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Targeting Akt signaling in breast cancer : expression of phosphorylated Akt in breast tumors and the… Kucab, Jill 2004

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TARGETING AKT SIGNALING IN B R E A S T C A N C E R : E X P R E S S I O N OF P H O S P H O R Y L A T E D AKT IN B R E A S T T U M O R S AND THE EFF ICACY OF C E L E C O X I B A N A L O G U E S A S POTENTIAL INHIBITORS OF AKT ACTIVATION by JILL K U C A B B . S c , North Carolina State University A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF S C I E N C E in THE FACULTY OF G R A D U A T E STUDIES (Department of Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA December 2004 © Jill Erin Kucab, 2004 1 ABSTRACT Constitutive activation of Akt, due to overexpression of receptor tyrosine kinases (RTKs) or loss of phosphatase and tensin homologue (PTEN), contributes to the development of breast cancer and confers resistance to conventional therapies. Therefore, the Akt signaling pathway is an attractive target for therapeutic intervention. Utilizing tumor tissue microarrays we show that 58% (225/390) of breast cancers express moderate to high levels of activated Akt (P-Akt), compared with 35% (9/26) of normal breast tissues. Additionally we find that P-Akt expression in primary breast cancer is significantly correlated with the expression of three RTKs, human epidermal growth factor receptor-2 (HER-2), insulin like growth factor receptor-1 (IGF-1R) and ephrin receptor EphA2, as well as integrin-linked kinase (ILK) and the transcription factors growth factor independence 1 (GFI-1) and Y box-binding protein-1 (YB-1). Further, we examined the potential of three celecoxib analogues for the treatment of breast cancers expressing P-Akt. We found that all three analogues, OSU-03008, OSU-03012, and OSU-03013, were able to disrupt Akt signaling in the MDA-MB-453 breast cancer cell line, which overexpresses HER-2 and has very high P-Akt levels. Treating the cells for two hours with the compounds inhibited Akt phosphorylation and kinase activity, as well as its downstream signaling through glycogen synthase kinase-3|3 (GSK3-6), at concentrations well below that of celecoxib (^10 u.M). Disruption of Akt phosphorylation by OSU-03012 and OSU-03013 was followed by an induction of apoptosis after 12 to 24 hours, whereas OSU-03008 did not cause cell death. When tested against a panel of three other breast cancer cell lines, OSU-03012 and O S U -iii 03013 (but not OSU-03008) were found to reduce viability in cell lines which did not constitutively express P-Akt (MDA-MB-231, MCF-7). Overexpression of constitutively activated Akt in the MDA-MB-453 or MCF-7 cells was not able to rescue cells from the cytotoxicity of OSU-03012 or OSU-03013. These data suggest that the celecoxib analogues are potentially useful for inhibiting Akt signaling in breast cancer, although it appears that the cytotoxic effects of OSU-03012 and OSU-03013 occur through additional targets. These inhibitors could hopefully be used in combination with other j therapies to bypass Akt-mediated drug resistance. iv TABLE OF CONTENTS Page A B S T R A C T ii T A B L E OF C O N T E N T S iv LIST OF T A B L E S vii LIST OF F IGURES viii LIST OF ABBREVIATIONS x Chapter 1 INTRODUCTION 1 1.1 Cancer of the Breast: Frequency, Prognosis and Origin 1 1.2 Standard Treatments for Breast Cancer 1 1.2.1 Chemotherapy 2 1.2.2 Hormone Therapy 2 1.3 Molecular Therapeutics: Targeting Receptor Tyrosine 4 Kinases 1.4 The PI3K7 P-Akt Pathway: Receptor Signaling 6 Convergence Point 1.5 The Role of Akt in Cancer Development, Progression, 11 and Drug Resistance 1.6 Expression of P-Akt in Primary Tumors 12 1.7 Akt as a Molecular Therapeutic Target: Current 12 Advances in Drug Development 1.8 Thesis Objectives 15 Chapter 2 MATERIALS AND METHODS 18 2.1 Tumor Tissue Microarray Construction and Patient 18 Information 2.2 Normal Tissues 18 2.3 P-Akt Immunohistochemistry 20 2.3.1 Validation of the P-AktS er473 IHC-Specific 20 Antibody 2.3.2 Protocol for P-Akt Immunohistochemistry 21 2.4 Immunohistochemistry for Other Proteins on the TMA 22 2.5 Scoring of P-Akt Staining 22 2.6 Statistical Analysis of IHC Data 22 2.7 Cell Culture Conditions 23 2.8 Cell Lysis and Protein Extraction 23 2.8.1 Whole Cell Lysis 23 V 2.8.2 Cytoplasmic and Nuclear Fractionation 24 2.8.3 Protein Quantification 24 2.9 Western Blotting 25 2.9.1 Preparation of samples 25 2.9.2 Electrophoresis 25 2.9.3 Transfer of Separated Proteins to Nitrocellulose 25 Membrane 2.9.4 Primary Antibodies 26 2.9.5 Detection of Primary Antibody: Protein 26 Complexes 2.10 Akt Immunoprecipitation and Kinase Assay 27 2.11 Drug Preparation 27 2.12 Determination of Effects of Celecoxib Analogues on 28 Cell Signaling 2.13 Cell Viability Analysis 28 2.14 Apoptosis Analysis 29 2.14.1 P A R P Cleavage 29 2.14.2 Nucleosomal Fragmentation Assay 30 2.15 Plasmids 31 2.16 Rescue Experiment 31 2.16.1 Transient Assay 31 2.16.1.1 Transfection 31 2.16.1.2 Treatment of Transfected Cells with 32 Analogues to Assess Viability; Analysis of Expression and Activity of Activated Akt Constructs 2.16.2 Stable Assay 33 Chapter 3 R E S U L T S 34 3.1 Validation of P-Akt Immunostaining 34 3.2 Frequency of P-Akt Expression in Tumor and Normal 34 Tissue 3.3 P-Akt Expression is Not Associated with Patient 36 Survival 3.4 Correlation of P-Akt Expression with Other Proteins 40 3.5 Expression of P-Akt in Breast Cancer Cell Lines 40 3.6 Analogues of Celecoxib Inhibit Akt Phosphorylation 42 3.7 Analogues of Celecoxib Inhibit Akt Kinase Activity and 42 Downstream Signaling 3.8 Effect of Analogues on Cell Viability 46 3.9 OSU-03012 and OSU-03013 Induce Apoptosis 48 3.10 Cell Confluency Protects Against Analogue-induced 51 Cell Death 3.11 Serum Protects Against Cytotoxic Effects of Celecoxib 51 and Low Doses of Celecoxib Analogues 3.12 Activated Akt Does Not Rescue Cytotoxic Effects of 54 vi Analogues Chapter 4 DISCUSSION 58 4.1 Introduction 58 4.2 Expression of Phospho-Akts er473 in Primary Breast 58 Tumors 4.2.1 Frequency of Expression and Relationship to 59 Patient Prognosis 4.2.2 Correlation of P-Akt with the Expression of Other 61 Proteins 4.3 Expression of Phospho-Aktser473 in Normal Breast 65 Tissue 4.4 Targeting Akt Signaling in Anticancer Therapy 66 4.5 Analogues of Celecoxib As Inhibitors of Akt Signaling 68 and Cytotoxic Agents 4.5.1 Summary of Inhibitory Effects on Akt 68 Phosphorylation 4.5.2 Summary of Effects on Cell Viability and 69 Apoptosis 4.6 Necessity of P-Akt Inhibition for Induction of Cell Death 69 4.6.1 Discrepancy Between Akt Inhibition and 69 Cytotoxicity 4.6.2 Attempt to Rescue the Cytotoxic Effects of 71 OSU-03012 and OSU-03013 by Over-expressing Activated Akt 4.7 Other Targets for Analogues of Celecoxib 71 4.8 Usefulness of the Celecoxib Analogues as a Breast 72 Cancer Therapy 4.9 Therapies for Downstream Targets of Akt: mTOR 74 4.10 Summary, Conclusions, and Future Work 75 R E F E R E N C E S 78 APPENDIX 1 95 APPENDIX 2 99 vii L IST O F T A B L E S Page Table 1. Characteristics of the TMA Study Population 19 Table 2. The Correlation of P-Aktser473 with Other Proteins in Breast 41 Tumors viii LIST OF FIGURES Page Figure 1. The PI3K/Akt Signaling Network 7 Figure 2. Structure and Design of the Celecoxib Analogues 16 Figure 3. Validation of the P-AktS er473 Antibody for 35 Immunohistochemistry Figure 4. Expression of P-Aktser473 in Tumor and Normal Tissues of 37 the Breast Figure 5. P-Aktser473 is Highly Expressed More Frequently in Breast 38 Cancer Compared with Normal Breast Figure 6. P-Aktser473 Expression Alone Does Not Predict Poor 39 Prognosis Figure 7. Levels of P-Akt and HER-2 in a Panel of Breast Cancer 43 Cell Lines Figure 8. Phosphorylation of Akt is Inhibited by Analogues of 44 Celecoxib Figure 9. Akt Kinase Activity is Decreased and Downstream 45 Signaling is Inhibited in MDA-MB-453 Cells Treated with Celecoxib Analogues Figure 10. Assessment of the Effects of Celecoxib Analogues on 49 Cell Viaibilty Figure 11. Induction of Apoptosis by Celecoxib Analogues 50 Figure 12. Confluency Protects Cells from Cytotoxic Effects of 52 Celecoxib and Analogues. Figure 13. Effect of Serum on Efficacy of Celecoxib and Analogues 53 Figure 14. Transient Overexpression of Activated Akt Does Not 56 Rescue Cells from Cytotoxic Effects of Treatment with OSU-03012 or OSU-03013 Figure 15. Stable Overexpression of Myr-Akt1 Does Not Rescue Cells from Analogues X LIST OF ABBREVIATIONS 4E-BP1 eukaryotic initiation factor 4E-binding protein 1 A F X ALL1 fused gene from chromosome X Akt v-akt murine thymoma viral oncogene homolog 1 Akt-DN dominant negative of Akt A M P K AMP-activated protein kinase API-2 Akt/protein kinase B signaling inhibitor-2 Bcr-Abl breakpoint cluster region - Abelson murine leukemia viral oncogene homolog BSA bovine serum albumin CDK2 cyclin dependent kinase 2 cDNA complementary DNA CHK1 cell-cycle-checkpoint kinase-1 C M L chronic myeloid leukemia C M V cytomegalovirus COX-2 cyclooxygenase-2 DMBA 7, 12-dimethylbenz(a)anthracene DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DNA-PK DNA dependent protein kinase E G F R epidermal growth factor receptor e lF4E eukaryotic initiation factor 4E xi ER estrogen receptor F B S fetal bovine serum FDA US Food and Drug Admininstration FKHR1 forkhead in rhabdomyosarcoma FOX01-4 forkhead box 01, 02, 03 or 04 GFI-1 growth factor independence 1 GIST gastrointestinal stromal tumor G P E C Genetic Pathology Evaluation Center GSK-3a /6 glycogen synthase kinase-3a/B HER1-4 human epidermal growth factor receptor 1,2,3 or 4 Hsp90 heat shock protein 90 IC50 concentration of compound that is required to produce a 50 percent inhibition of enzyme IGF-I insulin like growth factor-l IGF-1R insulin like growth factor receptor-1 IHC immunohistochemistry IKKa inhibitor kappa B kinase alpha ILK integrin-linked kinase KIT v-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog LDS lithium dodecyl sulfate MDM2 mouse double minute 2 homolog MDR-1 multidrug resistance-1 MMP-9 matrix metalloproteinase 9 xii mRNA messenger RNA mTOR mammalian target of rapamycin MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- trazolium) N F K B nuclear factor kappa B NSAID non-steroidal anti-inflammatory drug N S C L C non-small cell lung cancer p70S6K p70 S6 kinase P-Akt phosphorylated Akt P A R P poly(ADP-ribose) polymerase P B S phosphate buffered saline P D G F R platelet-derived growth factor receptor PDK-1 phosphoinositide-dependent kinase 1 PH pleckstrin homology PI(3,4,5)P3 phosphatidylinositol-3,4,5-triphosphate PI(4,5)P2 phosphatidylinositol-4,5-bisphosphate PI3K phosphatidylinositol 3-kinase PIAs phosphatidylinositol analogues PIN prostatic intraepithelial neoplasia P K B protein kinase B P K C a protein kinase C alpha P M S phenazine methosulfate PR progesterone receptor PTEN phosphatase & tensin homologue deleted on chromosome 10 RAPID Rapid Access to Preventive Intervention Development RNA ribonucleic acid RTK receptor tyrosine kinase S D S - P A G E sodium dodecyl sulfate polyacrylamide gel electrophoresis S E R M selective estrogen receptor modulators s iRNA small interfering RNA S R C symbol for the human gene homologous in sequence to the v-src gene of the Rous sarcoma virus T B S tris buffered saline TCN tricyclic nucleoside TMA tumor tissue microarray uPA urokinase plasminogen activator V E G F vascular endothelial growth factor YB-1 Y box-binding protein-1 1 CHAPTER 1 INTRODUCTION 1.1 Cancer of the Breast: Frequency. Prognosis and Origin In the year 2004 over 215,000 women will be diagnosed with breast cancer and 40,000 will die from the d isease (data from the Amer ican Cancer Society; http://www.cancer.org/). Several subtypes of breast cancer have been identified, with the majority of cases originating from the epithelial cells of breast ducts and lobules (invasive ductal carcinoma and invasive lobular carcinoma, respectively). The development of cancer is a complex process initiated by mutations in, and dysregulation of, genetic material. Mutations may be inherited or may develop during the course of one's life upon exposure to environmental insults or viruses [1]. Not all genetic mutations will lead to the development of cancer. However, it seems that cellular errors affecting processes such as proliferation, programmed cell death, invasion, or angiogenesis are most likely to generate what are considered cancer cells. Thus, cancers are populations of cells that exhibit self-sufficiency in growth signals, rapidly proliferate, can evade apoptosis, and are, in the worst cases, able to metastasize from the primary site of tumor development [2]. 1.2 Standard Treatments for Breast Cancer If breast cancer is detected early and does not metastasize, a woman can expect a 97% chance of surviving five years post-diagnosis. However, once the cancer has spread outside of the breast to distant organs, such as the lungs, liver or brain, there is only a 23% 5-year survival rate. This point underscores how important it is to eliminate the 2 primary tumor cells, and prevent them from spreading. The standard treatment options for patients include surgery, radiation, chemotherapy and hormone therapy. 1.2.1 Chemotherapy Chemotherapeutic agents commonly operate by preferentially inducing cell death in rapidly dividing cells. For example, anthracyclines, such as doxorubicin and epirubicin, induce apoptosis by preventing DNA synthesis (due to intercalation within DNA strands) and by causing double-strand breaks (stabilization of topoisomerase II in complex with DNA) [3]. Another class of drugs, known as taxanes (e.g., paclitaxel), functions by stabilizing microtubules. This effect causes improper spindle formation and aberrant mitosis, leading to cell death [4]. Disadvantages of these standard chemotherapies include the toxic effects on normal cells, leading to hair loss, neurotoxicity (taxanes) [5] and cardiotoxicity (anthracyclines) [6]. Additionally, many tumors either intrinsically possess or acquire drug resistance. The mechanisms for chemoresistance may include the action of ATP-binding cassette transporters, such as p-glycoprotein, which are responsible for the efflux of xenobiotics (such as chemotherapy) from cells [7]. Further, overexpression of anti-apoptotic proteins, such as Bcl2, or hyperactivation of survival signaling via growth factor receptors and Akt (discussed below), can also enable cancer cells to avoid death when confronted with cytotoxic drugs [8]. 1.2.2 Hormone Therapy Beyond the chemotherapies described above, hormone therapy is another standard option for some breast cancer patients, due to the importance of estrogen signaling in breast cancer. Approximately half of all breast cancers express estrogen 3 receptor a (ERa) [9], whereas only a small percentage of normal mammary epithelial cells are ERa positive [10]. ERa primarily becomes activated when bound to its ligand, the steroid hormone estrogen. It should be noted that ERa can also be activated in a ligand-independent fashion, through phosphorylation induced by growth factor receptor signaling [11]. In pre-menopausal women estrogen is produced in the ovaries, whereas in post-menopausal women aromatase enzymes synthesize estrogen from androgenic precursors in adipose tissues. It has been shown that prolonged estrogen exposure predisposes women to breast cancer occurrence. This is reflected by the fact that removal of the ovaries in pre-menopausal women decreases breast cancer risk, and high body fat increases this risk in post-menopausal women [12]. Estrogen binding to ERa exerts its effects by modulating the transcription of genes that stimulate proliferation and growth. There are two ways in which this can occur. Firstly, ligand binding to ERa leads to the dissociation of ERa from heat shock proteins, subsequent ERa dimerization, and association of these dimers with various cofactors. This complex binds to estrogen response element (ERE) sequences in the promoters of affected genes to stimulate or inhibit transcription [11]. Alternatively, estrogen binding to ERa may cause its association with transcription factors such as AP-1 and Sp1, and actually enhance the binding of these proteins to their respective promoter recognition sequences to affect transcription [13], [14]. Since the 1970's, selective estrogen receptor modulators (SERMs) , such as tamoxifen, have been used quite successfully to treat tumors which express the estrogen and/or progesterone receptor (ER or PR) [15], [16]. Tamoxifen exerts anti-estrogenic activity in the breast by competitively inhibiting estrogen binding to ERa, 4 leading to cytotoxic and cytostatic effects in hormone-dependent tumor cells. More recently, aromatase inhibitors (e.g., letrozole), which inhibit the synthesis of estrogen, have also proven to be an effective treatment against hormone responsive tumors [17]. Unfortunately, therapies targeting estrogen signaling are only useful for patients which express the ER, and most advanced breast cancer patients that initially responded to tamoxifen will become resistant [18]. Although the mechanism for tamoxifen resistance is unclear, it may be related to 1) loss of E R a expression, 2) changes in expression of E R a cofactors, or 3) crosstalk between E R a and growth factor signaling [11], [19]. 1.3 Molecular Therapeutics: Targeting Receptor Tyrosine Kinases Resistance to chemotherapy and anti-estrogen therapy, in part, is associated with upregulation of growth factor signaling and hyperactive survival networks within tumor cells. In an effort to reduce side effects of cancer therapy and bypass drug resistance, researchers are now focused on designing drugs to specifically target the disrupted cellular pathways of tumors [20]. As many kinases are commonly overexpressed in breast cancer and can promote tumor growth, metastasis, and resistance to chemotherapies and endocrine therapies, much effort has been expended in developing inhibitors to these particular molecules [21]. For example, receptor tyrosine kinases (RTKs) represent a major class of molecules for which small molecule inhibitors have recently been developed. The first clinically successful RTK inhibitor was STI571 (Gleevec, imatinib mesylate), which is active against the proteins Abl, Bcr-Abl, KIT, and the platelet-derived growth factor receptor (PDGFR) . STI571 has proven efficacious in treating patients with chronic myeloid leukemia (CML), as this cancer expresses and is dependent upon the Bcr-Abl fusion protein, [22], and also in treating 5 patients with gastrointestinal stromal tumors (GISTs) which commonly express mutated KIT or P D G F R proteins [23]. Other notable RTKs being targeted for therapy are members of the human epidermal growth factor (EGFR) family of transmembrane receptor tyrosine kinases (EGFR/HER-1 , HER-2 , HER-3 , HER-4) [24], which are expressed to varying degrees in many cancer types. HER-2, for example, is overexpressed in 25-30% of invasive ductal breast carcinomas. When HER-2 is overexpressed, it is associated with poor patient prognosis and increased risk of recurrence [25]. Additionally, overexpression of HER-2 in a cancer cell line model, using M C F - 7 cells, leads to increased resistance to chemotherapy [26]. However, a clinically applicable monoclonal antibody targeting HER-2 , called Herceptin, was developed to block the activation of the receptor [27]. This drug achieves a 30% response rate when used as a treatment of metastatic breast cancer, and demonstrates synergistic effect in combination with chemotherapy [28]. Further, targeting the tyrosine kinase domain of E G F R with small molecule inhibitors such as ZD-1839 (Iressa), has proven effective for 10% of non-small cell lung cancer (NSCLC) patients in phase II clinical trials [29]. It is possible that Iressa could benefit breast cancer patients as well, as it has been shown to inhibit growth of breast cancer cell lines [30]. E G F R is highly expressed in about 27% of primary breast cancers [31], although expression of the receptor alone does not seem to predict sensitivity to Iressa [32]. N S C L C patients whose tumors contain specific mutations around the ATP-binding pocket of the tyrosine kinase domain of E G F R , which leads to hyperactive signaling, appear to have the best clinical response to Iressa [33]. 6 Besides the EGF-receptor family, overexpression of another RTK, the insulin like growth factor receptor-1 (IGF-1R) is associated with malignant transformation in vitro and in vivo [34]. Signaling through the IGF-1R is also important for breast cancer invasion and metastasis [35]. Therefore, drugs are being sought to inhibit this receptor. It was recently demonstrated that targeting the IGF-1R with a human monoclonal antibody significantly inhibited the growth of breast tumors in vivo [36]. Another strategy for inhibiting IGF-1R is to use kinase inhibitors. Two such therapies have been developed, NVP-AEW541 [37] and NVP-ADW742 [38] that have both shown efficacy in vivo. Further studies will determine whether these drugs will be useful for cancer patients. By combining RTK inhibitors with standard chemotherapy, this removes one avenue for developing cancer drug resistance and hopefully will result in more success in the clinic. 1.4 The PI3K/ P-Akt Pathway: Receptor Signaling Convergence Point Researchers are also setting their sights on inhibiting the downstream intermediates of growth factor receptors. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been shown to be a major convergence point for RTK signaling and is activated by numerous receptors, including IGF-1R and HER-2 (see Figure 1) [39]. Actiavted RTKs recruit and activate PI3K. PI3Ks are actually a family of proteins which have been divided into three classes (I, II, and III), based on structure and substrate specificities. Growth factor RTKs primarily associate with the class IA PI3Ks [40]. Class IA PI3Ks are heterodimers composed of a regulatory (p85) and catalytic (p110) subunit [41]. The src-homology 2 (SH2) domains of the p85 subunit bind phospho-tyrosine residues on an activated receptor or on associated adaptor proteins and recruit 7 Growth Factor Receptor LY294002 PI-3,4,5-P3 AKT KD l-cr Thr308 PI-4.5-P2 ^ | o <«-Ser473 ILK, PDK2, DNA-PK, Akt mTOR, p27, GSK-3B, FKHR1 survival, proliferation, growth Figure 1. The PI3K/Akt Signal ing Network. This schematic illustrates the activation of the PI3K/Akt pathway by receptor tyrosine kinases (RTKs). Activated RTKs recruit and activate PI3K, which converts phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3). The PI(3,4,5)P3 lipids trigger attachment of Akt and PDK-1 to the plasma membrane (PM) by their PH domains. At the PM, PDK-1 phosphorylates threonine 308 on the kinase domain (KD) of Akt. To achieve full activation, Akt must also be phosphorylated in its regulatory domain (RD) at serine 473. This occurs either by ILK, DNA-PK, PDK-2 or autophosphorylation. Phospho-Akt then dissociates from the PM and proceeds to phosphorylate both cytoplasmic and nuclear target proteins, including mTOR, FKHR, GSK - 3 P and p27Kip-i. The PI3K7Akt system is also negatively regulated by the phosphatase PTEN, which converts PI(3,4,5)P3 lipids to PI(4,5)P2. 8 the enzyme to the membrane. At the membrane PI3K phosphorylates the lipid molecule phosphat idy l inos i to l -4,5-b isphosphate (PI(4,5 )P2) , to c r e a t e phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3) [42] in response to growth factor signals. The PI(3,4,5)P3 lipids trigger attachment of the protein Akt (also known as protein kinase B, or PKB) to the plasma membrane by specifically binding its pleckstrin homology (PH) domain. Akt is a serine/threonine kinase that contains a kinase domain and hydrophobic tail in addition to its PH domain. Once attached to the plasma membrane, Akt subsequently becomes phosphorylated at two key sites, threonine 308 (in the kinase domain) and serine 473 (in the hydrophobic tail). Phosphorylation of serine 473 is required for full activation and has been shown to correlate with Akt kinase activity [43]. Threonine 308 is phosphorylated by phosphoinositide-dependent kinase 1 (PDK1), while the mechanism of phosphorylation at serine 473 is controversial. There are several theories to explain serine 473 phosphorylation, including the action of integrin-linked kinase (ILK) [44], DNA dependent protein kinase (DNA-PK) [45], autophosphorylation [46], or an as yet unidentified "PDK2" . Once Akt is fully activated (P-Akt) it dissociates from the plasma membrane and proceeds to phosphorylate both cytoplasmic and nuclear target proteins, which will be discussed below. The PI3K/Akt system is also negatively regulated by the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which is responsible for converting PI(3,4,5)P3 lipids to PI(4,5)P 2 [47]. In PTEN-null cells the PI3K/Akt pathway is constitutively activated [48]. Loss of P T E N expression as detected by immunohistochemistry has been reported in approximately 40% of breast cancers [49], [50], [51]. Only one study has found an association between loss of P T E N and disease-related death in breast cancer [49]. 9 However, P T E N mutations in HER-2 positive tumors predict resistance to Herceptin treatment [51]. There are three members of the Akt family, Akt1, Akt2 and Akt3. Akt1 is located on chromosome 14q [52], Akt2 is on chromosome 19q [53], and Akt3 maps to chromosome 1q [54]. They exhibit 80% amino acid sequence homology, share the same key phosphorylation sites (Thr308/309/305 and Ser473/474/472, respectively), and appear to be regulated by similar mechanisms in cell culture [55]. However, differences in tissue distribution and results from knockout experiments suggest that the isoforms are not redundant in function. At the level of mRNA, Akt1 and Akt2 are expressed in most normal tissues [54], whereas Akt3 is predominantly found in the brain and testis, with lower levels in the heart, kidney and placenta [56], [54]. Akt1 knockout mice exhibit enhanced apoptosis and stunted growth [57]. In contrast, Akt2 knockout mice display a diabetic phenotype, but normal size [58]. Double Akt1/Akt2 knockout mice have also been generated [59]. These mice die shortly after birth and display impeded adipogenesis, as well as deficiencies in growth, muscle cell size and bone development. An Akt3 knockout mouse has not been reported to date. This work implies that specific targets or functions of the isoforms exist, which cannot be compensated for by the other two. However, all three isoforms when constitutively activated can transform normal cells [43], [60], implying that many key substrates are shared. The diverse substrates of the Akt family include proteins that regulate the cell cycle, cell growth, invasion, angiogenesis, translation and, perhaps most importantly, cell survival. Akt phosphorylates and inactivates glycogen synthase kinase-36 (GSK-10 36), a protein responsible for controlling proteolysis of cyclin D1 [61]. Thus, activation of Akt allows levels of cyclin D1, a cell cycle promoter, to accumulate in the cell by preventing its degradation. Akt also regulates the cell cycle inhibitor p27(Kip1) by phosphorylating its nuclear localization motif and trapping it in the cytoplasm [62], [63]. Phospho-p27(Kip1) can no longer inhibit cyclin dependent kinase-2 (CDK2) in the nucleus, thus allowing cell cycle progression. Another important target of the PI3K/Akt pathway is the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 results in inactivation of mTOR [64] and one study has shown direct phosphorylation of mTOR by Akt [65]. mTOR is responsible for activating p70 S6 kinase (p70S6K), a translational enhancer, [66] and inhibiting the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a translational repressor [67]. Finally, Akt mediates cell survival through several other targets. By phosphorylating members of the F O X O subclass of forkhead transcription factors, F O X 0 1 / F K H R 1 (forkhead in rhabdomyosarcoma) [68] and F O X 0 4 / A F X (ALL1 fused gene from chromosome X) [69], Akt prevents their nuclear localization and thus inhibits the transcription of proapoptotic genes, such as Fas ligand. Akt also phosphorylates inhibitor kappa B kinase alpha (IKKa) [70], which indirectly leads to activation and nuclear translocation of nuclear factor kappa B (NFKB) , a survival-promoting transcription factor. N F K B appears to play a role in drug resistance by upregulating expression of multidrug resistance-1 (MDR-1) [71]. Additionally, N F K B has been shown to negatively regulate P T E N expression, creating a feedback loop for Akt activation [72]. Another notable target of Akt is MDM2 (mouse double minute 2 homolog). Phosphorylation of MDM2 by Akt promotes its nuclear translocation [73]. In the nucleus MDM2 binds to, and promotes the 11 degradation of, p53, a key regulator of apoptosis. Thus, through wide-ranging effectors, the activity of Akt provides a powerful growth and survival advantage. 1.5 The Role of Akt in Cancer Development. Progression, and Drug Resistance Evidence from experimental models suggests that Akt is a key regulator of tumor development and progression. Mammary epithelium-specif ic coexpression of constitutively active Akt1 and a mutant polyomavirus middle T antigen promotes tumor progression in mice [74]. It has also been shown that constitutive activation of Akt1 transforms NIH3T3 mouse fibroblast cells, which can then form tumors in nude mice [43]. Additionally, Akt promotes cancer cell invasion and metastasis. Overexpression of wild-type Akt1 doubles the rate of HT1080 cell migration and triples their rate of invasion through Matrigel. The observed increase in migration is correlated with upregulation of matrix metalloproteinase 9 (MMP-9) production [75]. Another study showed that the overexpression of Akt2 increased the ability of breast cancer cells to invade by upregulating 61 integrins. This study went on to show that Akt2-overexpressing xenografts exhibit increased incidence of metastases when compared with xenografts which developed from control cells expressing the empty vector [76]. Akt is also able to regulate blood vessel formation. Expression of activated Akt in the chorioallantoic membrane of chicken embryo induces angiogenesis [77]. Finally, in addition to its role in cancer development and migration, Akt promotes the survival of tumor cells when confronted with chemotherapeutics and radiation. For example, in breast cancer cells, expression of constitutively active Akt1 reduced the ability of doxorubicin [78] or ionizing radiation [79] to induce apoptosis. On the other hand, combination of the PI3K inhibitor, LY294002, or a dominant-negative Akt1 with common 12 chemotherapeutic agents increased the drug-induced apoptosis [78]. These data suggest that inhibiting P-Akt signaling could have important therapeutic applications. 1.6 Expression of P-Akt in Primary Tumors In support of P-Akt as a molecular target for therapy, several laboratories report that P-Akt is expressed in aggressive primary tumors. Akt is activated in a wide range of cancers, including carcinomas of the breast [80], kidney [81], lung [82], colon [83] and prostate [84]. High levels of P-Akt expression are associated with metastatic disease in renal cell carcinoma patients [81] and are also associated with high-grade, poorly differentiated prostate cancer [84]. In the context of breast cancer, a study of endocrine-treated breast cancer patients (n = 93) showed that P-Akt1 expression was linked to increased relapse with distant metastasis [80]. Additionally, Akt1 kinase activity was shown to be significantly increased in approximately 40-50% of tumor samples from breast (19/50 cases), ovary (11/28 cases) and prostate patients (16/30 cases) relative to normal tissue [43]. 1.7 Akt as a Molecular Therapeutic Target: Current Advances in Drug Development Currently, there are no Akt inhibitors available to patients. However, recent studies inhibiting Akt or Akt phosphorylation in preclinical models have attempted to answer several important questions regarding the utility of such inhibitors in cancer treatment. It was previously unclear whether inhibition of Akt would itself induce apoptosis or rather would sensitize cells to apoptosis induced by other agents. It was also unknown whether all cells would respond the same to inhibition of Akt signaling, or whether there would be differences in response based on pathway activation. One way to inhibit Akt specifically was achieved by retroviral expression of dominiant negative 13 Akt (Akt-DN) [85]. Akt-DN inhibited the growth of tumor cell lines and induced apoptosis preferentially in cell lines expressing high levels of P-Akt. The injection of Akt-DN-adenovirus into mice containing breast cancer cell (ZR75-1) xenographs lead to 90% inhibition of tumor growth. This group also showed that Akt-DN does not cause apoptosis in normal cells, presumably because all of the normal cell lines they used express low levels of P-Akt. Other studies have recently reported the development or identification of small molecule inhibitors to inhibit Akt signaling. Castillo et al. described the synthesis and biological analysis of phosphatidylinositol analogues (PIAs) used to specifically inhibit Akt activation [86]. These PIAs were originally developed by modeling the interaction of phosphatidylinositol 3,4 bisphosphate with the PH domain of Akt. Subsequent modifications to the inositol ring improved stability and showed even greater potency for inhibiting Akt phosphorylation (IC5o < 5 \xM) than the first generation analogues [87], [88]. In cell culture PIAs inhibit Akt phosphorylation and downstream signaling targets within 2 hours. Treatment for 24 hours results in 40 to 80% apoptosis induction in cell lines in which Akt is highly activated, whereas cells with low levels of Akt activation show only 10 to 20% apoptosis. PIAs may not be useful in vivo, however, because of potential poor bioavailability and toxicity [89]. A novel Akt inhibitor was also recently identified from the National Cancer Institute Diversity Set, and is promising based on pre-clinical evidence [90]. This inhibitor, termed API-2 (Akt/protein kinase B signaling inhibitor-2), is a tricyclic nucleoside that, again, selectively inhibits cancer cells that express high levels of activated Akt. Ant i-cancer activity of API-2 was demonstrated both in cell culture and in an animal model. API-2 was identified previously as an anticancer agent that inhibits DNA synthesis, and was designated TCN 14 (tricyclic nucleoside) [91]. API-2/TCN was used in phase I and II clinical trials where high doses resulted in side effects such as hepatotoxicity, thrombocytopenia, and hyperglycemia, which suggest that it may have limited clinical use [92], [93]. It is unclear if these side effects are related to inhibition of Akt signaling or alternative mechanisms of API-2 action. A s a final example, the non-steroidal anti-inflammatory drug (NSAID) celecoxib has been shown to inhibit Akt phosphorylation and induce apoptosis in prostate cancer cells [94]. In addition to its effect on Akt phosphorylation, the known tolerability of celecoxib in patients made it a superior lead compound for the development of more potent analogues. Celecoxib is a cyclooxygenase-2 (COX-2) inhibitor traditionally used to treat the symptoms of patients with osteoarthritis and rheumatoid arthritis because of its anti-inflammatory properties [95]. It was further shown that celecoxib is chemopreventative against colon cancer [96] and was approved by the FDA for the treatment of patients with familial adenomatous polyposis [97]. In a rat model of breast cancer, celecoxib treatment can both prevent and regress the formation of tumors induced by 7, 12-dimethylbenz(a)anthracene (DMBA) [98], [99]. The effectiveness of celecoxib against cancer was initially attributed to the anti-apoptotic roles of COX-2 . Celecoxib induces the mitochondrial apoptosis pathway [100], however, apoptosis can be induced in cells that do not express COX-2 , indicating COX-2 independent mechanisms for cell death [94]. Several targets were proposed, including Akt [94]. Phosphorylation of Akt is downregulated in response to celecoxib treatment, which has been attributed to, at least in part, the inhibition of PDK-1 activity [101]. However, the doses of celecoxib which inhibit Akt phosphorylation in cell lines [50 to 100 u,M] are well above the 2 u.M serum 15 concentrations achievable in patients [102]. Nevertheless, the observation that celecoxib inhibited P-Akt set the course for the development of analogues that optimally disrupt this pathway at lower concentrations. Through structure-activity analysis, Zhu et al. determined the structural features of celecoxib necessary for COX-2 inhibition and for apoptosis induction [103]. By manipulating the 4-methylphenyl moiety of celecoxib, and maintaining the pyrazole ring, they created a series of celecoxib analogues (see Figure 2). The new analogues have no C O X - 2 inhibitory ability, increased PDK-1 inhibition, and improved apoptosis induction [104]. In particular, the compounds designated OSU-03012 and OSU-03013 could inhibit PDK-1 activity in vitro with an IC 5 o of 2 to 5 u . M , and inhibit Akt phosphorylation at doses less than 10 u.M in cell culture. Upon longer exposure, these inhibitors were shown to induce apoptosis in PC-3 prostate cancer cells. As a part of the Rapid Access to Preventive Intervention Development (RAPID) program at the National Cancer Institute, (http://www3.cancer.gov/prevention/rapid/). a panel of 60 cancer cell lines was screened for response to OSU-03012 and OSU-03013. They determined that the compounds were potent inhibitors of tumor cell growth with an average IC5o= ~1 to 2 u M The characterization of the celecoxib analogues thus far indicates that they could be very useful for treating many types of cancer. 1.8 Thesis Objectives Aim 1: Expression Profiling of P-Akt in Normal and Cancer Tissues of the Breast Significance Although the key role of Akt in cancer aggression has been definitively shown in experimental models, the expression and activity of the P-Akt signaling network in 16 N - N fc N - N B. Analogue Design Strategy A " ^ s c y i H , ^ S O ^ J H , OSU02067 rs. N - N k> N - N N - N 0 N - N c- AlSB°t9n o» b_«^ cv^v by; OSU03008 OSU03012 OSU03013 Figure 2. Structure and Design of the Celecox ib Ana logues. (A) The structure of the parent compound, celecoxib, is shown. This molecule includes three notable features; (1) the heterocyclic pyrazole ring with a tri-fluoromethyl moiety, (2) a sulfonamide group and (3) a terminal phenyl ring. (B) An initial strategy to increase the cytotoxic capability of celecoxib generated OSU-02067. This compound has a large hydrophobic, tricyclic aromatic ring replacing the terminal phenyl ring of celecoxib. (C) The latest strategy to further optimize apoptosis induction and strengthen PDK-1 inhibition, lead to the creation of OSU-03008, OSU-03012 and OSU-03013. The sulfonamide moiety of OSU-02067 was replaced with a series of heteroatom-rich functional groups to potentially increase hydrogen bonding of the compounds within the kinase pocket of PDK-1. 17 primary breast tumors is not well characterized. Additionally, the expression of P-Akt in normal breast tissue has not been explored. Experimental Design In order to better understand the function of activated Akt in breast cancer, we evaluated its expression on a tissue microarray containing tumor samples from 481 breast cancer patients. We also assessed P-Akt in normal breast tissues to help establish the potential significance of its expression in tumors. Moreover, we examined the association of P-Akt expression with 30 other proteins previously immunostained on the same TMA series. Aim 2: Characterization of Small Molecule Inhibitors to the Akt Signaling Pathway Significance Pre-clinical models targeting P-Akt signaling clearly show that tumor cells depend on this pathway for survival and drug-resistance. However, there are currently no inhibitors to P-Akt available for treating patients. Experimental Design The effectiveness of celecoxib analogues was evaluated for the treatment of breast cancer. Three compounds, OSU-03008, OSU-03012 and OSU-03013, were examined for their ability to inhibit Akt phosphorylation and decrease viability in breast cancer cells. The necessity for P-Akt inhibition, in regards to efficacy of analogue treatment, was also determined. Additionally, the effects of serum concentration and cell confluency during analogue treatment were analyzed. 18 CHAPTER 2 MATERIALS AND METHODS 2.1 Tumor Tissue Microarrav Construction and Patient Information For construction of the TMA, 481 primary breast cancer samples were obtained from archival cases at Vancouver General Hospital dating between 1974 and 1995. Patient information and tumor pathology are summarized in Table 1. Tumor samples were taken prior to initiation of cancer treatment, formalin-fixed and embedded in paraffin [105]. A hematoxylin and eosin stained section of each tumor block was used to define and mark representative tumor regions. This section served as a guide for the selection of two 0.6 mm punches from each original tumor block which were transferred to three composite recipient array blocks using a Tissue Micro Arrayer (Beecher Instruments; Sliver Springs, MD) as previously described [105]. Serial 4 u,m-thick sections of the arrays were cut using a Leica microtome (Leica Microsystems; Nussloch, Germany) and mounted onto charged polylysine-coated glass slides. This work was done at the Genetic Pathology Evaluation Centre (GPEC) (Vancouver, British Columbia). 2.2 Normal Breast Tissues Twenty-six samples of normal breast tissue were obtained from patients undergoing reduction mammoplasty at Vancouver General Hospital from 2000 to 2001. The tissue was formalin-fixed and paraffin-embedded. Serial 4 jxm-thick sections were cut from each tissue block. One section of each sample was stained with hematoxylin and eosin and then assessed by a pathologist (N. Makretsov) to ensure presence of 19 Characteristics Data Correlation to P-Akt (Chi-Square) Diagnostic Age, yr Mean Median Range 61.01 63.07 59.03 Lymph Node Status, n (%) Negative Positive Unknown 266 (60.7) 126 (28.8) 46 (10.5) 0.957 ER" status, n (%) Negative Positive Unknown 93 (21.2) 216 (49.3) 129 (29.4) 0.170 Tumor Grade, n (%) 1 2 3 94 (21.5) 236 (53.9) 108 (24.6) 0.276 Tumor Size, n (%) <= 5mm <= 1cm <= 2cm > 2cm Unknown 4(2.2) 64 (14.6) 142 (32.4) 147 (33.6) 81 0.900 Histology, n (%) in situ carcinoma IDCb, NOS c IDC, variants ILCd IDC and ILC 16 (3.6) 353 (80.5) 24 (5.5) 43 (10) 2 (0.4) 0.143 Total Follow-up, yr Mean Median Range 14.47 15.4 20.34 P-Akt Expression, n (%) 0 1 2 3 unscorable 43 (11.0) 122 (31.3) 120 (30.8) 105 (26.9) 48 Table 1. Characteristics of the TMA Study Population. Listed are the known characteristics of patients, and their tumors, for the 438 cases of invasive breast carcinoma included on the TMAs. The correlation of P-Akt to the clinicopathologic features was determined by Chi-Square analysis (p values less than 0.05 are considered significant). a E R , estrogen receptor; b IDC, invasive ductal carcinoma; c N O S , not otherwise specified; d ILC, invasive lobular carcinoma. 20 normal breast epithelium. Ages of patients and general pathology are described in Figure 5. 2.3 P-Akt Immunohistochemistry 2.3.1 Validation of the P-Aktw7* IHC-Specific Antibody Two controls were performed to verify the specificity of the phospho-Akt (S473) IHC specific antibody (Cat#9277; Cell Signaling Technology (CST); Beverly, MA). For the first control, P-Akt was induced in T47D cells and analyzed by both western blot and immunohistochemistry. To induce P-Akt T47D cells were first grown to 90% confluence in normal growth medium and then grown in medium without F B S overnight (refer to section 2.7). The next day the cells were treated with or without IGF-I (100 ng/ml) (Diagnostic Systems Laboratories, Inc.; Webster, TX) for 30 minutes. IGF-I is known to stimulate the phosphorylation of Akt on serine 473 [106]. Initially, the expression and localization of P-Akt were verified by western blot. The treated T47Ds were lysed to separate cytoplasmic and nuclear proteins (see section 2.8). Protein extracts (100 u,g each) were subjected to S D S - P A G E and transferred to a nitrocellulose membrane (see section 2.9). The membrane was probed with phospho- and total-Akt primary antibodies (CST Cat#9271 and #9272, respectively). Next, the T47D cells treated with and without IGF-I were examined by immunohistochemistry. The treated cells were pelleted by gentle centrifugation (1000 rpm, 5 minutes) and then fixed in 10% formalin for 20 minutes. The fixed cells were mixed with warm (37°C) Histogel (Richard-Allan Scientific; Kalamazoo, Ml) and then placed on ice to form a solid clot. The clots were paraffin-embedded in the Pathology lab at Vancouver General Hospital, sectioned 4 \im thick, mounted onto charged polylysine-coated slides, and processed by IHC (section 21 2.3.2). As a final control, the IHC-Specific Phospho-Akt antibody was incubated for two hours on ice with its specific blocking peptide (Cat#1140; CST) , prior to the application of the antibody for immunohistochemistry on a section of breast cancer tissue (obtained from CST). 2.3.2 Protocol for P-Akt Immunohistochemistry This protocol was used to detect P-Akt (S473) expression in the TMAs, normal breast tissue, and T47D cell blocks. Sections of each were first deparafinized in xylene and rehydrated in graded ethanol solutions. Antigen retrieval was achieved by incubating the slides for 30 minutes in 10 mM citrate buffer pH 6.0 (Appendix 1a) at 90°C in a vegetable steamer. Endogenous peroxidases were quenched by incubating the sections for 10 minutes in 3% hydrogen peroxide diluted in water. Prior to application of the primary antibody, non-specific interactions were blocked for 30 minutes using a non-serum (0.25% casein in PBS) blocking reagent (Cat# X0909; DAKO, Denmark), followed by twenty minutes with an avidin/biotin blocking solution (DAKO). The primary antibody (Phospho-Akt S473 IHC Specific, Cat# 9277; CST) was diluted 1:250 with a 1% bovine serum albumin solution in T B S (Appendix 1b), applied to the slides and incubated overnight at 4°C. For signal amplification, we then used the LSAB+ System (DAKO), which involved incubation with a biotinylated secondary antibody (30 minutes) followed by peroxidase-labelled streptavidin treatment (30 minutes). Signal was visualized by addition of NovaRed substrate-chromogen solution (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin. Additionally, a negative control reaction with no primary antibody was performed for each TMA slide, normal breast section or cell block, in parallel. 22 2.4 Immunohistochemistry for Other Proteins on the TMA Immunohistochemistry for 30 other proteins was also performed on the TMAs using methods with minor modifications to the above P-Akt IHC protocol. Antibody details, including dilutions and antigen retrieval methods, for the other proteins are described on the website: www.gpec.ubc.ca. and in Appendix 2. 2.5 Scoring of P-Akt Staining Two pathologists and a basic scientist arrived at a consensus score for each core on the TMA and normal breast section. The scoring system for P-Akt expression was as follows: 0) negative, 1) weak, 2) moderate and 3) high staining intensity. Cores on the TMA with staining that could not be interpreted (i.e., no tumor tissue present) were scored as "X". In the occasional instance of heterogeneous staining, the overall score was determined based on 75% or more of epithelial cells expressing the same level of P-Akt. In any case where duplicate TMA cores had conflicting scores, the higher score was taken. Raw scores were entered into a standardized electronic spreadsheet (Excel for Windows, Microsoft, Redmond, WA). 2.6 Statistical Analysis of IHC Data Data was processed using Deconvoluter software designed for management of TMA data [107], and then analyzed with the S P S S for Windows statistical software package (SPSS version 11; S P S S , Chicago, IL). For some analyses P-Akt scores were grouped as low (0 or 1) versus high (2 or 3) expression to create binary categories. Univariate analysis between proteins on the T M A was calculated by Spearman correlation coefficient and Fisher's Exact Test using S P S S . Data was considered significant when p < 0.05. We did not use the Bonferroni multicomparison test because 23 this aspect of the project was simply a hypothesis-generating exercise. The difference in P-Akt expression between normal and tumor breast tissue was calculated using Chi-Square analysis. The Kaplan-Meier method was used for survival analysis, and significance of differences between survival curves was assessed by the log-rank test. 2.7 Cell Culture Conditions MDA-MB-453, MDA-MB-231, MCF-7 and T47D breast cancer cells were obtained from ATCC. The 184htrt preneoplastic breast cell line and their growth conditions were described previously [106]. The MDA-MB-453, MCF-7 and T47D cells were grown in RPMI-1640 without phenol red (Cat# 11835, Invitrogen), supplemented with 5% fetal bovine serum (FBS), and 100 units/ml of penicillin and streptomycin (Invitrogen; Carlsbad, CA). The MDA-MB-231 cells were grown in DMEM (Cat# 11995, Invitrogen), also supplemented with 5% fetal bovine serum and antibiotics. All cells were grown at 37°C with 5% CO2. Growth medium was replaced every three days and stock plates were trypsinized and replated 1:10 once a week. Cell numbers for experimental plating were based on maintaining the same confluency between cell lines. Thus, cell lines of smaller size (MDA-MB-453 and T47D) were plated at higher number than larger cell types (MDA-MB-231, MCF-7, 184htrt) to achieve the same final confluency. 2.8 Cell Lysis and Protein Extraction 2.8.1 Whole Cell Lysis Cells were harvested by scraping, rinsed in ice-cold PBS (Appendix 1c), and pelleted by centrifuging at 3000 rpm for 5 minutes. The pellets were resuspended in four to five packed cell volumes of whole cell lysis buffer (Appendix 1d). Cells were 24 incubated in the lysis buffer for 30-45 minutes on ice, with finger-vortexing every 10 minutes. The lysates were sonicated (amplitude = 40 watts; cycle = 1 second; 5 cycles per 100 u.l volume) and then clarified by centrifuging for 10 minutes at 12000 rpm, 4°C. The supernatants were transferred to a fresh microtube, quantified (section 2.8.3), and stored at -80°C. 2.8.2 Cytoplasmic and Nuclear Fractionation Cells were harvested and pelleted as described in section 2.8.1. To extract cytoplasmic proteins, cells were resuspended in 4-6 packed cell volumes of cytoplasmic extraction buffer (Appendix 1e), incubated for 8 minutes on ice, and then passed through a 22 gauge needle 10 times. Nuclei were pelleted by centrifuging the lysate at 10000 rpm for 2 minutes at 4°C, and the supernatant (the cytoplasmic fraction) was transferred to a fresh tube. The pelleted nuclei were rinsed in a Wash Buffer B (Appendix 1f) and then resuspended in one packed cell volume of nuclear extraction buffer (Appendix 1g). The nuclei were incubated on ice for 30 minutes, ruptured by passing through a 26 gauge needle 10 times, and then incubated for another 10 minutes on ice. Nuclear debris was pelleted by centrifuging for 20 minutes at 13000 rpm at 4°C. Both fractions were quantified as described in section 2.8.3 and stored at -80°C. 2.8.3 Protein Quantification Proteins were quantified using the Bio-Rad Protein Assay (Cat#500-0006; Bio-Rad, Hercules, CA) . Standard curves were created by 1) incubating increasing amounts of bovine serum albumin (1 pg, 2 \ig, 5 u,g, 10 u.g, 20 ng) in 1 ml of dye reagent for 20 minutes, 2) reading the absorbance of each standard at 595 nm with a 25 spectrophotometer and 3) plotting the absorbance values versus the protein concentration and performing linear regression. 1-2 \x\ of cell protein extract were then incubated with 1 ml of dye reagent for 20 minutes and absorbance values read at 595 nm were used to extrapolate concentrations from the standard curve. Quantifications were performed in at least duplicate. 2.9 Western Blotting 2.9.1 Preparation of samples An amount of 50-100 jxg of whole cell, cytoplasmic, or nuclear extracts was combined 3:1 with 4X LDS sample buffer (Cat# NP0008, Invitrogen; Appendix 1h) with 2-mercaptoethanol and boiled for 5 minutes, to denature the proteins. 2.9.2 Electrophoresis Proteins were separated using the S D S - P A G E gel and buffer system. Denatured samples and 5 uJ of prestained broad range standards (Cat#161-0318; Bio-Rad) were loaded onto a 12% acrylamide gel submerged in running buffer (running buffer: Appendix 1i, stacking gel: Appendix 1j, separating gel: Appendix 1k). Samples were compressed in the stacking gel using 70 Volts of current and then separated using 120 Volts of current for 1.5 to 2.5 hours. 2.9.3 Transfer of Separated Proteins to Nitrocellulose Membrane Proteins were transferred from the separating gel to a 0.45 \im pore-size nitrocellulose membrane using the Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad). First, gels and membranes were pre-equilibrated in transfer buffer (Appendix 11) for 15 minutes. Transfers occurred for 1 hour at 110 Volts in chilled transfer buffer. After transfer, blots were stained with Ponceau-S (0.1% in 5% acetic acid solution; Cat# 26 P3504, Sigma) to inspect for proper separation and transfer of proteins and to confirm even loading of samples. 2.9.4 Primary Antibodies Nonspecific protein binding sites on the nitrocellulose membranes were blocked with 5% bovine serum albumin in P B S with 0.1% Tween (Appendix 1c) for one hour at room temperature before addition of primary antibodies. Antibodies against the following proteins were used to probe the western blots: Actin (goat polyclonal; Cat# sc-1616; Santa Cruz Biotechnology, Inc., CA) , total Akt (rabbit polyclonal; Cat# 9272, CST) , phospho-AktSer473 (rabbit polyclonal; Cat# 9271, CST) , phospho-Akt t h r308 (rabbit polyclonal; Cat# 9275, CST) , total 4E-BP1 (rabbit polyclonal; Cat# 9452, CST) , phospho-4E-BP1 (Ser65) (rabbit polyclonal; Cat# 9451, CST) , phospho-GSK-3a /B (rabbit polyclonal; Cat# 9331, CST) , total GSK-3p (mouse monoclonal; Cat# sc-7291, Santa Cruz Biotechnology), H E R - 2 (rabbit polyclonal; Cat# ab-2428, Abeam; Cambridge, UK), and cleaved P A R P (Asp214) (rabbit polyclonal; Cat# 9541, CST). All primary antibodies were diluted 1:1000 in 5% B S A (in phosphate buffer solution with 0.1% Tween), with the exception of anti-Actin (1:500), and were incubated with the membrane overnight on a rocking platform at 4°C. 2.9.5 Detection of Primary Antibody: Protein Complexes After incubation with primary antibody, membranes were washed three times in P B S with 0.1% Tween. In order to visualize the protein/antibody complexes, an anti-mouse/rabbit/or goat IgG horseradish peroxidase-l inked antibody (Amersham; Piscataway, NJ) was next incubated with the membranes. Secondary antibody was diluted 1:7500-10000 in 5% non-fat milk in P B S with 0.1% Tween. Membranes were 27 incubated with the secondary antibody for 1 to 1.5 hours at 4°C on a rocking platform. The nitrocellulose was again washed three times with P B S with 0.1% Tween before subjection to the enhanced chemiluminescence (ECL) detection system (Amersham) and exposure to film. 2.10 Akt Immunoprecipitation and Kinase Assay Nonradioactive Akt kinase assays were performed as described by Castillo et al. [86], with the following modifications to the protocol. Briefly, 500 \ig of whole cell extracted protein (section 2.8.1), were immunoprecipitated with the 5G3 pan-Akt antibody (mouse monoclonal; Cat# 2966, CST) , rotating overnight at 4°C in a 500 \i\ volume of whole cell lysis buffer. As a negative control, 500 u,g of protein was incubated with normal mouse IgG (Upstate) in place of 5G3 antibody. The following day the antibody:protein complexes were incubated with protein G-coated agarose beads (Cat# P4691; Sigma), rotating for 4 hours at 4°C. The beads were washed two times in whole cell lysis buffer, one time in Sato wash buffer (Appendix 1m) [108] and two times in kinase assay buffer (Appendix 1n). To assay for Akt kinase activity, the beads were incubated for 30 minutes at 30°C in kinase assay buffer with 1 uxj of recombinant GSK-3 protein (CST) and 200 \iM A T P . 4X LDS sample buffer with 6-mercaptoethanol was added to stop the reaction. The assays were boiled for 5 minutes, spun at 10000 rpm for 1 minute to pellet the beads, and then the supernatants were frozen at -20°C. For further analysis, one third of each reaction was separated on a 12% acrylamide gel and western blotted (section 2.9). 2.11 Drug Preparation 28 LY294002 was dissolved in DMSO to a stock concentration of 30 mM. Celecoxib was obtained from Pharmacia and was dissolved in D M S O to a stock concentration of 40 mM. The analogues of celecoxib, OSU-03008, OSU-03012 and OSU-03013, were synthesized at Ohio State University in the laboratory of Dr. Ching-Shi Chen (Figure 2) [104]. The analogues were also dissolved in DMSO to a stock concentration for each of 10 mM. All drugs were stored at -20°C in single use aliquots. For the treatment of cells, the drugs were mixed in warm medium before applying to the monolayers. Appropriate treatment concentrations for the celecoxib analogues were determined based on the work of Zhu et al. (Zhu, 2004). 2.12 Determination of Effects of Celecoxib Analogues on Cell Signaling For signaling studies, cells were plated to 60% confluence on 100 mm dishes. To achieve 60% confluence, the following number of cells were plated: MDA-MB-453, 5x10 6; T47D, 3x10 6 ; MCF-7 , 2.25x10 6; MDA-MB-231, 2x10 6 Cells were then treated with the inhibitors in RPMI-1640 medium supplemented with 5% F B S for two hours and harvested by scraping. Drugs were used at the following concentrations: LY294002, (30 nM); celecoxib, (50 and 75 nM); OSU-03008, OSU-03012, OSU-03013, (5 and 10 nM). 2.13 Cell Viability Analysis To determine effects of drugs on cell viability, cells were plated to 60% confluence on 96 well plates. To achieve 60% confluence, the following number of cells were plated: MDA-MB-453, 3x10 4 ; T47D, 2x10 4 ; M C F - 7 , 1.5x10 4; MDA-MB-231, 1.25x104; 184htrt, 2.5x10 4. To test the effect of confluency on drug treatment, the MDA-MB-453 cells were plated at the following confluencies: 60% (3x10 4 cells); 80% (4x10 4 29 cells); 100% (5x10 4 cells); >100% (6x10 4 cells). Cells were treated with the inhibitors in RPMI-1640 medium supplemented with either 0.1% F B S or 5% F B S for 24 hours. Drugs were used at the following concentrations: LY294002 (30 u,M); celecoxib, (25, 50, 75 or 100 fiM); OSU-03008 (5, 10, or 20 uM); OSU-03012 and OSU-03013 (2.5, 5, 7.5, or 10 |xM). Treatments were in quadruplicate. After 24 hours the cells were subjected to the CellTiter 96® Aqueous Non-Radioactive Cel l Proliferation Assay (Promega). This assay involves adding a tetrazolium compound, MTS (Appendix 1p), and an electron coupling reagent, P M S (Appendix 1q), to treated cells. Cells that are metabolically active will bioreduce MTS (by dehydrogenase enzymes) into a formazan product. This formazan product is soluble in cell culture medium and has an absorbance of 490 nm. Thus, spectrophotometrically-measured formazan is directly proportional to the number of metabolically active cells. For a 96 well plate, 2.0 ml of MTS was mixed with 100 uJ of P M S . This solution was then added to 9.6 ml of RPMI-1640 (without F B S or antibiotics) and 120 [i\ of the mixture was applied to each well. The cells were given between 40 and 120 minutes to metabolize the MTS and then the plate was read at 490 nm. The average absorbance for each treatment was background subtracted and then divided by the absorbance of control cells (treated with DMSO) to calculate "% cell viability". 2.14 Apoptosis Analysis 2.14.1 PARP Cleavage Poly(ADP-ribose) polymerase (PARP) , an enzyme involved in DNA repair in response to stress, is cleaved by caspase-3 during the execution of apoptosis. In human cells it is cleaved between Asp214 and Gly215, creating a 24 kDa and 89 kDa 30 cleavage product. To measure the effect of celecoxib and its analogues on the cleavage of P A R P , the MDA-MB-453 cells were plated to 60% confluency (7.5x10 5 cells) on 6 well plates. When confluency was an additional variable for drug efficacy, the cells were also plated at 80% confluence (1.0x10 6 cells) and 100% confluence (1.25x10 6 cells). The next day cells were treated in growth medium for 12 or 24 hours with the following: DMSO, LY294002 (30 u.M), celecoxib (50 or 100 u.M), OSU-03008 (10 or 20 nM), OSU-03012 (5, 7.5 or 10 u.M), OSU-03013 (5, 7.5, or 10 nM). After treatment both attached and floating cells were harvested. Cell lysates were prepared for each treatment using the lysis buffer included in the Cell Death Detection E L I S A P L U S kit (see section 2.14.2). Each lysate (50 ng) was analyzed by western blot using a cleaved P A R P antibody (see section 2.9). 2.14.2 Nucleosomal Fragmentation Assay In addition to P A R P cleavage, endogenous endonucleases are activated during apoptosis. These endonucleases cleave double stranded DNA at the exposed regions between nucleosomes. This yields fragments of DNA complexed with histones H2A, H2B, H3, and H4. These fragments can be found in the cytoplasm of apoptosing cells prior to the later event of plasma membrane destruction. The Cell Death Detection E L I S A P L U S assay (Roche; Penzberg, Germany) measures this release of fragmented nucleosomes into the cytoplasm during apoptosis. The same fresh, unfrozen extracts used to measure P A R P cleavage (section 2.14.1) were used to measure nucleosome fragmentation, in duplicate. Each sample (5 ng total protein) was diluted to 20 \i\ in lysis buffer and processed according to the manufacturer's instructions. In brief, nucleosomes in the cytoplasmic extracts are 31 bound to anti-histone-biotin and anti-DNA-POD antibodies on a streptavidin-coated 96 well plate. This creates a sandwich of streptavidin:biotin:histones:DNA:POD. Thus, the amount of nucleosomes in the cytoplasmic extract can be measured photometrically by reacting the peroxidase of POD (present in the sandwich) with an A B T S substrate. This reaction is read at 405 nm and unreacted A B T S is read at 490 nm. For each sample, the absorbance at 490 nm is subtracted from that at 405 nm. The resulting number is divided by the amount measured in the DMSO control cells, to show the fold increase in cytoplasmic nucleosome enrichment. 2.15 Plasmids The myr-Akt1 plasmid and its corresponding pUSEamp (+) empty vector were obtained from Upstate (Cat# 21-151). The myr-Akt1 construct contains mouse Akt1 cDNA with the myristoylation sequence of c-src at the 5' end and a Myc-His tag at the 3' end of the sequence, all under control of a C M V promoter. The DD-Akt1 construct was a gift from Dr. James Woodgett (Ontario Cancer Institute, Toronto). It consists of Akt1 cDNA, with the Thr308 and Ser473 sites mutated to aspartic acid residues, inserted into the pcDNA3 vector from Invitrogen. Both the myr-Akt1 and DD-Akt1 constructs contain a neomycin resistance gene for selection purposes. 2.16 Rescue Experiment 2.16.1 Transient Assay 2.16.1.1 Transfection Optimized transfection conditions were used for each cell line. MDA-MB-453 cells were plated on 6 well dishes at low density (7.5x10 5 cells). M C F 7 cells were plated on 6 well dishes at high density (5.0x10 5 cells). The following day, cells were 32 transfected in duplicate wells, with either empty vector (EV), DD-Akt1, or myr-Akt1 (section 2.15) using Lipofectamine™ 2000 (Invitrogen) according to manufacturer's instructions. In brief, 3 u.g of each plasmid was added to 200 u.l of RPMI 1640, without F B S or antibiotics (Tube A). In a second tube (Tube B) 9 u.l of Lipofectamine™ 2000 was added to 200 \i\ of RPM11640, without F B S or antibiotics. The contents of Tubes A and B were combined (Tube C) and incubated at room temperature for 20 minutes. Another 600 \x\ of serum free RPMI 1640 was added to Tube C. The cells were rinsed with serum free media and then the DNA:Lipofectamine complexes (Tube C) were added to the cells dropwise. After a 6 hour incubation at 37°C, the complexes were removed from the cells, replaced with normal growth medium, and cells were allowed to recover overnight. 2.16.1.2 Treatment of Transfected Cells with Analogues to Assess Viability; Analysis of Expression and Activity of Activated Akt Constructs The next day, 18 hours post transfection, cells were counted and replated to 96 well dishes at 60% density (see section 2.13), with the remaining cells replated on a new 6 well dish. Cells were allowed to attach overnight. On the 96 well plate, cells transfected with either empty vector, DD-Akt1, or myr-Akt1 were treated in quadruplicate for 24 hours with the following drugs diluted in growth medium: DMSO, LY294002 (30 u,M), OSU-03012 (5, 7.5 or 10 u.M), OSU-03013 (5, 7.5, or 10 u.M). Viability of treated cells was determined using the MTS assay (see section 2.13). Cells which had been replated to the 6 well dishes were harvested to determine expression and activity of the transfected constructs. Whole cell extracts of harvested cells (section 2.8.1) were western blotted and probed for P-Akt expression (section 2.9). 33 Additionally, these extracts were subjected to an Akt kinase assay to verify levels of Akt activity in the transfected cells (section 2.10). 2.16.2 Stable A s s a y MDA-MB-453 and MCF-7 cells were transfected as described in section 2.16.1.1. The next day transfected cells were trypsinized and replated in normal growth media containing neomycin (600 ng/ml for MDA-MB-453 cells; 400 u.g/ml for MCF-7 cells). Cel ls were selected for resistance to neomycin over the course of two weeks. Neomycin resistant pooled clones were harvested and assessed for expression of DD-Akt1 and myr-Akt1, as described in section 2.16.1.2. Cells stably expressing activated Akt or empty vector were plated to 96 well dishes and treated as described in section 2.16.1.2. CHAPTER 3 34 R E S U L T S 3.1 Validation of P-Akt Immunostaining To determine whether the P-Akt antibody was specific, we first treated T47D cells with and without IGF-I for 30 minutes and then analyzed the cells by both western blot and immunohistochemistry. IGF-I stimulated the phosphorylation of Akt, as detected by western blot, where P-Akt was predominantly cytoplasmic (Figure 3A, lanes 1 and 2) though a small amount trafficked into the nuclear fraction upon IGF-I treatment (Figure 3A, lanes 3 and 4). The cells were fractionated to confirm localization that might be observed in tissues. Next, by immunohistochemistry, using the IHC-specific antibody, the T47D cells treated with IGF-I showed a significant increase in P-Akt staining compared with untreated cells (Figure 3B). The staining was noted to be predominantly cytoplasmic with some membranous stain. As a final control, we preincubated the IHC-specific P-Akt antibody with its competing peptide and reduced immunohistochemical staining to that of background levels on a section of breast cancer tissue (Figure 3C). These controls show that the antibody was specific to P-Akt. 3.2 Frequency of P-Akt Expression in Tumor and Normal Tissue Of the 481 cases on the array, 438 contained invasive carcinoma and the characteristics of these patients are described in Table 1. Following P-Akt staining, a total of 390/438 invasive carcinoma cases were interpretable for P-Akt expression and were the only cases considered in this study. P-Akt expression was primarily cytoplasmic, although nuclear staining was visible in a few cases. P-Akt was 35 cytoplasmic SF +IGF nuclear SF +IGF T-AKT B. serum starved/ -IGF-1 serum starved/ +IGF-1 no competing peptide + competing peptide Figure 3. Validation of the P - A k t S e r 4 7 3 Antibody for Immunohistochemistry. (A) The expression of P-Akt was first analyzed by western blot of T47D cells stimulated with and without IGF-I (SF (serum free) indicates no addition of IGF-I). Localization of P-Akt is shown by nuclear and cytoplasmic fractionation. (B) Using an IHC-specific antibody, P-Akt expression was detected by immunohistochemistry of formalin-fixed, paraffin-embedded T47D cells stimulated with and without IGF-I. (C) Finally, by preincubating the IHC-specific P-Akt antibody with a competing peptide, staining of breast cancer tissue is reduced to background level. 36 predominantly expressed in epithelial cells and also was noted in endothelial cells, but was not expressed in the stroma. The distribution of P-Akt expression in the 390 tumors was: (no staining) 43/390 (11%), (weak) 122/390 (31%), (moderate) 120/390 (31%), and (strong) 105/390 (27%) (Figure 4A-D, respectively). Thus, 58% (225/390) of the breast cancers on this array expressed moderate to high levels of P-Akt. A s shown in Figure 4E , some cores on the T M A contained normal ducts alongside cancer tissue. In this particular example, the cancer cells expressed high levels (score 3) of P-Akt whereas the normal ducts expressed low levels (score 1) of P-Akt. To extend this initial observation, we immunostained 26 normal breast tissue samples for P-Akt. A s seen in the tumor tissue, P-Akt was predominantly expressed in epithelial cells. The stroma was consistently negative for P-Akt, while endothelial cells (Figure 4H) were found to express P-Akt at weak to moderate levels. We found the normal tissue distribution to be: (negative) 6/26 (23.1%), (weak) 11/26 (42.3%), (moderate) 4/26 (15.4%), and (strong) 5/26 (19.2%) (Figure 4F-G) . Thus, only 35% (9/26) of the normal breast samples expressed moderate to high levels of P-Akt. The difference between P-Akt expression in normal versus tumor tissue was statistically significant (p «s 0.025) by Chi-Square analysis (Figure 5). 3.3 P-Akt Expression is Not Associated with Patient Survival We hypothesized that patients with tumors that expressed high levels of P-Akt would have poor survival. However, we found that there was not a significant difference (p =0.5839) in overall survival between patients that expressed high levels of P-Akt versus those which expressed low levels of the activated protein (Figure 6). We also evaluated the relationship of P-Akt expression on the TMA to other clinicopathologic 37 Figure 4. Express ion of P-AktSer473 in Tumor and Normal T issues of the Breast. (A-D) On the tumor tissue microarray, P-Akt staining of invasive ductal breast carcinomas (IDC) ranged in intensity from 0 (no stain), 1 (weak), 2 (medium), to 3 (strong stain) respectively. (E) Normal ducts (dashed arrow) adjacent to the IDC (solid arrow) expressed appreciably less P-Akt. (F) Likewise, P-Akt staining was weak when whole sections of normal breast tissue were stained. (G) Examples of intense P-Akt staining in normal breast tissue were also observed. (H) It was also noted that P-Akt was present in endothelial cells. Pictures were taken at a magnification of 200X (A-G) or 400X (H). 38 AGE (yr) ID P-Akt Score Ductal Epithelium Pathology 19 91-01 1 normal 21 206-00 0 normal 22 150-00 3 normal 24 35-00 2 normal 26 174-00 3 adenosis 26 185-00 1 FCD 27 95-00 1 normal 28 191-00 1 normal 29 108-01 2 normal 30 201-00 0 normal 30 109-01 3 adenosis 31 151-00 1 normal 34 105-00 1 adenosis 36 220-00 0 normal 37 99-00 0 normal - w/ duct 38 168-00 1 adenosis 38 110-01 3 FCD 40 51-01 0 normal 42 36-00 1 adenosis 42 71-00 2 normal 42 232-00 1 FCD 42 148-01 3 FCD 46 170-00 1 FCD 46 274-00 0 FCD 57 72-01 1 FCD 59 175-01 2 FCD mean: 35 median: 35 B. Distribution of P-Akt Expression in Normal and Tumor Tissues SO 7 0 6 0 50 4 0 3 0 2 0 10 0 58 • Low P-Akt • High P-Akt Chi Square Analysts Tumor Normal Tota l Low (0 ,1 ) 165 17 182 Hiqh (2 .3) 225 9 234 To ta l 390 26 416 p = 0.025 Figure 5. P-AktSer473 is Highly Expressed More Frequently in Breast Cancer Compared with Normal Breast. (A) The range and frequency of P-AktSer473 expression in normal breast tissue is listed along with ductal pathology and age of each patient (FCD: fibrocystic disease). (B) Frequency of low (score 0 or 1) or high (score 2 or 3) P-Akt expression in normal and tumor breast samples is shown by bar graph. P-Akt is highly expressed in 58% of tumors and only 35% of normal breast samples. This difference in expression was determined to be significant by Chi-Square analysis, as shown. 39 Figure 6. P-AktSer473 Expression Alone Does Not Predict Poor Prognosis. Kaplan-Meier analysis was used to assess the relationship of P-Akt expression in breast tumors with patient prognosis. The differences in survival were not statistically significant (p=0.5839). score ofO (green curve); score of 1 (pink curve); score of 2 (yellow curve); score of 3 (grey curve) 40 variables, such as grade, lymph node status and histology, and found no significant correlations (Table 1). 3.4 Correlation of P-Akt Express ion with Other Proteins We next determined what proteins are coordinately expressed with P-Akt in primary breast cancer by comparing it to the expression profiles of 30 other proteins. These 30 proteins, listed in Table 2, were previously examined on the same breast cancer TMA series for other studies within, or in collaboration with, G P E C (Vancouver, British Columbia). We determined that the expression of P-Akt was significantly correlated with the expression of six proteins, by univariate analysis (Table 2). Of the six proteins, three were tyrosine kinase receptors (IGF-1R (p=0.000), HER-2 (p=0.012), EphA2 (p=0.001)). We also discovered that the expression of ILK (p=0.004) correlated with P-Akt expression. Lastly, the expression of two transcription factors, GFI-1 (p=0.016) and YB-1 (p=0.004), were positively correlated with expression of P-Akt. 3.5 Express ion of P-Akt in Breast Cancer Cel l L ines A panel of breast cancer cell lines was screened for levels of P-Akt (S473 and T308) and total Akt expression during exponential growth (Figure 7). As the effects of the analogues were analyzed in 5% F B S , the levels of P-Akt were assessed accordingly. The four breast cancer cell lines express similar levels of total Akt protein, whereas the 184htrt preneoplastic breast epithelial cells express very little Akt. Although all cell lines were grown in the presence of serum, which contains many growth factors that should activate Akt, only the MDA-MB-453 and T47D cell lines expressed high levels of P-Akt in this condition. It should be noted, however, that the 184htrts, MDA-MB-231 s and MCF-7s can be induced to express higher levels of P-Akt 41 Spearman Protein Correlation Fisher's Exact Test Number of Patients C A 9 -0.085 0.192 310 C D 3 -0.004 1.000 298 CD20 -0.012 0.306 315 CD43 -0.025 0.795 297 CD68 -0.051 0.351 317 Chromogranin -0.005 1.000 319 Clusterin -0.090 0.103 320 Cox-2 -0.051 0.816 333 Cycl in E 0.053 0.443 280 E2F1 0.085 0.190 302 E-cadherin 0.053 0.323 335 * E P H A 2 0.201 0.001 299 E R 0.107 0.170 309 * GFI-1 0.144 0.016 292 lit HER2 0.139 0.012 344 HSP27 -0.058 0.146 291 IGF-1 0.060 0.274 266 * IGF-1R 0.265 0.000 327 IGFBP-2 -0.091 0.238 292 IGFBP-5 -0.049 0.387 290 * ILK 0.171 0.004 299 Ki-67 -0.046 0.621 308 MEP21 0.019 1.000 260 N S E 0.054 0.296 320 p53 -0.030 0.751 296 PR 0.054 0.314 336 Survivin 0.067 0.717 318 * YB-1 0.159 0.004 373 Table 2. The Correlation of P-AktSer473 with Other Proteins in Breast Tumors. P-A k t e x p r e s s i o n w a s c o m p a r e d w i th t he e x p r e s s i o n o f 3 0 o the r p r o t e i n s i m m u n o s t a i n e d o n the s a m e b r e a s t T M A s e r i e s ( c o l umn 1). P r o t e i n c o r r e l a t i on w a s d e t e r m i n e d by S p e a r m a n ' s c a l c u l a t i o n ( c o l u m n 2) a n d s i g n i f i c a n c e w a s a s s e s s e d b y F i s h e r ' s E x a c t Tes t ( c o l u m n 3). N e g a t i v e co r r e l a t i on s r e p r e s en t i n v e r s e r e l a t i on sh i p s . A n as t e r i s k d e s i g n a t e s c o r r e l a t i on s that a r e s ign i f i cant . " N u m b e r of pa t i en t s " ( c o l u m n 4) i n d i c a t e s the tota l n u m b e r of pa t i en t s that w e r e s c o r a b l e for bo th P -Ak t a n d t he c o m p a r e d p ro te in . 42 when stimulated with supplemented IGF-I [106], [109]. On the other hand, both the MDA-MB-453 and T47D cells can maintain Akt phosphorylation in serum starved conditions [78], [110]. The reason for high Akt activation in the MDA-MB-453s and T47Ds may be accounted for by the expression of the HER-2 receptor in these cell lines (see Figure 7). Overexpression of HER-2 can lead to constitutive activation of the PI3K/Akt pathway [78]. Additionally, MDA-MB-453 cells express low levels of P T E N compared with MDA-MB-231, MCF-7, and T47D cells [78], [110]. 3.6 Analogues of Celecoxib Inhibit Akt Phosphorylation The MDA-MB-453 cells, which express high levels of P-Akt in log-growing conditions, provided a useful model system in which to characterize the effects of the celecoxib analogues on Akt signaling. After treatment for two hours in 5% serum, O S U -03008, OSU-03012, and OSU-03013 all inhibited Akt phosphorylation at a concentration of 10 |xM, similarly to the PI3K inhibitor, LY294002 (30 u.M) (Figure 8, top four panels). Both Ser473 and Thr308 phosphorylation were affected. There was slight downregulation of P-Akt using 5 u,M of the analogues, and 50 JAM and 75 nM of the parent compound, celecoxib. It is important to note that studies which found complete inhibition of Akt phosphorylation using 50 \iM of celecoxib were done in serum free conditions [94]. We also examined the effects of the analogues, celecoxib and LY294002 at 2 hours in another cell line, T47D (Figure 8, bottom four panels). The inhibition of Akt phosphorylation by the analogues in the T47Ds was more robust, perhaps because there is less basal P-Akt in those cells compared with the MDA-MB-453s. 3.7 Analogues of Celecoxib Inhibit Akt Kinase Activity and Downstream Signaling 43 f & # £ f 0 Phospho-Akt S 4 7 3 Phospho-Akt T 3 0 8 Total-Akt Total-Akt (longer exposure) HER-2 Actin Figure 7. Levels of P-Akt and HER-2 in a Panel of Breast Cancer Cel l L ines. Four breast cancer cell lines (MDA-MB-231, MCF-7, T47D, MDA-MB-453) and a preneoplastic breast cell line (184htrt) were analyzed for constitutive P-Akt expression during exponential growth. These levels were also compared to expression of total Akt and the receptor tyrosine kinase, HER-2. A longer exposure for total Akt shows a small amount of this protein in the 184htrt cells. Actin was used as a loading control. 44 o Q S 3 O CO 1 I CM O O •<* cn CM >-© 3 1- w co o o co o C/> O oo o o co o 3 CO O CN O CO o Z> o o o CO o {/) o 5 o CO o w O 3 O o CO o =5 o 5 3 O m x O o a O 2 3 .a 'x o o £ ca O Phospho-Akt S 4 7 3 Phospho-Akt T 3 0 8 Total Akt Actin Phospho-Akt S 4 7 3 Phospho-Akt T 3 0 8 Total Akt Actin Figure 8. Phosphorylat ion of Akt is Inhibited by Ana logues of Celecoxib. Both MDA-MB-453 and T47D cells were treated for 2 hours with the indicated drugs in media containing 5% fetal bovine serum. Phosphorylation of Akt on Thr308 and Ser473, in addition to total levels of Akt, was assessed by western blotting whole cell lysates. Actin was used as a loading control. 45 Phospho-GSK3a/p Total-GSKa/p Phospho-4EBP1 Total-4EBP1 Actin Phospho-GSK Total-GSK Total Akt Input Figure 9. Akt K inase Activi ty is Decreased and Downstream Signal ing is Inhibited in MDA-MB-453 Cel ls Treated with Celecoxib Ana logues. (A) The phosphorylation status of a direct (GSK-3p) and indirect (4E-BP1) substrate of Akt was analyzed by western blot, using protein extracts from MDA-MB-453 cells treated for 2 hours with the indicated compounds. The blot was reprobed for total GSK-3p\ total 4E-BP1 , and Actin proteins. (B) Additionally, kinase activity of Akt was found to be attenuated after MDA-MB-453 cells were treated with celecoxib analogues (10 pM each) or the PI3K inhibitor (30 pM) for two hours. Akt was immunoprecipitated from treated cells and subjected to an in vitro nonradioactive kinase assay, using recombinant GSK-3{3 as a substrate. Levels of total recombinant GSK-3p\ phosphorylated recombinant GSK-3p\ and immunoprecipitated Akt were detected by western blot. 46 To confirm that the inhibition of Akt phosphorylation actually corresponded to suppression of the Akt signaling network, downstream targets of P-Akt were also examined in MDA-MB-453 cells treated for two hours with the compounds. Each of the three analogues, using 10 \iM, and LY294002 (30 nM) inhibited phosphorylation of GSK-3a/6 (Figure 9A). OSU-03013 at 5 u.M had moderate success in inhibiting this target as well. Although the effect of celecoxib was modest on P-Akt (Figure 8), the phosphorylation of GSK-3a /6 is quite attenuated by the parent compound, suggesting some difference in mechanism of drug action. The effect of analogue treatment on 4E-BP1 phosphorylation, however, was quite modest even at the 10 \x,M dose. LY294002, on the other hand, completely abolished phosphorylation, as detected using the Ser65 phospho-specific 4E-BP1 antibody. Reprobing the western blot with a total 4E-BP1 antibody resulted in three detectable bands in the DMSO control sample, indicative of the various phosphorylation states of 4 E - B P 1 . Of all the samples treated with signaling inhibitors, only the LY294002 treated sample displayed the complete downward band shift of a hypophosphorylated 4E-BP1 protein. The kinase activity of Akt was measured to verify that inhibition of Akt phosphorylation at Thr308 and Ser473 correlates with loss of activity of the enzyme. Immunoprecipitated Akt from MDA-MB-453 cells treated for two hours with each of the analogues (10 u,M) was indeed inhibited in its ability to phosphorylate recombinant G S K - 3 (Figure 9B), albeit not completely. Similar inhibition was observed with LY294002 treatment. 3.8 Effect of Analogues on Cell Viability 47 We have shown that the celecoxib analogues inhibit Akt phosphorylation after two hours of treatment. We next wanted to assess the effect of the drugs upon longer exposure, as Zhu et al. showed cytotoxic effects of OSU-03012 and OSU-03013 in the PC-3 cells within 24 to 72 hours of treatment [104]. Increasing doses of celecoxib and the analogues were added to the MDA-MB-453 cells for 24 hours. The treated cells were then subjected to an MTS assay. This assay does not distinguish between live and dead cells, it simply measures metabolic activity in the present cells. In this report MTS results are referred to as "cell viability" measurements, bearing in mind that a reading may be lower because growth has slowed or metabolic activity has decreased, not necessarily because the cells are dead. As shown in Figure 10, 24 hour treatment with the PI3K inhibitor LY294002 resulted in approximately 63(±3)% viable cells. Celecoxib decreased viability at doses of 75 and 100 \iM to 55(±8)% and 14(±5)%, respectively. Interestingly, OSU-03008, at doses as high as 20 uM, only reduced cell viability by 15(±3)% of the DMSO control. However, both OSU-03012 and OSU-03013 had a robust effect at doses of 7.5 and 10 u.M, resulting in less than 20% viable cells. These concentrations of the analogues are 10-fold lower than doses of celecoxib required to have similar effects. Further, as little as 5 u.M of OSU-03013 was able to inhibit cell growth by 53(±10)%. The effects of the analogues were also examined in three other breast cancer cell lines; MDA-MB-231, MCF-7 and T47D (Figure 10). Results of treating the panel of cells were similar to those of the MDA-MB-453 cells, with the following exceptions. Treatment with LY294002 affected viability of the T47D cells (as it did the MDA-MB-453s) but not the MDA-MB-231 s or MCF-7s , in keeping with others who found that 48 LY294002 preferentially kills cells with high levels of P-Akt [78]. OSU-03008 was most effective in the T47D cells, resulting in a 21 (±2)% reduction in MTS metabolism at 20 uM. The T47Ds were also the most sensitive cell line to OSU-03012 and OSU-03013 treatment, responding to a low dose (5 u.M) of OSU-03012 and OSU-03013 by 44(±14)% and 70(±14)%, respectively. MCF-7 cells, on the other hand, were the least sensitive to low doses of OSU-03012 and OSU-03013, showing only a 35(±3)% reduction in viability with 7.5 \iM of OSU-03012 and 25(±10)% reduction with 5 JAM of OSU-03013. In general, the MDA-MB-453 and T47D cells were the most sensitive to low doses (5 and 7.5 u,M) of OSU-03012, but all four cell lines responded similarly to the 10 u.M dose of both OSU-03012 and OSU-03013. 3.9 OSU-03012 and OSU-03013 Induce Apop tos is Treatment of the MDA-MB-453 cells with 7.5 to 10 piM of the celecoxib analogues OSU-03012 and OSU-03013 for 24 hours resulted in a >80% reduction of viable cells by the MTS assay. These cells also show visual characteristics of apoptosis, such as cell rounding and lifting from the plate and membrane blebbing. Taking into account these observations, we hypothesized that OSU-03012 and OSU-03013 induce apoptosis in breast cancer cells. MDA-MB-453 cells were treated with increasing doses of the analogues for 12 and 24 hours. Comparisons were also made to treatment with the parent compound as well as treatment with LY294002. Extracts from the treated cells were then analyzed for P A R P cleavage in addition to release of fragmented DNA. Compared with DMSO-treated control cells, treatment with LY294002, celecoxib (100 u.M), OSU-03012 (7.5 and 10 u.M), and OSU-03013 (7.5 and 10 u.M) all induced P A R P cleavage by 12 hours, which continued to occur through the 24 hour timepoint (Figure 49 MDA-MB-453 <£> & 5 0 7 5 1 0 0 5 10 2 0 2 . 5 5 7 .5 1 0 2 . 5 5 7 5 1 0 <# Celecoxib OSU03008 OSU03012 OSU03013 MCF7 flu H B I I m ml , i J : I 11 i s III 1 I I m ,0 ,0- 5 0 7 5 1 0 0 5 10 2 0 2 . 5 5 7 .5 10 2 . 5 5 7 .5 10 ^ Celecoxib OSU03008 OSU03012 OSU03013 MDA-MB-231 ,X> & 5 0 7 5 1 0 0 5 10 2 0 2 . 5 5 7 .5 10 2 . 5 5 7 .5 10 # c#" Celecoxib OSU03008 OSU03012 OSU03013 T47D eP & 5 0 7 5 1 0 0 5 10 2 0 2 5 5 7 .5 10 2 . 5 5 7 .5 1 0 <JP of* 5 Celecoxib OSU03008 OSU03012 OSU03013 Figure 10. Assessment of the Effects of Celecoxib Analogues on Cell Viaibilty. Four breast cancer cell lines, MDA-MB-453, MCF-7, MDA-MB-231 and T47D, were treated with the indicated compounds for 24 hours in medium containing 5% fetal bovine serum. Cell viability was measured using the MTS Proliferation Assay. Shown in the bar graphs are the percentages of viable cells after treatment compared with the DMSO control. 50 Celecoxib OSU-03008 OSU-03012 OSU-03013 A. DMSO LY 50uM 100uM 10uM 20uM 5uM 7.5uM 10uM 5uM 7.5uM 10uM cleaved PARP Actin cleaved PARP Actin Figure 11. Induction of Apoptos is by Celecoxib Analogues. (A) PARP cleavage increased substantially after MDA-MB-453 cells were treated for 12 to 24 hours with OSU-03012 [7.5 or 10 uM], OSU-03013 [7.5 or 10 u.M], Celecoxib [100 \iM], and LY294002 [30 u.M] as detected by western blot. Blots were reprobed for actin as a loading control. (B) In parallel to PARP cleavage, nucleosomal fragmentation also increased when MDA-MB-453 cells were treated as in (A). Release of nucleosomal fragments into the cytoplasm was assessed using a Cell Death Detection ELISA. 51 11A). Low dose celecoxib (50 u.M), all doses of OSU-03008, and low dose (5 uM) OSU-03012 and OSU-03013 failed to induce P A R P cleavage. A similar pattern was observed when nucleosomal fragmentation was analyzed (Figure 11B). Thus, two methods confirm the induction of apoptosis by celecoxib, OSU-03012, OSU-03013 and LY294002 in the MDA-MB-453 cells. 3.10 Cell Confluency Protects Against Analogue-induced Ceil Death It was observed that when cells were plated more densely, the cytotoxic effects of the analogues were abrogated. To investigate this further, increasing densities of MDA-MB-453 cells were plated and then treated with the inhibitors for 24 hours. These cells were analyzed for MTS metabolism, P A R P cleavage and P-Akt expression. By the MTS assay, more confluent cells were protected from 100 fxM of celecoxib and 10 nM of both OSU-03012 and OSU-03013 (Figure 12A). In addition, P A R P cleavage was more pronounced in the less confluent cells after treatment (Figure 12B). Higher levels of P A R P cleavage could be induced in confluent cells by increasing the dose of O S U -03013 to 15 u.M (data not shown). Notably, increased cell density did not change the effect of LY294002 on cell viability, as measured by the MTS assay, but P A R P cleavage was inhibited by confluency. Further, it was noted that basal P-Akt expression was increased in more confluent cells (Figure 12B). Expression of actin, as assessed by reprobing the western blots used to detect cleaved P A R P and P-Akt, showed equal loading of protein samples. 3.11 Serum Protects Against Cytotoxic Effects of Celecoxib and Low Doses of Celecoxib Analogues We also considered the fact that 5% serum in our treatment medium may protect against the effects of the analogues. This phenomenon was noted for celecoxib by 52 B. 60% confluent 80% confluent 100% confluent Cleaved PARP P-Akt (S473) Actin Cleaved PARP P-Akt (S473) Actin Cleaved PARP P-Akt (S473) Actin Figure 12. Conf luency Protects Cel ls from Cytotoxic Effects of Celecoxib and Analogues. MDA-MB-453 cells were plated at increasing densities (60% to >100% confluent) and then treated with the indicated compounds for 24 hours. (A) Cell viability in response to treatments was assessed by MTS assay. Increased confluency protected against high dose celecoxib [100 uM] and high dose OSU-03012 and OSU-03013 [10 u.M]. (B) Cleavage of PARP and inhibition of P-Akt was assessed by western blot. Blots were probed with actin as a loading control. Increased cell density inhibited the ability of the analogues to induce apoptosis or attenuate Akt phosphorylation. 53 (3 5% serum • 0.1% serum MCF7 cells DMSO LY [25uM] [50uM] [75nM] [100uM] [SuM] [10uM] [20uM] [2.5uM] [5uM] [7.5uM] [10uM] [2.5uM] [5i.M] [7.5i,M] [10uM] Celecoxib OSU-03008 OSU-03012 OSU-03013 Figure 13. Effect of Serum on Eff icacy of Celecoxib and Analogues. MDA-MB-453 and MCF-7 cells were treated with the indicated compounds for 24 hours in medium containing either 0.1% or 5% fetal bovine serum. Cell viability was measured by the MTS proliferation assay. Strong serum protection against celecoxib treatment was noted for both cell lines, except at 100 u.M. Low serum conditions (0.1%) slightly increased the sensitivity of the MDA-MB-453 cells to all doses of OSU-03008 and [5 u.M] of O S U -03013. There was a considerable increase in response of the MDA-MB-453 cells to [5 u.M] of OSU-03012 in low serum. The MCF-7 cells were much more sensitive in low serum media to all doses of OSU-03008, to 5 and 7.5 u.M of OSU-03012, and to 5 uM of OSU-03013. The highest dose [10 u.M] of OSU-03012 and OSU-03013 was not protected by 5% serum. 54 Levitt, et al. [111]. Thus MDA-MB-453 cells were again treated with the signaling inhibitors in medium containing only 0.1% serum, monitored for cell viability, and compared to the results seen in 5% serum conditions. Doses of celecoxib that were <10% effective in 5% serum (i.e., 50 \xM) resulted in 100% loss of cell viability when serum was deprived (Figure 13). The effects of OSU-03008 and OSU-03013 were only slightly improved upon serum withdrawal, whereas the 5 \iM dose of OSU-03012 became 30(±17)% more effective in serum-deprived medium. This experiment was also repeated in the MCF-7 breast cancer cell line, where similar results were measured (Figure 13). Notably, 5% serum provided the MCF-7 cells with more protection against the effects of OSU-03008, OSU-03012, and OSU-03013 than that seen in the MDA-MB-453 cells. Serum did not protect the MDA-MB-453 cells from LY294002. However, in serum-deprived conditions, the MCF-7 cells became 12(±3)% more sensitive to LY294002. 3.12 Activated Akt Does Not Rescue Cytotoxic Effects of Analogues In an attempt to show whether the inhibition of Akt phosphorylation was a necessary mechanism for the cytotoxic effects of the celecoxib analogues rather than a secondary result, rescue experiments were performed. Zhu, et al. observed a 20-30%) rescue of cell viability when PC-3 cells transiently expressing constitutively activated Akt were treated with OSU-03012 [104]. Therefore, MDA-MB-453 and M C F - 7 cells were transiently transfected with either of two constitutively activated Akt constructs. Myr-Akt1 contains the myristoylation tag of c-src, which leads to anchoring of Akt to the membrane independently of PI3K activity [112]. DD-Akt1 is an Akt construct where both Ser473 and Thr308 are mutated to aspartate residues, which mimics phosphorylation 55 [74], [113]. Western blot analysis of transfected cells shows that cells expressing activated Akt constructs indeed have higher levels of P-Aktser473 than vector-expressing cells (Figure 14A). The phospho-myr-Akt1 is larger than wild type phospho-Akt due to a Myc-His tag. Additionally, kinase assays of immunoprecipitated Akt from cells transfected with either myr-Akt1 or DD-Akt1 show that these constructs are highly active in their ability to phosphorylate recombinant G S K - 3 , proving the functional capacity of the constructs (Fig 14A). However, transient expression of neither DD-Akt1 nor myr-Akt1 was able to rescue MDA-MB-453 cells from the cytotoxic effects of OSU-03012 or OSU-03013 (Figure 14B). An attempt was also made to generate MDA-MB-453 and MCF-7 cell lines stably overexpressing DD-Akt1 or myr-Akt1. We were unable to maintain stable expression of DD-Akt1 in either cell line, and also could not retain myr-Akt1 expression in the MCF-7 cells. MDA-MB-453 cells stably expressing myr-Akt1 were established (Figure 15A), although expression was lost after six passages. While the cells were expressing myr-Akt1 (passage 3) they were treated with OSU-03012 and OSU-03013 for 24 hours alongside MDA-MB-453 control cells expressing an empty vector. There was no difference in viability of cells expressing an empty vector versus those with constitutively activated Akt (Figure 15B). 56 A . Whole Cell Extracts Kinase Assay MDA-MB-453 M MCF-7 M MYR -AM1 WT P-Akt/ DD-Akt1 Phospho-GSK3 (recombinant) M 1 2 3 4 5 6 Ponceau S B. MDA-MB-453 cells DMSO OSU-03012 M C F 7 cells OSU-03013 empty vector HDD-Aktl •Myr-Aktl I r I l i f e . DMSO [5nM] [7.5„M] [10nM] [S^M] [7SnM] [10M ] OSU-03012 OSU-03013 Figure 14. Overexpression of Act ivated Akt Does Not Rescue Cel ls from Cytotoxic Effects of Treatment with OSU-03012 or OSU-03013. MDA-MB-453 and MCF-7 cells were transiently transfected with either DD-Akt1 (D), myr-Akt1 (M), or an empty vector control (V). (A) Expression of transgene was confirmed by western blot using a phospho-Akt antibody. Both DD-Akt1 and myr-Akt1 have much higher levels of P-Akt expression compared with control cells. (Note: myr-Akt1 is larger than wild type(WT)- and DD- Akt due to a Myc-His tag). Equal loading was assessed by staining the blot with Ponceau S (respective lanes are labeled 1-6; M = size marker; 120,100, 55, 38, 29, 20 kDa (top blue band to bottom blue band)). Additionally, activity of Akt was confirmed by immunoprecipitation of Akt from each sample followed by a kinase assay, using recombinant GSK-3 as a substrate. Phospho- recombinant GSK-3 was detected by western blot. (B) Transiently transfected cells were treated for 24 hours with the indicated compounds and cell viability was subsequently measured by MTS assay. 57 Ponceau S B. MDA-MB-453 ceils 100 9 0 8 0 70 6 0 5 0 4 0 30 20 10 0 n empty vector • myr-Aktl n DMSO LY [5u.M] [7.5u.M] [10u.M] [5(xM] [7.5u.M] [lOu-M] OSU-03012 OSU-03013 Figure 15. Stable Overexpression of Myr-Akt1 Does Not Rescue Cel ls from Celecox ib Ana logues. MDA-MB-453 cells stably expressing myr-Akt1 or empty vector were generated as described in Materials and Methods. (A) Expression of myr-Akt l construct in cell lysates was verified by examining phosphorylated Akt on a western blot. Myr-Akt1 is larger than wild type (WT) Akt due to a Myc-His tag. Equal loading was assessed by staining the blot with Ponceau S (M = size marker; 120,100, 55, 38, 29, 20 kDa (top blue band to bottom blue band)). (B) Stably transfected cells were treated for 24 hours with the indicated compounds and cell viability was subsequently measured by MTS assay. Bars in graph represent the percentage of viable cells remaining in each treatment group compared with DMSO control. 58 CHAPTER 4 DISCUSSION 4.1 Introduction Our laboratory has spent the last several years researching the role of receptor tyrosine kinases and Akt in breast cancer. We have shown that signaling through the IGF-1 R/P-Akt axis is responsible for upregulating the expression of molecules important for invasion and angiogenesis, such as urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF) [114], [106]. Further, the expression of IGF-1R and uPA in primary breast cancer was found to be associated with poor survival [115]. The potential for therapeutic intervention of this pathway has also been examined. Dunn et al. found that inhibition of IGF-1 R in tumor-bearing mice, using a dominant-negative mutant of the receptor, significantly decreases metastases [116]. Additionally, Nielsen et al. used the heat shock protein 90 (Hsp90) inhibitor, 17-allylamino geldanamycin, to degrade the IGF-1 R and inhibit downstream signaling through Akt in breast cancer cells [115]. The project reported herein continues in the vein of our prior research. We determined the frequency and level of Akt activation in 390 breast tumors, and assessed coordinate expression of P-Akt with other signaling molecules, including RTKs, in the same tumors. Further, we explored the potential for using analogues of the anti-inflammatory drug celecoxib as inhibitors of Akt phosphorylation and therefore as a novel treatment for breast cancer. 4.2 Expression of Phospho-Aktw73 in Primary Breast Tumors 59 4.2.1 Frequency of Expression and Relationship to Patient Prognosis By screening tumor tissue arrays containing samples from 390 breast cancer patients, we determined that a majority (58%) of breast tumors express moderate to high levels of activated Akt. This frequency is in accordance with other studies, in various cancer types (50-66% in breast cancer [117], [80], [62], 57% in ovarian carcinomas [118], 59% in pancreatic cancer [119], 50% in N S C L C [120]. Considering this data, a high percentage of patients could potentially benefit from inhibition of the Akt pathway. Additionally, we hypothesized that women whose tumors expressed activated Akt would have poor survival. However, our data indicate that P-Akt status is not a prognostic marker for breast cancer. There have been conflicting reports as to whether P-Akt expression is prognostic or not. In breast cancer, at least three studies have indicated that P-Akt does not correlate with survival [121], [122], [117]. However, another study indicated that P-Akt predicted distant recurrence and shorter disease-free survival in endocrine treated breast cancer patients [80]. Further, a study of node-negative breast cancers found P-Akt status to be correlated with decreased overall survival [123]. The differing results extend to other cancer types as well. Although P-Akt is found in high gleason grade prostate cancers and predicts poor outcome [84], [124], another study showed that P-Akt was associated with reduced rate of disease progression in lung cancer and actually predicted sensitivity to Iressa [120]. These differences could be accounted for based on variations in immunohistochemical 60 methods, antibodies, tissue fixation or scoring, but may be associated with the variety of treatment options used to treat the patients. The lack of prognostic value of P-Akt expression for the patients in our study does not discount a role for Akt activation in tumorigenesis or in cancer progression. Phospho-Akt levels can be modulated in many ways and can change during the progression of a tumor. Measuring the phosphorylated state of a protein is a "snapshot" of a dynamic system of signaling, similarly, TMAs only capture one moment in the progression of many tumors. Therefore, detection of high P-Akt levels does not imply that the pathway is constitutively activated in those tumors, just as detection of low levels does not mean that the tumors will not later activate P-Akt. Firstly, the patients in our study were not treated with a standardized chemotherapeutic regimen. As P-Akt may provide resistance to some treatments and not others, a study examining the relationship of activated Akt to the survival of women given the same therapies may provide more insight. Secondly, the tumor samples on our array were taken prior to initiation of therapy. P-Akt levels might change once the therapy begins. For example, the development of drug resistance in experimental models suggests that Akt phosphorylation is upregulated in the acquisition of this phenotype [125]. Tumor cells that remain in the body throughout chemotherapy may be forced to develop new mutations activating an anti-apoptotic pathway in order to survive. A final important consideration could be that Akt signaling becomes activated in metastatic cells. Patients rarely die from a primary breast tumor, but rather from the resulting metastases. Akt activation prevents anoikis [126] and has been shown to be upregulated in metastatic variants of GI101A human breast cancer cells [127]. A s the 61 cases on our TMA were all primary cancers, a future array containing both primary and metastatic tumors, if possible, could prove or disprove this theory. 4.2.2 Correlation of P-Akt with the Expression of Other Proteins The TMAs we employed for this study were previously immunostained for 30 other proteins (by us and others, see Appendix 2). This allowed us to explore the network of proteins coordinately expressed with P-Akt, some of which may be involved in the activation of Akt and/or may be a consequence of its signaling. By univariate analysis we found that activated Akt was positively correlated (significantly) with the expression profiles of six other proteins; HER-2 , IGF-1 R, EphA2, ILK, Y box-binding protein-1 (YB-1), and growth factor independence 1 (GFI-1). Bearing in mind that association does not necessarily imply causation, we developed two theories to explain this data. First, it is possible that the seven proteins are regulated by a common upstream pathway or common phase of the cell cycle, resulting in their coordinate expression in these breast tumors, but without direct links to one another. Secondly, perhaps coordinate expression does indicate regulatory or functional links between P-Akt and the other molecules. The second theory was given merit by the fact that coexpression of HER-2, IGF-1R and ILK with P-Akt in primary tumors corroborates previous data in experimental models, showing that expression of these molecules can activate the Akt signaling pathway. For example, MCF-7 breast cancer cells engineered to overexpress HER-2 exhibit an increase in Akt kinase activity [26]. Cell lines that endogenously overexpress HER-2 also exhibit upregulated P-Akt [128]. Similarly, stimulation of the IGF-1 R with IGF-I rapidly activates Akt [106] and transgenic mice overexpressing IGF-1 R in the 62 heart have a hyperactivated Akt pathway [129]. Finally, a role for ILK in Ser473 phosphorylation of Akt was demonstrated by ILK knock-out, using either R N A interference or the Cre-Lox system. Cells or mice lacking ILK expression showed a significant decrease in P-Akt and also Akt kinase activity [130]. In addition to in vitro evidence, our finding that activation of Akt is correlated with HER-2 expression in primary breast tumors is also supported by previous reports [123]. However, our study is the first to demonstrate a link in primary breast cancer between IGF-1R and ILK with P-Akt. Despite the significant correlations we observe between P-Akt and HER-2, IGF-1R and ILK, they are not complete correlations. Meaning, some tumors with P-Akt were negative for ILK, and some tumors positive for IGF-1R and HER-2 were negative for P-Akt. Thus, based on our data, the relationships between these molecules are not necessarily linear, but rather more complex. As there is evidence to explain some of the correlations with P-Akt, perhaps the T M A s could serve as a hypothesis-generating tool for exploring novel signaling relationships. In this study, activated Akt was also significantly correlated with the expression of the RTK EphA2, as well as the transcription factors GFI-1 and Y B - 1 . Several characteristics of these molecules make functional or regulatory relationships with P-Akt a logical scenario. EphA2 is upregulated in 40% of breast cancers and has been shown to impart tumorigenic and metastatic potential when overexpressed in MCF-10A normal breast epithelial cells [131]. The mechanism of EphA2 function in cancer remains unclear, and appears to be dually involved in cel lxel l adhesion and repulsion. For example, overexpression of EphA2 mediates cell:cell contact repulsion, and allows for survival independent of basement membrane attachments [132]. As Akt 63 is also involved in anchorage independent growth, it is possible that EphA2 overexpression may positively regulate the activation of Akt, creating a survival signal for tumor cells and promoting cell migration. The relationship between P-Akt and the expression of the transcription factors GFI-1 and YB-1 also has interesting implications. Although the function of GFI-1 in the malignant transformation of T-cells and lymphoma progression is well established, GFI-1 has never before been studied in the context of breast cancer. In T-cells, GFI-1 operates as a transcriptional repressor of Sax, which correlates with suppression of apoptosis [133]. GFI-1 also regulates the expression of cell-cycle factors cMyc and P21 in myeloid cell lines [134]. YB-1 expression, on the other hand, has been previously examined in breast cancer patients and is related to poor prognosis [135]. This protein has diverse functions in both the cytoplasm, where it binds to RNA [136], and in the nucleus where it acts as a transcription factor [137] and regulates mRNA transport [138]. Overexpression of YB-1 in HBL100 pre-neoplastic breast epithelial cells promotes their resistance to doxorubicin [139]. One hypothesis to explain the observed correlations on the TMA is that perhaps P-Akt signals upregulate the expression of these two transcription factors, as a novel mechanism for resisting drug-induced apoptosis or promoting tumor growth. It is also possible that the relationships are inverted, where GFI-1 or YB-1 signaling indirectly upregulates Akt phosphorylation. Further, the possibility exists that the activation of Akt leads to upregulation of the RTKs HER-2, IGF-1 R or EphA2. Future research could determine whether the correlations observed on the TMA relate to novel, functional arms of the Akt signaling network. 64 This study highlights the power of analyzing signaling networks in a large number of patients, which sharply contrasts the traditional method of measuring individual molecules of a signaling cascade in cell culture. Molecular profiling of tumors by cDNA, protein, or tissue microarray technologies, presents many opportunities for translational research [140]. As a clinical application, profiling makes it possible to 1) classify tumor types for diagnosis by identifying tumor-specific markers, 2) predict response to therapy or 3) project the likelihood of metastasis. From a research standpoint the ability to discern global expression patterns allows us to 1) comprehend the network of molecular interactions occurring within a cell and/or tumor, 2) decipher the differences between normal and cancerous cells and 3) rationally design therapies that exploit signaling pathways which cancer cells depend on for survival and metastasis. The ability to analyze signaling networks in primary tumors was demonstrated by Choe, et al., who determined the expression of multiple molecules of the PI3K pathway in glioblastoma using TMAs [141]. Their study was able to validate, in patients, relationships between P-Akt and the expression of molecules previously studied only in in vitro models (phospho-FKHR and phospho-mTOR). Similar to the Choe study, our work used TMAs to further explore the P-Akt signaling axis in patients. However, because we examined a total of 30 different proteins, most of which had no prior connection with P-Akt, we were able to identify potential novel relationships in this pathway. Hopefully, using TMAs to establish patient profiles for expression levels of particular growth/survival pathway proteins (i.e., RTKs and P-Akt) will aid in the development of tailored therapies. For example, identifying patients which overexpress P-Akt seems to predict positive lung cancer response to Iressa [120]. 65 4.3 Expression of Phospho-Aktsnr^ in Normal Breast Tissue In addition to determining the level of P-Akt in breast tumors, we also examined this protein in a set (n=26) of normal breast tissues. We found that the frequency of Akt activation in normal breast tissue is significantly lower than that seen in breast tumors (58% in tumor versus 35% in normal). However, the range of expression in normal tissue was similar to that of the tumors, with some tissues expressing no P-Akt (score 0) and some tissues expressing very high levels (score 3) of P-Akt. The expression of P-Akt in normal tissue is not surprising. Akt is phosphorylated in hippocampal neurons to facilitate survival [142], in skeletal muscle during exercise [143], and in cardiomyocytes where it regulates cell size and contractile function [144]. The range of P-Akt expression in the normal breast, however, leads to questions regarding its regulation and necessity in mammary tissue. There has not been much attention placed on normal breast cells and Akt in the research community, thus its role and regulation can only be speculated upon. One important question is whether the expression of P-Akt represents normal function of the Akt signaling pathway, or is actually representative of an adverse event. Although we did not find P-Akt expression to be prognostic of poor survival on the breast TMA, we did not rule out a role for P-Akt in cancer development, most likely in combination with another mutation. In fact many studies have shown that constitutive P-Akt is transforming and causes mammary tumor formation when coexpressed with HER-2 or mutant polyomavirus middle T antigen [145], [74]. However, 9 of 26 (1 in 2.8) women in our study had moderate to high levels of P-Akt, and only 1 in 7 women will develop breast cancer in their lifetime. Therefore, statistically speaking, it is more likely that the levels of P-Akt we observed are due to 66 normal processes of the mammary gland. This apparently is not due to age or fibrocystic disease, as we observed a relatively random distribution of P-Akt among those variables in our study (Figure 5). Normal fluctuations in hormones which can activate Akt, such as estrogen and progesterone, or growth factors, such as insulin or leptin, may be responsible for variations in P-Akt [146], [147]. For example, changes in ductal proliferation during menstrual cycling are associated with variations in estrogen and progesterone secretions from the ovary, which could lead to increases in Akt phosphorylation. Thus, it is still unclear whether high P-Akt levels in our sample of 26 normal breast tissues is indicative of healthy mammary function or represents patients at high risk for developing breast cancer. By analyzing these same normal tissues for P T E N mutation or overexpressed RTKs, we could rule in or out adverse mechanisms for Akt activation. 4.4 Targeting Akt Signaling in Anticancer Therapy Perhaps the most important question is, since P-Akt can be expressed in both normal and tumor t issues at high levels, will inhibiting Akt in cancers result in deleterious side effects? The current dogma for Akt phosphorylation is that 1) in a normal setting, P-Akt increases and decreases in response to extra- and intra-cellular signals in a regulated manner and 2) in a mutant cell, the P-Akt signal can also be modulated, but it is often constitutive because of overexpressed RTKs or P T E N mutation. Thus, without stimulation, a normal cell would have little P-Akt, whereas a cancer cell might express very high levels. For example, the preneoplastic breast cell line, 184htrt, expresses undetectable P-Akt in log growing conditions, suggesting that it does not require P-Akt for normal growth. However, upon addition of IGF-I, one can 67 achieve a spike in Akt phosphorylation [106]. The 184htrt cells use Akt to transmit specific signals at particular times, but they don't necessarily depend on these signals for survival. Could the requirement for P-Akt be what makes a cell sensitive to its inhibition? The key role for Akt in survival signaling entertains the thought that any inhibition of this molecule in cells will result in certain death. How can a cell survive without it? It might be hypothesized that cells with low levels of P-Akt would be the most sensitive to Akt inhibition, since the target would be destroyed quickly; whereas cells with higher levels of P-Akt would be more resistant, as there is more target to inhibit. But the results of several studies using either LY294002 or more specific inhibitors of Akt phosphorylation, suggest that it is actually the cells with the most P-Akt that are most sensitive [85], [86]. Cells with low or undetectable levels of P-Akt barely respond to inhibitors of the pathway. During the development of cancer, there is a selection for mutations in pathways protecting cells from programmed cell death (including p53, Bcl2, Akt). These same mutations may simply be crutches which, if removed, leave the tumor cells particularly vulnerable to death. Drugs targeting Akt signaling are attractive because they attack cancer cells at a point of resistance to apoptosis. The recognized dependency of many tumors (albeit, not all) on P-Akt, is hopefully not shared by the normal t issues of the body. Current studies with potential Akt pathway inhibitors suggest that this could indeed be the case [85], [90]. Thus, inhibitors of Akt signaling should sensitize tumors which overexpress P-Akt to standard chemotherapies, without affecting normal cells or tumors cells which do not depend on the Akt pathway for survival. 68 4.5 Analogues of Celecoxib As Inhibitors of Akt Signaling and Cytotoxic Agents 4.5.1 Summary of Inhibitory Effects on Akt Phosphorylation Although much work has been done developing new inhibitors to Akt signaling, currently there are still no therapies that can be given to patients. Because celecoxib is already used clinically and is known to cause few side effects, this was a superb lead compound for new inhibitors of Akt signaling. Unfortunately, it was determined that while celecoxib can inhibit Akt phosphorylation, this only occurs at superphysiological concentrations. To potentially identify a new treatment for breast cancers dependent on P-Akt signaling, we characterized a new class of small molecule inhibitors derived from celecoxib. The three inhibitors, OSU-03008, OSU-03012 and OSU-03013 have the ability to inhibit PDK-1 kinase activity in vitro, with an I C 5 o reported of 16 u , M , 5 L I M and 2 u , M , respectively [104]. By treating breast cancer cells with the analogues for 2 hours, we found that, at 10 u . M , all of the inhibitors were able to significantly reduce Thr308 phosphorylation of Akt, the target site of PDK-1 . Additionally, the phosphorylation of Ser473 was abrogated. The inhibition of Ser473 phosphorylation indicated one of three things: 1) PDK-1 plays a role in Ser473 phosphorylation, 2) Ser473 phosphorylation requires T308 to also be phosphorylated or 3) the analogues act on molecules other than PDK-1 . This point will be discussed further below. The inhibition of Thr308 and Ser473 phosphorylation also corresponded to decreased Akt kinase activity against recombinant GSK-36 substrate. Further, in treated cells we were able to show that phosphorylation of the direct Akt substrate, G S K - 3 6 , was strongly attenuated. Phosphorylation of the indirect Akt substrate 4E-BP1 (which is a direct substrate of mTOR) was only somewhat downregulated, however, indicating that other pathways 69 which inactivate 4E-BP1 (and activate mTOR) are unaffected by the analogues. These data suggest that the Akt signaling pathway is functionally inhibited by the three analogues, albeit not completely. On the other hand, the parent compound celecoxib had a very mild inhibitory effect on P-Akt, despite the much higher doses used. 4.5.2 Summary of Effects on Cell Viability and Apoptosis We also examined the effect of the analogues on cell viability. For these assays we treated the cells for 24 hours, and observed that OSU-03012, OSU-03013, high dose celecoxib and LY294002 all had a negative effect on cell viability, based on an MTS cell proliferation assay. The cells responded in a dose-dependent manner to these drugs, again with the analogues potent at much lower doses than celecoxib. To determine the mechanism for a decrease in cell viability, we examined P A R P cleavage and nucleosomal fragmentation, classic results of apoptosis. We found that doses of 7.5 u.M and 10 nM, for OSU-03012 and OSU-03013, as well as LY294002 treatment, induced programmed cell death. The decrease in cell viability seen with the 5 nM dose of OSU-03012 and OSU-03013 was not due to apoptosis (at least in the in the MDA-MB-453 cells), but could indicate cell cycle arrest. Although OSU-03008 does not induce apoptosis at the doses and times examined in this study, this compound does inhibit Akt phosphorylation. Future work could analyze the effects of long term treatment (more than 24 hours) with OSU-03008, to determine if apoptosis is eventually induced, specifically in cell lines overexpressing P-Akt. 4.6 Necessity of P-Akt Inhibition for Induction of Cell Death 4.6.1 Discrepancy Between Akt Inhibition and Cytotoxicity 70 A discrepancy arises, however, between the ability of the drugs to induce apoptosis and their ability to inhibit Akt phosphorylation. For example, OSU-03008 is equally effective at inhibiting Akt phosphorylation as OSU-03012 and OSU-03013, however it has a minimal effect on cell viability. We wondered if perhaps this compound is less stable than the other two analogues. Meaning, perhaps the effects of O S U -03008 are similar to the other compounds at 2 hours, but longer time points might show a relapse in Akt phosphorylation. If OSU-03008 cannot maintain inhibition of P-Akt, it could allow the MDA-MB-453 cells to recover, whereas a sustained inactivation of P-Akt by OSU-03012 and OSU-03013 would not allow the cells to recover and apoptosis could occur. However, when P-Akt levels are compared after 24 hours between the treatment groups, no difference is detected, despite the increase in P A R P cleavage in the OSU-03012 and OSU-03013 treated cells (Figure 12B). Another discrepancy is that although LY294002 completely inhibits Akt phosphorylation at 2 hours and induces apoptosis at 12 to 24 hours, 60% of cells remain viable after 24 hours. This suggests that cell death initiated by the inhibition of PI3K and Akt phosphorylation should not be complete by 24 hours. However, the 10 u.M dose of both OSU-03012 and OSU-03013, the dose required to best inhibit Akt phosphorylation, results in almost no remaining viable cells. These observations introduced the idea that the cytotoxic properties of OSU-03012 and OSU-03013 might be due to other targets not related to Akt phosphorylation. This theory seems even more likely considering the fact that O S U -03012 and OSU-03013 kill MDA-MB-453, T47D, M C F - 7 and MDA-MB-231 cells similarly. As has been shown in other studies, cells expressing the most constitutive P-Akt levels should be the cells most sensitive to its inhibition. In fact, in our experiments 71 the MDA-MB-453 and T47D cells were the most responsive to LY294002 treatment, showing proof of principle. Thus if OSU-03012 and OSU-03013 were specific inhibitors of the Akt signaling pathway, MDA-MB-453 and T47D cells should have been preferentially sensitive over the MCF-7 and MDA-MB-231 cells. 4.6.2 Attempt to Rescue the Cytotoxic Effects of OSU-03012 and OSU-03013 by Overexpressing Activated Akt Our data seemed to indicate that inhibition of Akt phosphorylation is not the only function of the celecoxib analogues. To further explore the question of Akt inhibition as a necessary or sufficient target for analogue-induced apoptosis, a rescue experiment was performed. Transient transfection of activated Akt has been shown to rescue cytotoxic effects of drugs which act by inhibiting Akt signaling. For example, transient overexpression of myr-Akt1 decreased apoptosis induced by UCN-01 , a drug that inhibits PDK-1 [148]. Further, transient transfection of myr-Akt1 also decreased apoptosis induced by phosphatidylinositol ether lipid analogues, which inhibit Akt activation [86]. In our rescue experiment, however, transient transfection with either of two constitutively activated Akt constructs did not rescue the MDA-MB-453 or MCF-7 cells. There was not even a partial rescue, which was apparently found in the PC-3 cells by our collaborators [104]. Additionally, we did not observe a rescue when MDA-MB-453 cells stably overexpressing myr-Akt1 were treated with the celecoxib analogues (compared with empty vector expressing cells). This data further suggests that although Akt phosphorylation is inhibited by OSU-03012 and OSU-03013, this is not the primary mechanism for inducing cell death. 4.7 Other Targets for Analogues of Celecoxib 72 Based on our data, several possibilities arise, including: 1) Targets of PDK-1 , other than P-Akt, mediate the pro-apoptotic response initiated by PDK-1 inhibition, 2) The OSU-03012 and OSU-03013 analogues target other proteins in addition to PDK-1. The first scenario seems unlikely considering that expression of PDK-1 antisense oligonucleotides in human glioblastoma cells (which express mutant PTEN) only results in 10% apoptosis induction [149]. It would be an important experiment to express antisense or s iRNA to PDK-1 in our breast cancer cell line panel and observe effects on cell viability, in order to determine if selective inhibition of PDK-1 mediates apoptosis. Further, it is quite possible that OSU-03012 and OSU-03013 have additional cellular targets, which causes cell death independently of P-Akt signaling. Such targets would likely be common to all of the cell lines tested in this study, as there were only slight cell-type specific responses to the analogues. Perhaps the structure of the celecoxib analogues allows them to bind to kinase domains of multiple proteins, particularly those with kinase pockets similar to that of PDK-1 . Drugs that potently inhibit PDK-1 have previously been shown to inhibit the activity of other k inases. For example, staurosporine and UCN-01 both strongly inhibit P D K - 1 . However, when screened against a panel of 29 different kinases, these drugs also inhibit the activity of cell-cycle-checkpoint kinase-1 (CHK1), protein kinase C a (PKCa) , AMP-activated protein kinase (AMPK), cyclin-dependent kinase 2 (CDK2/cyclinA), and others with similar potency [150]. Further screening of OSU-03012 and OSU-03013, perhaps against such a kinase panel as used by Komander, et al., is necessary to determine their effects on other kinases and signaling pathways. 4.8 Usefulness of the Celecoxib Analogues as a Breast Cancer Therapy 73 We have found that the celecoxib analogues OSU-03008, OSU-03012 and O S U -03013 have the ability to inhibit Akt phosphorylation in breast cancer cells, including cells which overexpress HER-2. Thus, it is conceivable that these agents could be used in combination with other therapies that are susceptible to drug resistance due to upregulated Akt signaling. For example, HER-2 overexpressing tumors often either do not initially respond to Herceptin treatment or later develop resistance. At this point, the underlying mechanism for recalcitrance to Herceptin is not understood. One possible explanation for resistance is expression of high levels of P-Akt, possibly through IGF-1R stimulation [151]. This idea is supported by a study showing that transfection of constitutively activated Akt into BT-474 cells, a cell line known to be sensitive to HER-2 inhibition, rendered the cells insensitive to Herceptin treatment [152]. Thus, it would appear that inhibiting P-Akt could improve responsiveness to Herceptin. This is consistent with another report showing that inhibiting P-Akt with LY294002 enhances the cytotoxic effect of Herceptin [78]. Further, inhibiting P-Akt with LY294002 or a dominant negative inhibitor to the p85 subunit of PI3K also prevents the anchorage independent growth of breast cancer cell lines that overexpress HER-2 , such as the MDA-MB-453 cells [153]. The ability of the celecoxib analogues OSU-03012 and O S U -03013 to inhibit P-Akt and ultimately kill Herceptin-resistant MDA-MB-453 cells suggests their potential clinical utility. This is timely given a recent report showing that the less potent parent compound, celecoxib, did not benefit patients with HER-2 overexpressing tumors resistant to Herceptin [154]. The analogues could feasibly be used to treat any tumor that is drug resistant due to hyperactivation of the Akt signaling network. Future experiments in cell culture using OSU-03008, OSU-03012 and OSU-03013 in 74 combination with other chemotherapeutic agents, or with Herceptin, could determine the potential synergistic effects. It is also important to begin preclinical testing of the analogues in a mouse model of breast cancer. Determining the bioavailability of the compounds, as well as their capacity to inhibit Akt phosphorylation in tumors or xenographs in vivo are key objectives for future work. 4.9 Therapies for Downstream Targets of Akt: mTOR Another mechanism for treating tumors with hyperactivated Akt signaling is to target the downstream effectors of P-Akt, rather than the upstream regulators or Akt itself. In an example of this theory coming to fruition, inhibitors to the protein mTOR are already in Phase l-lll trials, exhibiting some success against a range of tumor types [155], [156]. Regulation of mTOR is complex and not fully understood, but it is linked to activity of the PI3K/Akt pathway either through direct phosphorylation by Akt on Ser2448 [65], or through Akt's inactivation of the tuberous sclerosis complex [157], [158], an inhibitor of mTOR. Regardless of the route, activation of Akt is associated with activation of mTOR [159], which is responsible for phosphorylating the further downstream proteins, ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Phosphorylated 4E-BP1 dissociates from eukaryotic initiation factor 4E (elF4E), an R N A cap-binding protein, allowing the formation of the e lF4F complex. This complex is responsible for initiating translation of mRNAs such as cyclin D1 [160], in turn regulating cell proliferation and cell cycle [161]. mTOR was first found to be inhibited by rapamycin, an anti-fungal, immunosuppressive agent isolated from the bacteria Streptomyces hygroscopicus [162]. However, because of stability and solubility issues, analogues of rapamycin were recently developed that can be delivered 75 orally or intravenously, namely CCI-779 and RAD-001. Rapamycin and its analogues have been shown to chemosensitize tumors dependent on P-Akt/mTOR signaling. For example, combining rapamycin with doxorubicin results in complete remission of mouse lymphomas which express constitutively activated Akt [163]. This observation is particularly noteworthy since these lymphomas were resistant to treatment with chemotherapy alone. Other studies have also shown that sensitivity to mTOR inhibition is dependent on an activated Akt pathway, such as through loss of P T E N [164], [165]. Further, in a mouse model of prostate cancer, mTOR inhibition with RAD-001 was able to completely reverse a prostatic intraepithelial neoplasia (PIN) phenotype generated by overexpressing activated Akt1 in the ventral prostate [166]. This work suggests that, as with other inhibitors to P-Akt signaling, mTOR inhibition is very useful for cancer intervention, but predominantly for cancers which overexpress and are dependent on this pathway. Although the integral role of mTOR in mediating P-Akt signals is now quite apparent, it remains to be seen whether inhibitors to other downstream P-Akt effectors would be quite as efficacious. 4.10 Summary. Conclusions, and Future Work In this study we have determined that Akt is moderately to highly activated in 58% of breast carcinomas. In contrast, only 35% of normal breast tissues express moderate or high levels of P-Akt. Thus, Akt is activated more frequently in cancerous versus normal breast tissue. Future objectives to expand this study include 1) Determine level of P T E N mutation and RTK activation in coordination with P-Akt expression in tumors, 2) Assess whether coordinate expression of P-Akt with either mutant P T E N or overexpressed/ hyperactivated RTKs is related to poor survival, 3) 76 Obtain matched samples of primary breast tumors with metastases to determine whether P-Akt levels change during cancer spread. Additionally, we found that levels of P-Akt in breast cancer are significantly correlated with the expression of HER-2, IGF-1R, EphA2, ILK, GFI-1 and YB-1 . Future work will determine the significance of these associations by 1) Overexpressing activated Akt or dominant negative Akt in cell culture and measuring changes in expression of the associated proteins, 2) Modulating the expression of the associated proteins and assessing changes in Akt phosphorylation, or 3) Determining if these proteins are associated on TMAs at other laboratories. We also characterized analogues of celecoxib for their usefulness in treating breast cancer. The three compounds, OSU-03008, OSU-03012, and OSU-03013, were able to inhibit Akt phosphorylation within 2 hours of treatment in cell culture. Two compounds, OSU-03012 and OSU-03013, induced apoptosis 12 to 24 hours after treatment, whereas the compound OSU-03008 did not cause cell death. All three analogues could be used in the presence of serum, whereas the parent compound, celecoxib, was severely inhibited by 5% serum. However, the ability of the analogues to inhibit Akt phosphorylation or induce apoptosis appears to be limited by cell density. We further show that the mechanism for apoptosis induction is not dependent on inhibition of Akt phosphorylation as evidenced by: A) OSU-03012 and OSU-03013 kill breast cancer cell lines with low and high levels of P-Akt similarly, and B) Overexpression of activated Akt does not rescue cells from analogue induced cytotoxicity. Future work will focus on 1) Identifying other kinases which may be inhibited by the celecoxib analogues, 2) Assessing the usefulness of combining these 77 inhibitors of Akt phosphorylation with standard chemotherapy in an attempt to bypass drug resistance, and 3) Applying the analogues to a mouse model for breast cancer, whereby bioavailability and capacity for inhibition of Akt phosphorylation in vivo can be assessed. 78 REFERENCES 1. Vogelstein, B. and Kinzler, K. W. Cancer genes and the pathways they control. Nat Med, 10: 789-799, 2004. 2. Hanahan, D. and Weinberg, R. A. The hallmarks of cancer. Cel l , 100: 57-70, 2000. 3. Minotti, G. , Menna, P., Salvatorelli, E., Cairo, G. , and Gianni, L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev, 56:185-229, 2004. 4. Abal, M., Andreu, J . M., and Barasoain, I. Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action. 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L Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A , 98:10314-10319, 2001. 166. Majumder, P. K., Febbo, P. G. , Bikoff, R., Berger, R., Xue, Q., McMahon, L. M., Manola, J . , Brugarolas, J . , McDonnell, T. J . , Golub, T. R., Loda, M., Lane, H. A., and Sel lers, W. R. mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat Med, 10: 594-601, 2004. APPENDIX Appendix 1. Buffers and Reagents 1a. 10 mM citrate buffer. pH 6.0 9 ml of 0.1 M citric acid 41 ml of 0.1 M sodium citrate dihydrate adjust pH to 6.0 1b. Tris Buffered Saline (TBS) 8 g NaCl 0.2 g KCI 3 g Tris Base deionized water to 1000 ml adjust pH to 7.4 1c. Phosphate Buffered Saline (PBS) 8 g NaCl 0.2 g KCI 1.44 g N a 2 H P 0 4 0.24 g K H 2 P 0 4 deionized water to 1000 ml adjust pH to 7.4 1d. Whole Cell Lysis Buffer 20 mM Tris (pH 7.5) 150 mM NaCl 1 mM EDTA 1 mM EGTA 1%Triton-X100 Protease Inhibitors: 2.5 mM Na-pyrophosphate 1 mM 6-Glycerophosphate 1 mM N a 3 V 0 4 1 (xg/ml leupeptin 1 mM P M S F 1e. Buffer A (Cytoplasmic Extraction Buffer) 96 10 mM HEPES (pH7.9) 1.5 mM MgCI2 10 mM KCI 1 mM EDTA 0.1% NP40 Protease Inhibitors (see whole cell lysis buffer) 1f. Buffer B (Nuclear Pre-extraction Wash Buffer) 10 mM Tris-Hcl (pH 7.2) 2 mM MgCI2 Protease Inhibitors (see whole cell lysis buffer) 1g. Buffer C (Nuclear Extraction Buffer) 0.42 M NaCl 20 mM HEPES (pH 7.9) 1.5 mM MgCI2 20% glycerol Protease Inhibitors (see whole cell lysis buffer) 1h. LPS Sample Buffer. 4X 4.00 g Glycerol 0.682 g Tris Base 0.666 g Tris HCI 0.800 g Lithium Dodecyl Sulfate 0.006 g EDTA 0.75 ml of 1% solution Serva Blue G250 0.25 ml of 1% solution Phenol Red Ultrapure Water to 10 ml Add 10 u.l of B-mercaptoethanol to 90 \i\ of 4X buffer before adding to protein samples. 1i. 10X Electrode Running Buffer 30.3 g Tris Base 144.0 g Glycine 10.0 g SDS deionized water to 1000 ml To make 1X buffer: Dilute 50 ml of 10X stock in 450 ml deionized water 1j- Stacking Gel for SDS-PAGE 97 4% acrylamide/bis 0.125 M Tris pH 6.8 0.1% S D S 0.05% ammonium persulfate 0.1% T E M E D 1k. Separating Gel for SDS-PAGE 12% acrylamide/bis 0.375 M Tris pH 8.8 0.1%) S D S 0.05%o ammonium persulfate 0.05% T E M E D 11. 10X Transfer Buffer 140 g Glycine 30.3 g Tris Base deionized water to 1000 ml To make 1X buffer (20% methanol): Dilute 100 ml of 10X Transfer Buffer in 700 ml deionized water and 200 ml of methanol. Chill before use. 1m. SATO Wash Buffer 20 mM Tris-HCl (pH 7.5) 0.2% NP40 10% glycerol 1 mM EDTA 1.5 mM MgCI 2 137 mM NaCl 1n. Akt Kinase Assay Buffer 25 mM Tris-HCl (pH 7.5) 5 mM B-Glycerophosphate 2 m M DTT 0.1 mM N a 3 V 0 4 10 mM MgCI 2 1o. Dulbecco's Phosphate Buffered Saline (DPBS) 0.2 g KCI 8.0 g NaCl 0.2 g K H 2 P 0 4 1.15 g Na2HP04 98 deionized water to 1000 ml adjust pH to 7.35 add the following and mix thoroughly: 100 mg MgCI2«6H20 133 mg CaCI2 • 2H20 1p. MTS Reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) Protect from light during preparation and storage. Add 42 mg MTS powder to 21 ml D P B S (section 5.1.15) Mix for 15 minutes. Adjust pH to 6.0-6.5. Filter sterilize through 0.2 \im filter. Store at -20°C. (stock concentration = 2 mg/ml) 1q. PMS Reagent (phenazine methosulfate) Protect from light during preparation and storage. Dissolve 92 mg P M S powder to 100 ml D P B S (section 5.1.15). Filter sterilize through 0.2 u.m filter. Store at -20°C. (stock concentration = 0.92 mg/ml) 99 Appendix 2: Antibody and Protocol Information for TMA Immunohistochemistry Protein Clone and Source Dilution Antiqen Retrieval Staininq Pattern Akt/ Protein Kinase B Cell Signaling Technologies, 9277; Beverly, MA 1 :250 Steam 30min Citrate Buffer (pH=6.0) cytoplasmic, occasionally nuclear carbonic anhydrase IX (CA9) Chia, S. K. et al. J Clin Oncol. 2001 Aug 15; 19(16) :3660-8. 1 :50 Steam 30min Citrate Buffer (pH=6.0) membranous CD3 Novocastra; United Kingdom 1 :600 Decloak 5 min, EDTA stromal T-cell CD20 DAKO; Denmark 1 :500 0.059? pronase stromal B-cell CD43 MTI, RVR Sci 1 :10 Decloak 5min TRS stromal T-cell CD68 DAKO; Denmark 1 :800 Decloak 5min TRS stromal macrophages Chromogranin A LC2H10Biogenex; San Rmon, CA 1 :1000 None cytoplasmic Clustering beta isoform Santa Cruz Biotechnology, SC-6420 goat polyclonal Santa Cruj, CA 1 :500 Steam 30min Citrate Buffer (pH=6.0) cytoplasmic cyclo-oxygenase 2 (Cox-2) CX229, Cayman Chemical; Ann Arbor, Ml 1 :100 Steam 20min Citrate Buffer (pH=6.0) cytoplasmic Cyclin E CYE05, Neomarker; Fremont, CA 1 :300 Microwave 30min Citrate Buffer (pH=6.0) nuclear E2F1 KH95, Neomarker; Fremoont, CA 1 :200 Microwave 15m in Citrate Buffer (pH=6.0) nuclear E-cadherin HECD-1, Zymed/lntermedico; Markham, CA 1 :150 Decloak 5min TRS cytoplasmic EphA2 B208; Med immune Dr. Michale Kinch, Gaithersburg, MD) 1 MOO None membranous Estrogen Receptor (ER) DAKO; Denmark 1 :100 Steam 20min EDTA nuclear Growth Factor Independence-1 (GFI-1) Dr. Lee Grimes, mouse monoclonal Institute for Cellular Therapeutics, Louisville, KV 1 :20 Steam 30min Citrate Buffer (pH=6.0) nuclear HER2/neu DAKO, A485; Denmark 1 :500 Steam 20min, TRS membranous Heat Shock Protein 27 (HSP27) Novocastra; United Kingdom 1 :800 Steam 30min Citrate Buffer (pH=6.0) membranous 100 Appendix 2: (continued) Protein Clone and Source Dilution Antigen Retrieval Staining Pattern Insulin-like Growth Factor 1 (IGF-0 Santa Cruz Biotechnology, SC-713; Santa Cruz, CA 1 :IOOO Steam 30min Citrate Buffer (pH=6.0) cytoplasmic Insulin-like Growth Factor-1 Receptor (IGF-1R) Cell Signaling Technologies, 3D21; Beverly, MA 1 :200 Steam 30min Citrate Buffer (pH=6.0) membranous and cytoplasmic Insulin-like Growth Factor Binding Protein-2 (IGFBP-2) Santa Cruz Biotechnology, goat polyclonal; Santa Cruz, CA 1 :200 Steam 30min Citrate Buffer (pH=6.0) cytoplasmic Insulin-like Growth Factor Binding Protein-5 (IGFBP-5) Santa Cruz Biotechnology, goat polyclonal; Santa Cruz, CA 1 :300 Steam 30min Citrate Buffer (pH=6.0) cytoplasmic Integrin-linked Kinase (ILK) Cell Signaling Technologies, rabbit polyclonal; Beverly, MA 1 MOO Steam 30min Citrate Buffer CpH=6.0) cytoplasmic Ki-67 MM1, Novocastra; United Kingdom 1 MOO Pressure cooker in MV, 2min nuclear Neuron-specific Enolase (NSE) DAKO; Denmark 1 MOO None cytoplasmic p53 DO-7, DAKO; Denmark 1 :400 Pressure cooker in MV, 2min nuclear Podocalyxin (MEP-21) 3D3, Dr. David Kershaw University of Michigan Ann Arbor, Ml 1 :80 Steam 30min Citrate Buffer (pH=6.0) cytoplasmic, nuclear, membranous Progesterone Receptor PR DAKO; Denmark 1 MOO Steam 20min EDTA nuclear survivin Santa Cruz Biotechnology, rabbit polyclonal, SC-10811; Santa Cruz, CA t :200 Steam 30min Citrate Buffer (pH=6.0) cytoplasmic, nuclear Y-box Binding Protein 1 (YB-0 Dr. Nelson Prostate Center VGH University of British Columbia Vancouver, BC 1 :2000 Steam 30rnin Citrate Buffer (pH=6.0) cytoplasmic, some nuclear 

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