Chem 275, 2399-2403. Su, L. J . , and Arab, L. (2001). Nutritional status of folate and colon cancer risk: evidence from NHANES I epidemiologic follow-up study. Ann Epidemiol 11, 65-72. Sugimoto, Y., Whitman, M., Cantley, L. C , and Erikson, R. L. (1984). Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc Natl Acad Sci U S A 81, 2117-2121. Suh, O., Mettlin, C , and Petrelli, N. J . (1993). Aspirin use, cancer, and polyps of the large bowel. Cancer 72, 1171-1177. Sun, H., King, A. J . , Diaz, H. B., and Marshall, M. S. (2000). Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak. Curr Biol 10, 281-284. Sun, H., Lesche, R., Li, D. M., Liliental, J . , Zhang, H., Gao, J . , Gavrilova, N., Mueller, B., Liu, X., and Wu, H. (1999). PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci U S A 96, 6199-6204. Suzuki, A., de la Pompa, J . L., Stambolic, V., Elia, A. J . , Sasaki, T., del Barco Barrantes, I., Ho, A., Wakeham, A., Itie, A., Khoo, W., et al. (1998). High cancer 310 susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice. Curr Biol 8, 1169-1178. Tachiiri, S., Sasai, K., Oya, N., and Hiraoka, M. (2000). Enhanced cell killing by overexpression of dominant-negative phosphatidylinositol 3-kinase subunit, Deltap85, following genotoxic stresses. Jpn J Cancer Res 97, 1314-1318. Tamura, M., Gu, J . , Danen, E. H., Takino, T., Miyamoto, S., and Yamada, K. M. (1999). PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 274, 20693-20703. Tan, C , Costello, P., Sanghera, J . , Dominguez, D., Baulida, J . , de Herreros, A. G., and Dedhar, S. (2001). Inhibition of integrin linked kinase (ILK) suppresses beta-catenin-Lef/Tcf-dependent transcription and expression of the E-cadherin repressor, snail, in APC- / - human colon carcinoma cells. Oncogene 20, 133-140. Tanaka, K., Adachi, H., Konishi, H., Iwamatsu, A., Ohkawa, K., Shirai, T., Nagata, S., Kikkawa, U., and Fukui, Y. (1999). Identification of protein kinase B (PKB) as a phosphatidylinositol 3,4,5-trisphosphate binding protein in Dictyostelium discoideum. Biosci Biotechnol Biochem 63, 368-372. Tanti, J . F., Grillo, S., Gremeaux, T., Coffer, P. J . , Van Obberghen, E., and Le Marchand-Brustel, Y. (1997). Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes. Endocrinology 738, 2005-2010. Tetsu, O., and McCormick, F. (1999). Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422-426. 311 Thompson, F. H., Nelson, M. A., Trent, J . M., Guan, X. Y., Liu, Y., Yang, J . M., Emerson, J . , Adair, L., Wymer, J . , Balfour, C , era / . (1996). Amplification of 19q13.1-q13.2 sequences in ovarian cancer. G-band, FISH, and molecular studies. Cancer Genet Cytogenet 87, 55-62. Thompson, H. J . , Briggs, S., Paranka, N. S., Piazza, G. A., Brendel, K., Gross, P. H., Sperl, G. J . , Pamukcu, R., and Ahnen, D. J . (1995). Inhibition of mammary carcinogenesis in rats by sulfone metabolite of sulindac. J Natl Cancer Inst 87, 1259-1260. Thompson, H. J . , Jiang, C , Lu, J . , Mehta, R. G., Piazza, G. A., Paranka, N. S., Pamukcu, R., and Ahnen, D. J . (1997). Sulfone metabolite of sulindac inhibits mammary carcinogenesis. Cancer Res 57, 267-271. Thun, M. J . , Calle, E. E., Namboodiri, M. M., Flanders, W. D., Coates, R. J . , Byers, T., Boffetta, P., Garfinkel, L , and Heath, C. W., Jr. (1992). Risk factors for fatal colon cancer in a large prospective study. J Natl Cancer Inst 84, 1491-1500. Thun, M. J . , Namboodiri, M. M., and Heath, C. W., Jr. (1991). Aspirin use and reduced risk of fatal colon cancer. N Engl J Med 325, 1593-1596. Tokino, T., Thiagalingam, S., el-Deiry, W. S., Waldman, T., Kinzler, K. W., and Vogelstein, B. (1994). p53 tagged sites from human genomic DNA. Hum Mol Genet 3, 1537-1542. Tonelli, F., Valanzano, R., and Dolara, P. (1994). Sulindac therapy of colorectal polyps in familial adenomatous polyposis. Dig Dis 12, 259-264. 312 Torrance, C. J . , Jackson, P. E., Montgomery, E., Kinzler, K. W., Vogelstein, B., Wissner, A., Nunes, M., Frost, P., and Discafani, C. M. (2000). Combinatorial chemoprevention of intestinal neoplasia. Nat Med 6, 1024-1028. Trent, J . M., Wiltshire, R., Su, L. K., Nicolaides, N. C , Vogelstein, B., and Kinzler, K. W. (1995). The gene for the APC-binding protein beta-catenin (CTNNB1) maps to chromosome 3p22, a region frequently altered in human malignancies. Cytogenet Cell Genet 71, 343-344. Trifan, O. C , Smith, R. M., Thompson, B. D., and Hla, T. (1999). Overexpression of cyclooxygenase-2 induces cell cycle arrest. Evidence for a prostaglandin-independent mechanism. J Biol Chem 274, 34141-34147. Troussard, A. A., Costello, P., Yoganathan, T. N., Kumagai, S., Roskelley, C. D., and Dedhar, S. (2000). The integrin linked kinase (ILK) induces an invasive phenotype via AP-1 transcription factor-dependent upregulation of matrix metalloproteinase 9 (MMP-9). Oncogene 19, 5444-5452. Troussard, A. A., Tan, C , Yoganathan, T. N., and Dedhar, S. (1999). Cell-extracellular matrix interactions stimulate the AP-1 transcription factor in an integrin-linked kinase- and glycogen synthase kinase 3-dependent manner. Mol Cell Biol 19, 7420-7427. Tsou, H. C , Ping, X. L., Xie, X. X., Gruener, A. C , Zhang, H., Nini, R., Swisshelm, K., Sybert, V., Diamond, T. M., Sutphen, R., and Peacocke, M. (1998). The genetic basis of Cowden's syndrome: three novel mutations in PTEN/MMAC1/TEP1. Hum Genet 102, 467-473. 313 Tsujii, M., and DuBois, R. N. (1995). Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 83, 493-501. Tsujii, M., Kawano, S., Tsuji, S., Sawaoka, H., Hori, M., and DuBois, R. N. (1998). Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93, 705-716. Tsujio, I., Tanaka, T., Kudo, T., Nishikawa, T., Shinozaki, K., Grundke-lqbal, I., Iqbal, K., and Takeda, M. (2000). Inactivation of glycogen synthase kinase-3 by protein kinase C delta: implications for regulation of tau phosphorylation. F E B S Lett 469, 111-117. Tuhackova, Z., Sloncova, E., Hlavacek, J . , Sovova, V., and Velek, J . (1999). Activity of glycogen synthase kinase-3beta is down-regulated during transient differentiation of human colon cancer HT-29 cells. Oncol Rep 6, 827-832. Tuveson, D. A., and Fletcher, J . A. (2001). Signal transduction pathways in sarcoma as targets for therapeutic intervention. Curr Opin Oncol 13, 249-255. Uthoff, S. M., Eichenberger, M. R., McAuliffe, T. L., Hamilton, C. J . , and Galandiuk, S. (2001). Wingless-type frizzled protein receptor signaling and its putative role in human colon cancer. Mol Carcinog 31, 56-62. Van Aelst, L , Barr, M., Marcus, S., Polverino, A., and Wigler, M. (1993). Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A 90, 6213-6217. 314 van Hengel, J . , Nollet, F., Berx, G. , van Roy, N., Speleman, F., and van Roy, F. (1995). Assignment of the human beta-catenin gene (CTNNB1) to 3p22->p21.3 by fluorescence in situ hybridization. Cytogenet Cell Genet 70, 68-70. van Weeren, P. C , de Bruyn, K. M., de Vries-Smits, A. M., van Lint, J . , and Burgering, B. M. (1998). Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J Biol Chem 273, 13150-13156. Vanhaesebroeck, B., and Alessi, D. R. (2000). The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 346 Pt 3, 561-576. Vanhaesebroeck, B., and Waterfield, M. D. (1999). Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253, 239-254. Virbasius, J . V., Guilherme, A., and Czech, M. P. (1996). Mouse p170 is a novel phosphatidylinositol 3-kinase containing a C2 domain. J Biol Chem 277, 13304-13307. Vojtek, A. B., Hollenberg, S. M., and Cooper, J . A. (1993). Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74, 205-214. Waddell, W. R., and Loughry, R. W. (1983). Sulindac for polyposis of the colon. J Surg Oncol 24, 83-87. Wang, Q., Wang, X., Hernandez, A., Kim, S., and Evers, B. M. (2001). Inhibition of the phosphatidylinositol 3-kinase pathway contributes to HT29 and Caco-2 intestinal cell differentiation. Gastroenterology 720, 1381-1392. Warne, P. H., Viciana, P. R., and Downward, J . (1993). Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature 364, 352-355. 315 Wei, W., Mosteller, R. D., Sanyal, P., Gonzales, E., McKinney, D., Dasgupta, C , Li, P., Liu, B. X., and Broek, D. (1992). Identification of a mammalian gene structurally and functionally related to the CDC25 gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 89, 7100-7104. Welsh, G. I., Miller, C. M., Loughlin, A. J . , Price, N. T., and Proud, C. G. (1998). Regulation of eukaryotic initiation factor elF2B: glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin. F E B S Lett 427, 125-130. Welsh, G. I., Stokes, C. M., Wang, X., Sakaue, H., Ogawa, W., Kasuga, M., and Proud, C. G. (1997). Activation of translation initiation factor e lF2B by insulin requires phosphatidyl inositol 3-kinase. F E B S Lett 410, 418-422. Whitman, M., Kaplan, D. R., Schaffhausen, B., Cantley, L., and Roberts, T. M. (1985). Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature 375, 239-242. Whittemore, A. S., Wu-Williams, A. H., Lee, M., Zheng, S., Gallagher, R. P., Jiao, D. A., Zhou, L., Wang, X. H., Chen, K., Jung, D., and et al. (1990). Diet, physical activity, and colorectal cancer among Chinese in North America and China. J Natl Cancer Inst 82, 915-926. Willert, K., and Nusse, R. (1998). Beta-catenin: a key mediator of Wnt signaling. Curr Opin Genet Dev 8, 95-102. Willett, W. C. (2000). Diet and cancer. Oncologist 5, 393-404. 316 Williams, C. S., Smalley, W., and DuBois, R. N. (1997). Aspirin use and potential mechanisms for colorectal cancer prevention. J Clin Invest 100, 1325-1329. Williams, C. S., Tsujii, M., Reese, J . , Dey, S. K., and DuBois, R. N. (2000). Host cyclooxygenase-2 modulates carcinoma growth. J Clin Invest 105, 1589-1594. Woodgett, J . R. (1994). Regulation and functions of the glycogen synthase kinase-3 subfamily. Semin Cancer Biol 5, 269-275. Wu, C. (1999). Integrin-linked kinase and PINCH: partners in regulation of cell-extracellular matrix interaction and signal transduction. J Cell Sci 112, 4485-4489. Wu, C , Keightley, S. Y., Leung-Hagesteijn, C , Radeva, G., Coppolino, M., Goicoechea, S., McDonald, J . A., and Dedhar, S. (1998a). Integrin-linked protein kinase regulates fibronectin matrix assembly, E-cadherin expression, and tumorigenicity. J Biol Chem 273, 528-536. Wu, J . S., Paul, P., McGannon, E. A., and Church, J . M. (1998b). A P C genotype, polyp number, and surgical options in familial adenomatous polyposis. Ann Surg 227, 57-62. Wu, X., Senechal, K., Neshat, M. S., Whang, Y. E., and Sawyers, C. L. (1998c). The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Natl Acad Sci U S A 95, 15587-15591. 317 Wymann, M. P., and Pirola, L. (1998). Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 7436, 127-150. Yamada, T., Takaoka, A. S., Naishiro, Y., Hayashi, R., Maruyama, K., Maesawa, C , Ochiai, A., and Hirohashi, S. (2000). Transactivation of the multidrug resistance 1 gene by T-cell factor 4/beta-catenin complex in early colorectal carcinogenesis. Cancer Res 60, 4761-4766. Yamamoto, H., Hinoi, T., Michiue, T., Fukui, A., Usui, H., Janssens, V., Van Hoof, C , Goris, J . , Asashima, M., and Kikuchi, A. (2001). Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A. J Biol Chem 276, 26875-26882. Yamamoto, H., Kishida, S., Kishida, M., Ikeda, S., Takada, S., and Kikuchi, A. (1999a). Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability. J Biol Chem 274, 10681-10684. Yamamoto, H., Kishida, S., Uochi, T., Ikeda, S., Koyama, S., Asashima, M., and Kikuchi, A. (1998). Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3beta and beta-catenin and inhibits axis formation of Xenopus embryos. Mol Cell Biol 78, 2867-2875. Yamamoto, Y., Yin, M. J . , Lin, K. M., and Gaynor, R. B. (1999b). Sulindac inhibits activation of the NF-kappaB pathway. J Biol Chem 274, 27307-27314. Yan, M., and Templeton, D. J . (1994). Identification of 2 serine residues of MEK-1 that are differentially phosphorylated during activation by raf and MEK kinase. J Biol Chem 269, 19067-19073. 318 Yao, R., and Cooper, G. M. (1995). Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267, 2003-2006. Yao, R., and Cooper, G. M. (1996). Growth factor-dependent survival of rodent fibroblasts requires phosphatidylinositol 3-kinase but is independent of pp70S6K activity. Oncogene 13, 343-351. Yip-Schneider, M. T., Lin, A., Barnard, D., Sweeney, C. J . , and Marshall, M. S. (1999). Lack of elevated MAP kinase (Erk) activity in pancreatic carcinomas despite oncogenic K-ras expression. Int J Oncol 15, 271-279. Yuan, H., Mao, J . , Li, L., and Wu, D. (1999). Suppression of glycogen synthase kinase activity is not sufficient for leukemia enhancer factor-1 activation. J Biol Chem 274, 30419-30423. Yuan, Z. Q., Sun, M., Feldman, R. I., Wang, G., Ma, X., Jiang, C , Coppola, D., Nicosia, S. V., and Cheng, J . Q. (2000). Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer. Oncogene 19, 2324-2330. Zandomeni, R., Zandomeni, M. C , Shugar, D., and Weinmann, R. (1986). Casein kinase type II is involved in the inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription. J Biol Chem 261, 3414-3419. Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J . , Perry, W. L., 3rd, Lee, J . J . , Tilghman, S. M., Gumbiner, B. M., and Costantini, F. (1997). The 319 mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell 90, 181-192. Zhang, J . , Banfic, H., Straforini, F., Tosi, L , Volinia, S., and Rittenhouse, S. E. (1998). A type II phosphoinositide 3-kinase is stimulated via activated integrin in platelets. A source of phosphatidylinositol 3-phosphate. J Biol Chem 273, 14081-14084. Zhang, L , Yu, J . , Park, B. H., Kinzler, K. W., and Vogelstein, B. (2000). Role of BAX in the apoptotic response to anticancer agents. Science 290, 989-992. Zhang, X., Morham, S. G., Langenbach, R., and Young, D. A. (1999). Malignant transformation and antineoplastic actions of nonsteroidal antiinflammatory drugs (NSAIDs) on cyclooxygenase-null embryo fibroblasts. J Exp Med 190, 451-459. Zheng, C. F., and Guan, K. L. (1994). Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. Embo J 73, 1123-1131. Zuo, Z., Dean, N. M., and Honkanen, R. E. (1998). Serine/threonine protein phosphatase type 5 acts upstream of p53 to regulate the induction of p21(WAF1/Cip1) and mediate growth arrest. J Biol Chem 273, 12250-12258. 320 

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