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Characterization of genetic alterations in lung cancer Cleveland, Krista Chereen 2004

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CHARACTERIZATION OF GENETIC ALTERATIONS IN LUNG CANCER by KRISTA CHEREEN CLEVELAND B.Sc, The University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 2004 © Krista Chereen Cleveland, 2004 THE UNIVERSITY OF BRITISH COLUMBIA FACULTY OF G R A D U A T E STUDIES Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Name of Author (please print) Date (dd/rrim/yyyyi Title of Thesis: CA^QUC^C^^AOK\ < p £ Gtn^jflc^ A/J?tehohS ^ L-VtS\<=j Cot/} nj£ ^ D e 9 r e e : MaS-U>^ & A Scfd^^JZ. Year: 2~&of-D e p a r t m e n t o f ^bbUio/ocjt CTL^J LaJLorodk^ {LtzMc<^ The University of British Columbia U/ / Vancouver, BC Canada grad.ubc.ca/forms/?formlD=THS page 1 of 1 last updated: 7-Oct-04 ABSTRACT Lung cancer is the leading cause of cancer-related deaths in North America and Europe for both men and women. In order to improve the survival rate o f lung cancer, early detection and treatment approaches need to be developed. Markers of genetic instability in preinvasive stages wi l l facilitate treatment design. Conventional techniques used to identify genomic areas of instability such as microsatellite analysis and comparative genomic hybridization require amounts o f D N A that exceed the yield from preinvasive lesions. In our laboratory, we have developed a PCR-based genome scanning method called scanning of microdissected archival lung lesions ( S M A L L ) - P C R in order to detect recurrent genomic alterations. D N A was isolated from microdissected formalin fixed paraffin embedded squamous cell carcinomas, preinvasive stages and normal lung samples. Fol lowing whole genome screening with S M A L L - P C R , altered genomic segments were cloned, sequenced and localized to chromosomal regions and the known genes within these regions were identified. One region identified by S M A L L - P C R was investigated by fluorescent in situ hybridization and loss o f heterozygosity to verify the findings of S M A L L - P C R . The expression levels o f selected genes were evaluated by reverse transcription-PCR using a panel o f squamous cell lung carcinomas and paired normal samples from the same patient. Expression of a selected gene was evaluated in a developing mouse embryo as well as from lungs dissected from different embryonic stages. Attempts were made to investigate protein expression by immunohistochemistry. Sixty-four normal, preinvasive stages and tumour D N A samples originating from 16 patients were analyzed by S M A L L - P C R . Scanning the genome using S M A L L - P C R i i identified six recurrent chromosomal abnormalities detected in multiple patients. Four of the regions contain genes belonging to or interacting with the Wnt signaling pathway. Microsatellite analysis confirmed genomic instability at one of the regions, 1 lq l4 .2 , discovered by S M A L L - P C R . Expression analysis offzd4, a gene near the S M A L L - P C R alteration, verified a statistically significant altered level o f expression in tumour samples. Another region, 9q21.33 contained 2 genes that were evaluated for expression levels in tumour samples compared to normal samples, dapk and gasl were both differentially expressed when normalized against gapdh expression levels. Only dapk was differentially expressed in tumour samples when using B-actin for normalization. fzd4 expression was confirmed in the developing embryo at different time points. Expression offzd4 was also evident in lungs dissected from mice embryos as early as 11.5 days post coitum. Genome wide scanning using S M A L L - P C R has identified recurring chromosomal alterations in premalignant and malignant lesions of the lung. These alterations point to the involvement of important developmental signaling pathways in the progression o f lung cancer. i i i TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vii List of Figures viii Abbreviations x Acknowledgements xi Chapter 1: Introduction 1 1.1 Background 1 1.1.1 Lung Cancer 1 1.1.2 Diagnosis 2 1.1.3 Lung Cancer Staging 2 1.2 Molecular Basis of Lung Cancer 5 1.2.1 Molecular Alterations Associated with Non-Small Cell Lung Cancer 6 1.2.2 Chromosomal Regions Associated with Lung Cancer 6 1.2.3 Genes Associated with NSCLC 7 1.2.3.1 RAS Oncogenes 7 1.2.3.2 MYC Family of Oncogenes 8 1.2.3.3 p53 Tumour Suppressor 8 1.2.3.4 RB/pl 6INK4A Pathway 9 1.2.3.5 3p Tumour Suppressor Genes (TSGs) 10 1.3 Current Methods for Genome-Wide Scanning of Alterations 12 1.3.1 Comparative Genomic Hybridization 12 1.3.2 Loss of Heterozygosity 13 1.3.3 Scanning of Microdissected Archival Lung Lesions (SMALL)-PCR 16 1.4 Involvement of Developmental Pathways in Cancer 19 1.4.1 Wnt Signaling 19 1.4.2 Wnt Signaling in the Lung. 26 1.4.2.1 Lung Development 26 1.4.2.2 Wnt Signaling in Lung Development. 26 1.4.3 Wnt Pathway in Human Carcinogenesis 28 1.5 Thesis Hypotheses and Objectives 31 Chapter 2: Materials and Methods 33 2.1 Sample Selection 33 2.1.1 DNA Isolation 33 2.1.2 DNA Quantification 33 2.2 Identification of Genomic Alterations. 34 2.2.1 SMALL-PCR. 34 2.2.1 Cloning SMALL-PCR Alterations 36 2.2.2 Sequencing SMALL-PCR Alterations 39 2.2.3 Localization of SMALL-PCR Alterations to Chromosomal Regions 39 2.3 Fluorescent in situ Hybridization (FISH) 40 iv 2.3.1 Probe Preparation 40 2.3.2 Slide Preparation 40 2.3.2.1 Blood Metaphase Slides 40 2.3.2.2 Paraffin-Embedded Tissue Sections 41 2.3.3 Hybridization 41 2.3.4 Post-Hybridization Washes 41 2.3.4.1 Blood Metaphase Slides ". 41 2.3.4.2 Paraffin-Embedded Tissue Sections 41 2.3.5 Imaging. 42 2.4 Loss of Heterozygosity Analysis 42 2.5 Expression Analysis of Selected Genes 43 2.5.1 mRNA Isolation : 43 2.5.2 Reverse Transcription (R T) -PCR 43 2.5.3 Gene Expression Analysis using Semi-Quantitative RT-PCR 45 2.5.4 Statistical Analysis of Gene Expression 45 2.6 Expression Analysis offzd4 from Embryonic Lung Tissue 46 2.7 Immunohistochemistry 46 Chapter 3: Results..... 49 3.1 SMALL-PCR Identifies Recurrent Chromosomal Alterations 49 3.1.2 Cloning 49 3.1.3 Localizing to Chromosomal Regions 56 3.2 Fluorescent in situ Hybridization 60 3.3 Map Description ofllql4.2 64 3.4 Loss of Heterozygosity Analysis 67 3.5 Gene Expression Analysis, Semi-Quantitative RT-PCR 69 3.5.1 Frizzled 4 (fzd4) at llql4.2. 69 3.5.2 Growth Arrest Specific 1 (gasl) at 9q21.33 76 3.5.3 Death Associated Protein Kinase (dapk) at 9q21.33 80 3.6 Embryonic Expression of fzd4 83 3.7 Immunohistochemistry 86 Chapter 4: Discussion 90 4.1 Summary of Results 90 4.2 Validation for the SMALL-PCR Approach to Identify Recurrent Alterations in Lung SCC. 91 4.3 Significance of Regions Discovered by SMALL-PCR 91 4.3.1 Novel llql 4.2 Alteration in SCC of the Lung 93 4.3.1.1 Description of the DNA Analysis of llql4.2 Alteration 94 4.3.1.1.1 F ISH Analysis at l l q l 4 . 2 94 4.3.1.1.2 L O H Analysis at 1 l q l 4 . 2 94 4.3.1.2 Description of the Expression Analysis offzd4 at llql 4.2 95 4.3.2 Candidate genes at 9q21.33: gasl and dapk 96 4.3.2.1 GAS1 97 4.3.2.1.1 G A S 1 a Role in Cancer 98 4.3.2.2 DAPk 99 4.3.2.2.1 D A P k Structure 99 4.3.2.2.2 D A P k Function 101 4.3.2.2.3 D A P k a Role in Cancer 102 4.3.3 Candidate genes at 20pl3: angpt4, tcfl5, soxl2 and csnk2al 105 4.3.3.1 ANGPT4 105 4.3.3.2 TCF15 106 4.3.3.3 SOX12 106 4.3.3.4 CSNK2A1 107 4.3.4 Candidate gene at 13q33.1: ercc5 108 4.3.5 Candidate genes at 14q24.2: psenl and numb 109 4.3.5.1 PSEN1 109 4.3.5.2 NUMB. 110 Chapter 5: Conclusions.. 112 5.1 Conclusions 112 5.2 Significance of Findings 113 5.3 Future Directions. 114 References 117 Appendix I: Non-small Cell Lung Cancer TNM Staging Guide 134 Appendix II: List of Patient Profiles that were Available from the Archival Samples 135 Appendix III: Patient Cohort Available for SMALL-PCR 136 Appendix IV: Lung SCC Lesion Grading System 137 Appendix V: Representation of Microsatellite Analysis Performed at D11S1780 138 Appendix VI: Case Alteration Frequency Identified by SMALL-PCR .....139 vi LIST OF TABLES Table 1. W H O Classification of Lung Tumours 4 Table 2. Wnt Secreted Factors from Different Lineages and Their Knockout Phenotype or Other Functions Know To Date 22 Table 3. A l l Frizzled Receptors from Different Lineages and Their Binding Capabilities and Functions 24 Table 4. Chromosomal Location of Wnt Signaling Molecules 30 Table 5. List o f A l l Primers and Their Sequence Used In This Thesis 35 Table 6. Panel o f Frozen Squamous Cel l Lung Carcinoma Samples Used for L O H and Expression Analysis 44 Table 7. Summary of S M A L L - P C R Alterations 50 Table 8. Successfully Cloned and Mapped Recurrent Chromosomal Alterations Discovered by S M A L L - P C R 59 Table 9. Numerical Description o f the STS Markers Used for L O H Analysis at l l q l 4 . 2 66 Table 10. Summary of L O H Analysis Performed at 1 lq l4 .2 and Control at 9p 68 Table 11. Output Intensities from ImageQuant Software for fzd4 and gapdh R T - P C R Analysis......... 70 Table 12. Successfully Cloned and Mapped Recurrent Chromosomal Alterations Discovered by S M A L L - P C R 92 vn LIST OF FIGURES Figure i . Progression of Squamous Cell Carcinoma of the Lung 3 Figure 2. Principle of Detecting Loss of Heterozygosity (LOH) by Microsatellite Analysis 15 Figure 3. Principle of S M A L L - P C R D N A Fingerprinting 18 Figure 4. Canonical and Non-canonical Wnt Signaling Pathways 20 Figure 5. Representative Examples of Lungs Dissected from CD1 Mice Embryos at Different Time Points.... 47 Figure 6. Reamplificatioh of Alteration G using Different Primer Combinations 52 Figure 7. Colony-PCR of Alteration G to Determine if the Correct Size Fragment was Cloned 54 Figure 8. Colony Fingerprint to Illustrate Appropriate Sequence Pattern of Cloned Inserts from Alteration G 55 Figure 9. Sequence Analysis of a S M A L L - P C R Alteration as determined by NAPS 57 Figure 10. Display Screen from UCSC Genome Browser 58 Figure 11. Verifying FISH Probe on a Normal Blood Metaphase Spread 61 Figure 12. FISH Analysis of Formalin Fixed Squamous Lung Cell Carcinoma. 62 Figure 13. FISH Performed Using an Increased Concentration of Nick Translated B A C on Formalin Fixed Squamous Lung Cell Carcinoma. 63 Figure 14. Map Description of 1 lql4.2 to Illustrate STS Marker Positions and B A C Assembly 65 Figure 15. Normalized fzd4 Expression Levels Evaluated by RT-PCR. 71 Figure 16. Relative Expression Levels offzd4 Using Paired Lung SCCs and Normal Tissue from the Same Patient 73 Figure 17. Mean Relative Expression Levels offzd4 in Lung SCCs Compared to Normal Tissue using gapdh or B-actin as Housekeeping Genes 74 Figure 18. Mean Relative Expression Levels offzd4 in Lung SCCs Compared to Normal Lurig Parenchyma and Normal Lung Brushings Using gapdh or B-actin as Reference Genes... 75 v i i i Figure 19. Map Description o f 9q21.33 77 Figure 20. Relative Expression Levels o f gasl Using Paired Lung SCCs and Normal Tissue from the Same Patient 78 Figure 21. Mean Relative Expression Levels of gasl in Lung SCCs Compared to Normal Tissue Using gapdh or $-actin as Reference Genes 79 Figure 22. Relative Expression Levels o f dapk Using Paired Lung S C C s and Normal Tissue from the Same Patient 81 Figure 23. Mean Relative Expression Levels of dapk in Lung SCCs Compared to Normal Tissue using gapdh or $-actin as Reference Genes 82 Figure 24. Embryonic Expression of mfzd4 from Different Time Points of a Developing Mouse Embryo 84 Figure 25. Embryonic Expression of mfzd4 from Different Time Points o f Dissected Lungs from CD1 Mice Embryos 85 Figure 26. IHC Optimization Using Target Antigen Retrieval Solutions 87 Figure 27. IHC Antibody Concentration Optimization 88 Figure 28. I H C Negative Control Using Normal IgG Purified from Goat 89 Figure 29. D A P k Structural Protein Domains 100 ix ABBREVIATIONS A l = Al le l ic imbalance B A C = Bacterial artificial chromosome C G H = Comparative genomic hybridization CIS = Carcinoma in situ DAPI = 4', 6-diamidino-2-phenylindole D P C = Days post coitum F ISH = Fluorescent in situ hybridization F ITC = Fluorescein-5-isothiocyanate F Z D = Frizzled L O H = Loss of heterozygosity N S C L C = Non-small cell lung cancer P A G E = Polyacrylamide gel electrophoresis P C R = Polymerase chain reaction R E = restriction endonuclease S C C = Squamous cell carcinoma S C L C = Small cell lung cancer S M A L L - Scanning of microdissected archival lung lesions T S G = Tumour suppressor gene ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Wan Lam for his guidance, support and generosity throughout my journey as a graduate student. As well, the other members of the lab have always been there to discuss problems and ideas. In particular I would like to thank Baljit Kamoh for her help, patience and her ears that are always open to discussion. I would like to thank Dr. Stephen Lam for all his hard work and dedication in cancer research. The specimens he collected were the source of most data in this thesis. Thank you to Dr. James Hogge and Dr. Peter Pare at St. Paul's Hospital for their generous supply o f tissue specimens from the tissue repository. A special thanks to Dr. J.C. Cutz who collected specimens on behalf o f our lab, his laughter, knowledge and conversation made it an incredible experience in the lab. Dr. Margaret Sutcliffe has been another invaluable person throughout my experience at the C R C . The close relationship our lab has with the L ing lab is certainly an asset to those who seek it. They are kind and very generous with their time and helpfulness. As well the Vielk ind lab has brought much laughter, enjoyment and helpfulness to my project. M y committee members, Dr. Calum MacAulay and Dr. Pamela Hoodless have been excellent. They have helped in everyway possible from the collection o f data to continual ideas and time spent guiding me. I would like to thank my family for all their support, trust, guidance and friendship through every aspect o f my life. They have made it a blessing. And to my husband who I treasure and love in everyway possible. x i Chapter 1: Introduction 1.1 Background 1.1.1 Lung Cancer Lung cancer is the leading cause of cancer related deaths in North America for both men (32%) and women (25%). It is estimated in the United States that 173,000 new cases wi l l be diagnosed and 160,440 deaths from lung cancer wi l l occur in 2004 (Jemal et al. 2004). Lung cancer is a very heterogeneous disease and can be subdivided into two main categories: non-small cell lung cancer ( N S C L C ) and small cell lung cancer (SCLC) . N S C L C can be further described as large cell carcinoma (LCC) , adenocarcinoma (AdC) or squamous cell carcinoma (SCC). N S C L C comprises the majority (80%) of lung tumours and S C L C constitutes 20% of the population (Travis 2002). It is important to classify lung tumours into these categories as they have a very different outcomes as wel l as different responses to therapy. S C L C has a five year survival rate o f less than 5% and is a very aggressive form of the disease as it has often metastasized at the time of diagnosis. Treatment strategies for S C L C are both radiation therapy as wel l as chemotherapy as S C L C tends to respond to both forms of treatment. N S C L C does not usually respond as well to chemotherapy but i f caught at an early stage is typically cured with surgery by tumour resection. N S C L C has a poor long term survival rate and patients who have had complete surgical resection still only have a 40-50% five year survival rate (Scagliotti and Novello 2003). The overall five year survival rate o f lung cancer is 15%). 1 1.1.2 Diagnosis The reason for such poor outcome of the disease is that patients are not typically diagnosed until late invasive stages of the disease whereby their tumour has metastasized to other regions o f the body. Traditional methods of tumour detection such as chest radiography, sputum cytology and white light bronchoscopy are limited in their ability to detect preinvasive lung lesions. Chest radiographs have misdiagnosed 65-90% of early lung tumours (Monnier-Cholley et al. 2001). Sputum cytology sensitivity is very low and white light bronchoscopy can detect less than 40%) of carcinomas in situ (McWil l iams et al. 2002; Lam et al. 2000). More innovative methods of tumour detection such as a low dose spiral computed tomography (CT) scans, automated sputum cytology and fluorescent bronchoscopy are more effective in early detection o f lung cancer. Unfortunately, these methods are still in development and are not routinely used for the screening of lung cancer. 1.1.3 Lung Cancer Staging Squamous cell carcinoma develops through a series o f histopathological stages progressing from a normal bronchial epithelium to dysplastic degrees, carcinoma in situ, an invasive phenotype and finally to a metastatic form of the disease (Figure 1). Most efforts o f disease staging have come in the form of diagnosing invasive stages of the disease. The World Health Organization (WHO) has grouped all preinvasive forms of lung lesions into one category (Table 1) (Mountain 1997). See Appendix I for T N M staging criteria. The current staging system does not recognize a multi-step preinvasive form o f the disease whereby therapy can be most advantageous. If a lesion is detected at an early stage it has the potential to revert to a normal phenotype, with only 10% of moderate 2 A. B. C. Figure 1: Progression of Squamous Cell Lung Carcinoma from Normal Epithelium to an Invasive Carcinoma. (A) Normal epithelium. (B) Hyperplasia. (C) Squamous metaplasia. (D) Dysplasia. (E) Carcinoma in situ. (F) Invasive carcinoma. Images were generously provided by Dr. C. MacAulay, previously presented in Minna et al.(2002). 3 Table 1: WHO Classification of Lung Tumours Stage TNM Subsets 0 Tis NO MO IA T l NO MO IB T2 NO MO IIA T l N l MO HB T2 N l MO T3 NO MO IIIA T3 N l MO T1-T3 N2 MO IIIB Any T N3 MO T4 Any N MO IV Any T Any N M l Table Key T= primary tumour N= lymph node M= distant metastasis 4 dysplasias progressing to an invasive phenotype and 40-85% of severe dysplasias progressing to an invasive phenotype (Mc Wil l iams et al. 2002). Therefore, early diagnosis is the key to improving lung cancer mortality. Another problem associated with early diagnosis is in fact the diagnosis itself. If a lung lesion is detected there is little disagreement among pathologists in the diagnosis o f invasive N S C L C , unfortunately there is significant disagreement in the staging o f preinvasive lesions (Holiday et al. 1995; MacAulay et al. Unpublished). With limitations in current diagnostic imaging and problems arising with preinvasive diagnosis, genomic markers o f lung cancer progression may be more practical for cost effectiveness as well as their potential to elucidate an etiological role in cancer progression. 1.2 Molecular Basis of Lung Cancer In the last decade, there has been a shift in research focus to discover the underlying molecular changes occurring within tumour cells. The essential changes that a tumour cell must acquire to become successful are 1) self-sufficient growth signals 2) insensitivity to anti-growth signals 3) evasion of apoptotic signals 4) limitless replicative potential 5) sustained angiogenesis and 6) tissue invasion and metastasis (Hanahan and Weinberg 2000). These alterations that occur within a tumour cell are a result o f actions directly or indirectly affecting the genome of the tumour cell (Hanahan and Weinberg 2000). Examples o f which are genomic mutations which act directly to alter the genome or epigenetic factors such as D N A methylation or covalent modifications of histone proteins which act to control the genome indirectly. As a lung lesion progresses from a normal phenotype to an invasive carcinoma there is the accumulation o f genetic alterations occurring in these cells. 5 1.2.1 Molecular Alterations Associated with Non-Small Cell Lung Cancer The majority o f lung cancer cases are attributed to cigarette smoking, but puzzling is that only about 15% of smokers acquire lung cancer. Questions have arisen concerning the possibility of a hereditary form of lung cancer (Sekido et al. 1998; L i and Hemminki 2003). Are there genetic defects that predispose people to lung cancer? Assuming that important genetic alterations wi l l occur in early tumourogenesis a late stage tumour wi l l contain these alterations as well. Most studies have used late stage tumours and even tumour cell lines to identify alterations that are present within the cells. By the time a tumour has progressed to an invasive phenotype there are many alterations present. By charting al l the events people have reported in N S C L C there starts to emerge a pattern of chromosomal alteration, but unfortunately, most of the alterations are not specific to lung cancer alone and have been implicated in a wide variety o f cancer types. Those alterations specific to N S C L C and where possible specifically to S C C wi l l be discussed. The alterations fall into 2 main categories; chromosomal regions associated with lung cancer and the genes associated with lung cancer. Each wi l l be discussed in detail in the following pages. 1.2.2 Chromosomal Regions Associated with Lung Cancer From the literature virtually every chromosome arm has had evidence of alteration in N S C L C with the exception of a few (13p, 14p, 15p, 21p and 22p). There are however genetic alterations that are more prevalent and recurrent in N S C L C . Most o f these chromosomal alterations have been detected by genome wide comparative genomic hybridization (CGH) as well as loss o f heterozygosity (LOH) studies. The main chromosome arms affected in lung cancer are lp , 3p, 5q, 6p, 6q, 8p, 9p, 13q, 17p, 19p, 22q and X q (Sanchez-Cespedes 2003). Since the progression of lung cancer involves the accumulation 6 of genetic alterations, identifying early genetic alterations from premalignant lesions could yield the most important events which start the progressional stages of N S C L C . It is thought that genetic alterations affecting chromosome 3p are the earliest genetic events in the development o f N S C L C . Consistently throughout the literature there is a trend of 3p alteration which involves approximately 80% of N S C L C patients (Yokota and Kohno 2004; Braithwaite and Rabbitts 1999; Minna et al. 2002; Wiest et al. 1997; Hirsch et al. 2001). Alterations at 3p are then followed by loss of chromosomes 9p, 5q as well as 17p which are also genetic alterations that have been identified in premalignant lesions. 1.2.3 Genes Associated with NSCLC 1.2.3.1 RAS Oncogenes The family o f ras proto-oncogenes (k-ras, h-ras and n-ras) has been widely associated with N S C L C . Receptor tyrosine kinases are responsible for sending signals to the R A S protein along with interacting molecules such as SOS and G R B 2 . In its active state, R A S binds G T P . ras proto-oncogenes have intrinsic GTPase activity which hydrolyzes G T P to G D P which renders the molecule inactive. In the active state R A S begins a signal transduction cascade which includes the M A P kinase pathway to send growth signals to the nucleus. Mutations to the ras proto-oncogene render it constitutively active, a form which is not capable o f hydrolyzing G T P . Common mutations involve codon 12, 13 or 61 and are mostly associated with adenocarcinoma (20-30%) but can be found in other forms of N S C L C s (15-20%) (Toyooka et al. 2003; Sekido et al. 1998). The main mechanism for mutations in ras is G->T transversions which have been associated with tobacco smoke damage. 7 1.2.3.2 MYC Family of Oncogenes The expression of myc family proto-oricogenes (c-myc, l-myc and n-myc) is strongly correlated with cell growth and proliferation. M Y C proteins are predominantly localized to the nucleus and are transcription factors shown to recognize the hexameric D N A sequence C A C G T G when coupled with the M A X protein (Grandori et al. 2000). Gene amplification is the most common mechanism of overexpressing the M Y C family proteins, myc overexpresion is more common to S C L C with 15-30% of tumours containing this alteration but it has been associated with 5-10% of N S C L C (Mitsuuchi and Testa 2002). 1.2.3.3 p53 Tumour Suppressor P53 is a transcription factor which possesses a vast array of tumour suppressor activities. P53 activity is largely associated with response to D N A damage to ultraviolet light, irradiation and carcinogens. Some examples include regulating cell cycle progression, apoptosis, differentiation, gene amplification, D N A recombination, chromosomal segregation, and cellular senescence. It had also been shown to facilitate in D N A repair, and most recently to accelerate aging when constitutively expressed (Tyner et al. 2002). A l l o f these roles are common events involved in cancer progression. p53 inactivation has been shown in virtually all cancer types including approximately 50%) of N S C L C (Mitsuuchi and Testa 2002). The main mechanism for inactivation is missense mutations and less frequently loss o f the P53 protein by nonsense or frameshift mutations resulting in a truncated protein (Hofseth et al. 2004). As previously reported with ras oncogenes, p53 mutations are primary G - ^ T transversions due to tobacco smoke carcinogens. 8 1.2.3.4 RB/pl6INK4A Pathway Retinoblastoma (RB) is a nuclear phosphoprotein that regulates a cell entering into cell cycle from a quiescence state. Upon phosphorylation by cyclins and their cycl in depedent kinases ( C D K ) , R B is released from E2F1 transcription factor allowing for the progression into S phase of the cell cycle. cdkn2a locus on 9p21.3 encodes for two gene products, p l 6 I N K 4 A and p l 4 A R F , due to differential splicing of the transcript. Briefly, p l 6 I N K 4 A has tumour suppressor capabilities due to its role in inhibiting the cell cycle kinases (CDK4/Cyc l inD and CDK6/Cyc l inD) . Thus p l 6 I N I C 4 A is capable of disabling a cell from entering the G l phase of the cell cycle. R B and p l 6 I N K 4 A are not often deleted together in the same tumour due to their overlapping roles at the same checkpoint of the cell cycle. The rb locus is located on chromosome 13ql4 and is a frequent and early target for disruption in S C L C affecting approximately 90% of tumours (Sekido et al. 2003; Hiroshima et al. 2004). R B is not frequently affected in N S C L C (15-30%) but rather p i 6 1 N K 4 A is more commonly associated with approximately 70% of N S C L C s (Sekido et al. 2003). This suggests that this pathway is a very highly associated with all forms of lung cancer as one would expect because tumour cells require the entry into cell cycle to become successful. 9 1.2.3.5 3p Tumour Suppressor Genes (TSGs) A s previously stated, loss of 3p is thought to be the earliest and most common event in the development of lung cancer. Ninety percent o f S C L C s contain loss o f heterozygosity at 3p and consistently throughout the literature there is evidence that 80% of N S C L C s possess alterations at this chromosomal arm (Kok et al. 1987; Sanchez-Cespedes 2003; Minna et al. 2002). Several distinct regions of 3p loss have been identified, 3p l2 , 3pl4, 3p21.1-21.2, 3p21.3, and 3p24-26 (Zabarovsky et al. 2002). Within 3p l2 one T S G , robol has been studied in lung and other cancer types. X ian et al. (2001) showed through homozygous deletion of exon 2 of robol that mice frequently die at birth due to reduced breathing capabilities as well, survivors acquire extensive bronchial epithelium abnormalities including hyperplasia associated with early lung cancer. 3p l4 harbors a well documented T S G fragile histidine triad (fhif) gene. FHIT protein expression is frequently lost in lung cancer (50%) and is mostly due to epigenetic inactivation by promoter hypermethylation (Maruyama et al. 2004). Just centromeric to fhit at 3pl3 is another potential T S G known as foxpl, forkhead box P I . FOXP1 is a transcriptional repressor that was shown to have decreased m R N A expression in 51%> of lung tumour cases and more interestingly F O X P 1 was shown to be lost in histologically normal tissue surrounding a tumour (Banham et al. 2001). Lung cancer T S G region otherwise known as LUC A is the most frequently affected region in epithelial tumours. Aberrations due to benzo[a]pyrene diol epoxide (BPDE) , a product o f tobacco smoke, are more prone to affect 3p21.3 than control regions, indicating this region as a target for B P D E damage in lung cancer cases (Wu et al. 1998). LUCA (3p21.3) spans a critical region o f 630 kb which was defined by homozygous deletions in 10 lung cell lines. Since then the region has been narrowed down further to 120 kb (Sekido et al. 1998). This smaller region was identified due to a homozygous deletion in a breast cancer cell line. The similar patter of 3p loss previously seen between lung and breast cancer encouraged the authors to search breast cells lines for a homozygous deletion (Sekido et al. 1998). Mult iple candidate TSGs reside in this region but only 3 w i l l be discussed briefly in this thesis. Firstly, the calcium channel a2d2 subunit gene (cacna2d2) has decreased m R N A and protein levels in tumours which are more prominent in N S C L C (64%) than S C L C (17%) (Lerman and Minna 2000). Knockout mouse studies thus far have not revealed any predisposition for these mice to acquire tumours (Bri l l et al. 2004). Ras association domain family 1A (rassfld) is another candidate T S G within the LUCA region. Promoter hypermethylation is the main mechanism of inactivation for this gene and 70-80% of S C L C s and 30-40% o f N S C L C s are affected (Pfeifer et al. 2002). Shivakumar et al. (2002) demonstrated that R A S S F 1 A can induce cell cycle arrest by engaging the R B family cell cycle check point. These results indicated that rassfla is a T S G most likely involved in the majority of lung tumours. Lastly, semaphorin 3B and 3F (sema3b and sema3f) are well documented TSGs that reside within the LUCA region o f 3p. S E M A 3 B and S E M A 3 F secreted proteins bind to neuropilin (NP) - l and NP-2 receptors (Giger et al. 1998). These receptors also serve as co-receptors for isoforms o f vascular endothelial growth factor ( V E G F ) (Fujisawa and Kitsukawa 1998). Thus S E M A 3 B and S E M A 3 F may involve inhibition o f tumour angiogenesis through interference with V E G F function (Tse et al. 2002; Tomizawa et al. 2001). Kurok i et al. (2003) confirmed that 4 1 % of N S C L C primary tumours and 50% 11 N S C L C cell lines showed hypermethylation in the promoter region of sema3f. Sekido et al. (1996) reported that sema3b is not expressed in many S C L C (18/23) nor is it expressed in very many N C S L C (7/16) cell lines and that mutations were an infrequent event (3/40). Other candidate genes that reside on 3p are wnt5a at 3pl4.3, wntla at 3p25.1 as well as ctnnbl on 3p22.1 which encodes p-catenin. These genes wi l l be discussed in more detail in section 1.4.3. The (3-retinoic acid receptor gene (rarft) at 3p24.2, tgfrbll at 3p24.1 as well as other genes within the LUCA region which haven't been wel l define are also candidate genes on this chromosome arm. 1.3 Current Methods for Genome-Wide Scanning of Alterations A variety o f techniques are now available for genome-wide screening of alterations in copy number, structure, and D N A sequences as well as expression of genes. These include molecular cytogenetic techniques such as comparative genomic hybridization, spectral karyotyping, and multicolor fluorescent in situ hybridization. Molecular genetic techniques used for genome-wide expression screening are representational difference analysis, differential display, as well as serial analysis o f gene expression and microarray analyses. The more common methods of genomic screening wi l l be introduced. 1.3.1 Comparative Genomic Hybridization Comparative genomic hybridization (CGH) permits the detection of chromosomal copy number changes without the need for cell culturing and gives a global overview o f chromosomal gains and losses throughout the entire genome of a tumour. C G H is based on a co-hybridization o f normal and tumour D N A probes that are differentially labeled with fluorochromes to metaphase chromosomes. Slides are then visualized with an image analysis 12 system and signal intensities ratios o f the normal and tumour D N A probes are calculated. Gains or amplifications are seen as an increased fluorescent ratio while deletions or losses are seen as a reduced fluorescent ratio (Forozan et al. 1997). C G H is a powerful tool for screening the genome for imbalances but it has limitations. Firstly, the resolution o f the technique is limited by the metaphase chromosomes. Alterations are only detected when the region o f imbalance exceeds 5-10 Mb , or there is a 5-10 fold amplification of approximately 1 Mb . This technique may be beneficial in analyzing cell lines where alterations are abundant and quite often over exaggerated due to cell culturing. But identifying early genetic events in premalignant lesions or tumour suppressor genes in primary tumours is not likely using this methodology. Another limitation of the technique is that C G H typically requires 500 ng - 5 pg of D N A extracted from formalin fixed paraffin-embedded tissue for each hybridization (Harvell et al. 2004; Brandal et al. 2003). This technique would not be appropriate in analyzing premalignant lesions due to the minute amount o f total D N A (20 - 200 ng) that is typically extracted from the lesions. Due to the limitations o f C G H , the resolution it is capable o f and D N A consumption it utilizes, there is a requirement for improved genome-wide scanning procedures. 1.3.2 Loss of Heterozygosity Loss o f heterozygosity (LOH) is a P C R based technique that takes advantage o f short nucleotide repeats in the human genome. In each of our somatic cells there is a maternal and a paternal set of chromosomes. This technique requires the loci being investigated to have a different number of short nucleotide repeats between the paternal and maternal chromosomes. L O H uses primers that are specific to unique D N A that flank a region o f 13 short nucleotide repeats. Amplif ication o f both alleles wi l l result through P C R and the products are visualized on polyacrylamide gels. If the alleles are heterozygous the maternal and paternal fragments wi l l resolve on the gel. If both alleles are the same size then only one band wi l l resolve on the gel and this is termed an uninformative locus for testing purposes. B y comparing tumour and normal D N A from the same patient, allelic imbalance at a specific locus can be determined (Figure 2). Inactivation of TSGs can occur by a few mechanisms: 1) mutation occurs on one allele and the second wi ld type allele is deleted or 2) through mitotic recombination the wi ld type allele is replaced by a duplicated copy of the mutant allele or 3) hypermethylation of a promoter region. L O H is not capable of determining i f amplification or deletion at a specific locus has occurred because it is the ratio o f the two disease alleles that are compared to the ratio o f the wi ld type alleles from the same patient. Testing specific loci for allelic imbalances in multiple patients wi l l reveal regions of instability and may implicate genes in those regions that are required for the development o f carcinogenesis. L O H has been used extensively to identify novel regions of interest in lung cancer. Because it is a P C R based assay it requires the use of very little D N A . But like C G H , L O H also has its limitations. Firstly, because it is a P C R based assay it cannot tolerate normal contamination o f cells within the test material. A solid tumour population is very heterogeneous containing inflammatory cells, stromal cell as well as necrotic tumour cells. In order to ensure reproducibility o f results microdissection is mandatory, which is labour intensive and sometimes not possible with frozen tumour sections. 14 A. B. Figure 2: Principle of Detecting Loss of Heterozygosity (LOH) by Microsatellite Analysis. Polymorphic microsatellite markers are used to detect allelic imbalances at specific chromosomal regions. (A) A schematic representation of a single chromosome pair from a normal and a tumour cell. Blue arrows indicate a chromosomal region of retention. Red arrows represent a region of loss. (B) A schematic representation of the fragments as they are visualized on a polyacrylamide gel. Blue fragments show both alleles retained in both the normal and the tumour cells. Red fragments show that the normal cell contains both alleles but the tumour cell has lost one allele. 15 Secondly, L O H is inappropriate for high throughput discovery o f novel markers associated with lung cancer. The technique requires a large patient set and generates information from only one locus therefore in order to screen the entire genome would require the use of a lot o f D N A . In fact, fine mapping one region of interest is very labour intensive and requires a tremendous amount o f D N A . Furthermore, detection o f allelic imbalance depends upon the frequency of heterozygosity in the population for a locus to be informative. And lastly, there is biased towards markers that are already discovered in other cancer types. Since the technique relies on previously discovered polymorphic markers the identification o f novel regions implicated in cancer progression is rare. There is a need for new assays to be employed in the detection o f novel regions involved in lung cancer progression. 1.3.3 Scanning of Microdissected Archival Lung Lesions (SMALL)-PCR The techniques mentioned above, in particular L O H , have been successful in identifying chromosomal alterations in lung cancer. But because o f their limitations employing a method that screens multiple regions of the genome simultaneously using a minute amount o f D N A would be advantageous. S M A L L - P C R is based on the genomic scanning approach called randomly amplified polymorphic D N A ( R A P D ) - P C R (Will iams et al. 1990) and closely related to arbitrarily primed P C R (AP-PCR) (Welsh and McClel land 1990). Our lab developed S M A L L - P C R to screen formalin-fixed paraffin embedded premalignant lesions. Some o f these lesions are very small and the amount of D N A that can be extracted from them is minimal. Therefore, S M A L L - P C R was developed in order to screen multiple regions of premalignant lesions using as little as 0.5 ng of D N A . S M A L L -P C R procedure uses randomly chosen decanucleotide primers to simultaneously amplify a large number o f regions within the genome. These fragments are resolved on a 16 polyacrylamide gel to create a fingerprinting pattern specific for the primer set chosen. If tumour D N A and normal D N A from the same patient are run simultaneously a comparison of fingerprint banding patterns can be detected. Polymorphisms in the population that occur between tumour and normal D N A as wel l as between multiple patients wi l l be detected by S M A L L - P C R and result in different banding patterns (See Figure 3). Changes can also occur as relative differences in the intensity o f the fingerprinting bands due to tumour aneuploidy or as alterations that have accumulated somatically in the tumour D N A . The alterations detected by S M A L L - P C R could be amplifications or deletions of genomic material. Other chromosomal alterations that can be detected by S M A L L - P C R include translocations and inversions. There are three ways in which these alterations can be detected by S M A L L - P C R ; 1) altering the relative amplification targets or 2) by altering the distance between the two primers. By altering the relative targets by amplification, deletion or translocation the genomic material that binds the primer may no longer be present in its native position and thus the primer is no longer capable o f binding. This would result in the loss o f a fingerprint band. By altering the distance between the primers by inversion or amplification a different P C R product size could be produced and would not resolve at the same position on the S M A L L gel and again a difference would be seen. S M A L L - P C R amplifies many regions of the genome using one set o f primer pairs but i f multiple primer sets are utilized then this technique may in fact be capable o f full genomic coverage. S M A L L - P C R can be performed using minute amounts o f D N A and allows the unbiased cloning of genomic regions of instability within a tumour cell which may not have been previously examined. 17 1 2 3 4 5 6 Figure 3: Principle of SMALL-PCR DNA Fingerprinting. Red arrows represent the decanucleotides that are used as random primers to land in multiple regions of the genome. In the above example, the pair of decanucleotides used produce six PCR fragments from one chromosome. The blue fragment is used to illustrate how this technique detects regions of chromosomal loss. Six patients containing regions of different size deletions (dotted lines) are represented. The fragment generated in that region (indicated by the yellow arrow) will be amplified in 2 of 6 samples where no deletion is present. This occurs in all normal samples (N) and in tumour samples (T) from patients 1 and 6. 18 1.4 Involvement of Developmental Pathways in Cancer 1.4.1 Wnt Signaling The Wingless (Wnt) signaling pathway has crucial roles in development and disease. The Wnt pathway is a conserved pathway ranging from organisms such as the more simplistic worms to flies, fish, frogs, birds, mice and humans. Every genome from multicellular organisms sequenced to date contains information to produce components of this pathway. In humans there are nineteen Wnt secreted factors and eleven Frizzled (FZD) receptors. The Wnt ligands have been shown to bind multiple F Z D receptors and a receptor is capable o f binding multiple Wnt ligands so the number o f potential downstream targets is tremendous. For simplistic reasons Wnt signaling has been divided into 2 main pathways: 1) canonical Wnt signaling which involves P-catenin as a downstream target has been wel l described in the literature and 2) the non-canonical pathway which involves many downstream targets and is far less understood due to the complexity (Nusse 1999). Figure 4 A displays a simplified mechanism o f target gene activation by both the canonical and non-canonical Wnt signaling pathways. Brief ly the classical Wnt signaling pathway (Figure 4B) involves co-receptors lipoprotein receptor-related proteins 5 and 6 (LRP5/6) that bind to F Z D receptors upon activation by Wnt ligand binding to the cystein rich domain (CRD) on the F Z D receptor. Upon activation, the complex including A P C , Ax in and P-catenin is dissociated by G S K 3 P inhibition by Dishevelled (Dvl). This releases p-catenin from the complex and allows for the accumulation o f free (active) P-catenin in the cytosol which can then translocate to the nucleus. Upon entering the nucleus P-catenin can act as a co-transcription factor (TF) along 19 A. Canonical Wnt Signaling Wnt I Dvl I P-catenin I LEF/TCF Wnt/fi-catenin Non-canonical Wnt Signaling Wnt I Calcium PKC CamKII CaCN I NF-AT Wnt I Dvl I rhoA I JNK Wnt/JNK PCP DWnt-4 + Dvl I FAK Wnt/FAK B Figure 4: The Canonical and Non-canonical Wnt Signaling Pathways. (A) The canonical and non-canonical Wnt signaling pathways and the key downstream targets of the pathway. Type in bold is the common name the pathways are referred to as. (B) Canonical Wnt pathway components involved in p-catenin signaling. 20 with L E F / T C F transcription factors to activate target genes such as Cyc l inD, c - M Y C , V E G F , M M P s and many more (http://www.stanford.edu/~rnusse/pathways/targets.html). To add to the complexity o f the pathway there is evidence that crosstalk between the canonical and non-canonical P-catenin pathways occurs and that the cadherin pathway is affected by P-catenin signaling. P-catenin is a structural adapter protein that links the cadherins to the actin cytoskeleton in cell-cell adhesion at adherens junctions. Altering Wnt and cadherin expression is likely to lead to alterations in cell fate, adhesion, and migration, all o f which are hallmarks of cancer (Nelson and Nusse 2004). The pathway has been shown to contain both oncogenic potential as wel l as tumour suppressor potential depending on the ligand/receptor combination that is present at the cell surface. 21 Table 2*: Wnt Secreted Factors from Different Lineages and Their Knockout Phenotype or Other Functions Known to Date Gene Organism Phenotype of Knockouts or other Functions wnt-1 Mouse • Loss of midbrain and cerebellum (McMahon and Bradley 1990; McMahon et al. 1992) • Deficiency in neural crest derivatives (Ikeya et al. 1997) wnt-2 Mouse • Placental defects (Monkley et al. 1996) wnt-2b Mouse • Retinal cell differentiation (Kubo et al. 2003) wnt-3 Mouse • Early gastrulation defects due to axis formation (L iu et al. 1999) • Hair growth (Kishimoto et al. 2000) wnt-3a Mouse • Lack o f caudal somites and tailbud (Takada et al. 1994) • Deficiency in neural crest derivatives (Ikeya et al. 1997) • Loss o f hippocampus (Lee et al. 2000) • Segmentation oscillation clock (Aulehla et al. 2003) wnt-4 Mouse • Kidney defects (Stark et al. 1994) • Defects in female development (Vainio et al. 1999) • Side-branching in mammary gland (Brisken et al. 2000) • Repression o f the migration of steroidogenic adrenal precursors into the gonad (Lyuksyutova et al. 2003) wnt-5a Mouse • Truncated limbs, truncated anterior/posterior (AP) axis, reduced number proliferating cells (Yamaguchi et al. 1999) • Distal lung morphogenesis (L i et al. 2002) • Chondrocyte differentiation, longitudinal skeletal outgrowth (Yang et al. 2003) • Inhibits B cell proliferation and functions as a tumour suppressor (Liang eta l . 2003) • Defects in posterior growth of the female reproductive tract (Mericskay et al. 2004) wnt-5b Mouse wnt-6 Mouse wnt-7a Mouse • L imb polarity (Parr and McMahon 1995) • Female infertility (Parr and McMahon 1998) • Appropriate uterine patterning during development o f female reproductive tract (Mil ler and Sassoon 1998) • Delayed maturation synapses in cerebellum (Hall et al. 2000) • Promotes neuronal differentiation (Hirabayashi et al. 2004) wnt-7b Mouse • Placental development defects (Parr et al. 2001) • Respiratory failure, defects in early mesenchymal proliferation leading to lung hypoplasia (Shu et al. 2002) Continued... 22 Table 2*: Wnt Secreted Factors from Different Lineages and Their Knockout Phenotype or Other Functions Known to Date Gene Organism Phenotype of Knockouts or other Functions wnt-8a Mouse wnt-8b Mouse wnt-9a Mouse wnt-9b Mouse wnt-10a Mouse wnt- 10b Mouse • Overexpression inhibits adipogenesis (Ross et al. 2000) wnt-11 Mouse • Ureteric branching defects (Majumdar et al. 2003) • Cardiogenesis (Pandur et al. 2002) wnt-16 Mouse • Activated by E 2 A - P b x l fusion in Pre-B A L L (McWhirter et al. 1999) wingless Drosophila • Segment polarity, limb development, many others (Hays et al. 1999) dwnt-2 Drosophila • Muscle defects, testis development (Kozopas and Nusse 2002) • Pigment cells gonads (Kozopas et al. 1998) • Trachea (Llimargas and Lawrence 2001) dwnt-3/5 Drosophila • Axon guidance (Yoshikawa et al. 2003) dwnt-4 Drosophila • Cel l movement in ovary (Cohen et al. 2002) dwnt-6 Drosophila dwnt-8 Drosophila dwnt-10 Drosophila * modified from http://www.stanford.edu/~rnusse 23 Table 3*: All Frizzled Receptors from Different Lineages and Their Binding Capabilities and Functions Gene Organism Wnt Interaction Other Effects Mutant Phenotype fzdl Mouse fzd2 Mouse fzdS Mouse Wnt-4 in commissur al axons (Lyuksyuto va et al. 2003) P K C (Sheldahl et al. 1999) and P-catenin (Umbhauer et al. 2000) activation A P guidance o f commissural axons (Lyuksyutova et al. 2003) fzd4 Mouse P K C (Sheldahl et al. 1999) activation P-catenin activation with Wnt-5A (Umbhauer et al. 2000) Internalization with Wnt-5A (Chen et al. 2003) Cerebellar, auditory, and esophageal defects (Wang eta l . 2001) fzd5 Mouse Essential for yolk sac and placental angiogenesis (Ishikawa et al. 2001) fzd6 Mouse P K C activation (Sheldahl et al. 1999) Hair patterning, tissue polarity (Guo et al. 2004) fzd7 Mouse p-catenin activation with Wnt-5A (Umbhauer et al. 2000) fzd8 Mouse fzd9 Mouse Wnt-2 (Karasawa et al. 2002) fzdlO Mouse smo Mouse Arrest at somite stages with a small, linear heart tube, open gut and cyclopia. L / R asymmetry phenotype (Zhang et al. 2001) Continued... 24 Table 3*: AH Frizzled Receptors from Different Lineages and Their Binding Capabilities and Functions Gene Organism Wnt Interaction Other Effects Mutant Phenotype Drosophila wg, dwnt-2, dwnt-4 (Bhanot et al. 1996) (Wu and Nusse 2002) (Rulifson et al. 2000) Tissue polarity and segment polarity fz2 Drosophila wg, dwnt-2, dwnt-4 (Bhanot et al. 1996) (Wu and Nusse 2002) (Rulifson et al. 2000; Cadigan eta l . 1998) Tissue polarity and segment polarity fi3 Drosophila wg, dwnt-2 (Wu and Nusse 2002) (Sato etal . 1999) fz4 Drosophila dwnt-4 dwnt-8 (Wu and Nusse 2002) smo Drosophila No known Segment polarity, part o f hedgehog receptor complex modified from http://www.stanford.edu/~rnusse 25 1.4.2 Wnt Signaling in the Lung The focus of this thesis is on lung cancer and due to the complexity o f the Wnt signaling pathway; it wi l l focus on the role of Wnt signaling in lung development and its potential role in tumourogenesis. 1.4.2.1 Lung Development In a mature lung there are two main components: 1) proximal airways, which conduct air and 2) the distal airways, involved in gas exchange. The development o f which begins in mice at 9.5 days post coitum (dpc) as a lung bud from the floor o f the foregut endoderm. It then undergoes a series o f branching to give rise to the conducting airways. Alveolarization begins in the late stages of gestation and continues postnatally to give rise to millions of alveolar cells which is accompanied by the extensive vascularization in the lung. The periphery o f the lung is composed of alveolar type I and type II cells which comprise the epithelial component o f the alveoli. Type I cells make up the majority of the cell surface area (95%) and are involved in the gas exchange (CO2 and O2), between the capillaries and the alveoli. Type II cells serve multiple roles in the lung. They are the progenitor cells for type I cells, secrete pulmonary surfactant and produce molecules involved in innate host defense. Lung development is a highly regulated process and the role o f Wnt signaling in this process wi l l be examined. 1.4.2.2 Wnt Signaling in Lung Development The lung develops through an intricate network o f epithelial-mesenchymal interactions. Due to the variety o f differentiated cell types in the lung there are multiple pathways of molecular regulation (Warburton et al. 2000; Shannon and Hyatt 2004). One 26 pathway recently involved is the Wnt signaling pathway. There are at least seven fzd genes and thirteen wnt genes that are expressed in the lung throughout development (Bonner et al. 2003). fzd3, wnt-3a and wnt-4 have elevated levels o f expression in postnatal developing lungs. fzd6 and wnt-1 have elevated levels of expression in prenatal lungs (Bonner et al. 2003). wnt-7b which is expressed throughout the lung epithelium is responsible for proper lung mesenchymal growth and vascular development (Shu et al. 2002). Mice that were null for wnt-7b had hypoplastic lungs and died at birth o f respiratory failure (Shu et al. 2002). Differentiation o f type I alveolar cells was impaired in these mice and the lack ofv/nt-7b increased vascular smooth muscle cell apoptosis (Shu et al. 2002). wnt-5a knock out mice also died short after birth due to respiratory failure. The mice had the correct number of lobes but were slightly larger due to an increase in proliferation in both epithelial and mesenchymal compartments. Because wnt-5a null mice showed an increase in shh expression, it was proposed that wnt5a signaling is required for the normal downregulation of shh ( L i et al. 2002). P-catenin was shown to be required for specification o f proximal/distal cell fate during lung morphogeneis. Targeted knock out o f p-catenin during mouse lung development showed deficits in lung patterning at 13.5 dpc and lung hypoplasia at 18.5 dpc. The bronchiolar tubes were enlarged and elongated, and their constituitive cells expressed predominantly proximal epithelial markers. Epithelial cells expressing distal markers such as surfactant protein C were infrequently observed. This suggests that the deletion o f p-catenin inhibited the formation of the gas exchange region of the lung (Mucenski et al. 2003). 27 Transgenic mice that have constitutively active p-catenin had abnormal lungs composed of large air spaces lined with highly proliferative cuboidal epithelium. The authors suggest that these cells resemble differentiated cell types normally found in the intestine rather than in the lung. This result is interesting as it may be the first example o f intestinal metaplasia in the lung (Okubo and Hogan 2004). Metaplasia is often associated with the progression of epithelial tumours in particular squamous cell carcinoma of the lung. 1.4.3 Wnt Pathway in Human Carcinogenesis Adenomatous polyposis col i (ape) gene was originally discovered as the culprit o f the hereditary cancer termed familiar adenomatous polyposis (FAP) (Kinzler and Vogelstein 1996). The vast majority (80 %) o f sporatic colorectal cancers also carry mutations in the ape gene that allow for increased levels o f free P-catenin which is capable o f cell signaling. Those cases that do contain an intact ape gene 50 % of them were found to carry mutations in P-catenin gene (ctnnbl) causing the same effects as ape mutations (Kar im et al. 2004). Alterations in the components of the canonical Wnt signaling pathway are thought to be early genetic events in the progression of colorectal carcinoma. Other cancer types that have Wnt signaling aberrations are malignanat melanoma, female genital tract tumours, hepatocellular carcinoma, prostate cancer, gastric cancer, oesophageal carcinoma, pancreatic cancer, breast cancer, non-melanocytic skin tumours, anaplastic thyroid cancer, desmoid tumours, synovial sarcoma and renal cell carcinoma (Karim et al. 2004). Wnt pathway alterations have been reported in lung cancer. 5q at the ape locus is a frequent target o f L O H in lung cancer but previous studies have found no ape mutations. Ohgaki et al. (2004) reported ape mutations in 5 % of S C C and 3 % of S C L C while Wissman 28 et al. (2003) detected Wnt inhibitory factor (wif-1) reduction in 75 % of NSCLCs. dvl-3 overexpression has also been reported in N S C L C with 75 % of primary tumours showing this aberration (Uematsu et al. 2003). As previously stated loss of 3p is the most common genetic event in lung cancer. Three members of the Wnt signaling pathway have their genetic information located on chromosome 3p (Table 4), they are wnt-5a, wnt-7a and ctnnbl which encodes P-catenin. It was found that Wnt-7A and P-catenin have a positive effect on the levels of E-cadherin. Deletion of 3p would result in a loss of P-catenin and Wnt-7A which were shown to decrease the expression of E-cadherin inducing an epithelial-mesenchymal transition with increased tumourgenicity in lung cancer cells (Ohira et al. 2003). Wnt-5A has not been reported in lung tumourogenesis but has been reported in other types of cancer (Weeraratna et al. 2002; Liang et al. 2003; Taki et al. 2003; Saitoh et al. 2002; Holcombe et al. 2002). Wnt-5A has been reported to signal through the non-canonical pathway and to be an inhibitor of the canonical pathway so it has tumour suppressor potential which is expected as it resides on chromosome 3p which is frequently lost (Topol et al. 2003; Westfall et al. 2003; Slusarski et al. 1997). 29 Table 4: Chromosomal Location of Wnt Signaling Molecules Gene Chromosomal Location wnt-2b l p l 3 .2 wnt-4 lp36.12 dvll lp36.33 wntSa lq42.13 wnt-9a lq42.13 fzdl 2q33.1 fzd5 2q33.3 wnt6 2q35 wnt-10a 2q35 wnt-5a 3pl4.3 ctnnbl 3p22.1 wnt-7a 3p25.1 gsk3B 3ql3.33 dvl3 3q27.1 dkk2 4q25 lefl 4q25 ape 5q22.2 wnt-8a 5q31.2 fzd9 7q l l . 23 fzdl 7q21.13 wnt-16 7q31.13 wnt-2 7q31.2 smo 7q32'.l * Imprecisely placed in the human map Gene Chromosomal Location dkk4 8pl l .21 fzd3 8p21.1 fzd6 8q22.3 fzd8 lOp l 1.21 dkkl 10q21.1 wnt-8b 10q24.31 dkk3 l l p l 5 . 3 lrp5 l l q l 3 . 2 wnt-11 l l q l 3 . 5 fzd4 l l q l 4 . 2 lrp6 12pl3.2 wnt-5b 12pl3.33 wnt-1 12ql3.12 wnt-10b 12ql3.12 fzdlO 12q24.33 axinl 16pl3.3 cdhl 16q22.1 dvl2 17pl3.1 wnt-3 17q21.31 fzd2 17q21.31 wnt-9b 17q21.32 axin2 17q24.1 wnt-7b *(22ql3) 1.5 Thesis Hypotheses and Objectives The focus of this project was to identify genomic alterations occurring in premalignant lesions. Genetic alterations critical to cancer progression wi l l occur at early stages of carcinogenesis. Therefore, identifying these alterations wi l l be critical to understanding the molecular basis o f lung tumourogenesis and could be more beneficial for treatment strategies. The hypotheses o f this thesis are as follows: 1) High resolution scanning of tumour D N A wi l l identify multiple regions altered in lung cancer. The altered regions may be important to lung cancer progression i f they are recurrent in multiple patients. 2) If regions are important then they should contain genes differentially expressed between normal and tumour samples. 3) Genes involved in normal lung development may be differentially expressed in tumour samples. The following objectives were created in order to support theses hypotheses. The first objective o f this thesis was to perform a high density P C R based fingerprinting assay, S M A L L - P C R , to look at multiple regions of the genome simultaneously using minute amounts o f D N A isolated from archival formalin fixed paraffin embedded samples. Identifying recurrent alterations occurring in multiple is important to focus on key genetic events occurring in early stage lung cancer. Once these alterations have been identified they are cloned and mapped to genomic regions. Potential oncogenes and tumour suppressor genes within these regions identified. The second objective was to validate the findings of the S M A L L - P C R alterations by performing separate genomic assays. The initial assay performed was fluorescent in situ 31 hybridization (FISH) followed by microsatellite analysis on a separate patient set o f malignant tumours as these techniques require the use of much more D N A . The third objective o f this thesis was to evaluate the m R N A expression levels o f selected genes near the S M A L L - P C R alterations. Gene silencing or gene over expression may occur i f a gene is important to the success o f a tumour population. The fourth objective of this thesis was to determine a spatial and temporal role o f one of the genes identified to have altered m R N A expression levels in lung tumours. By defining a native role for a protein it may help to understand the advantage or disadvantage this protein wi l l possess to a tumour cell. 32 Chapter 2: Materials and Methods 2.1 Sample Selection More than 200 archival formalin fixed paraffin embedded lung samples were provided by Dr. S. Lam (British Columbia Cancer Agency [BCCA] ) . There were a number of normal, mild to severe dysplasia, carcinoma in situ (CIS) and invasive carcinomas. The majority o f samples are from the premalignant categories (>85%). The patient cohort consisted of smokers, non-smokers and previous smokers. A l l samples were histologically graded by Dr. J . LeRiche ( B C C A ) , see Appendix IV for grading system. Appendix II displays all sample profiles that were available. Serial sections were cut from each paraffin block and manual microdissection was performed by C. Dawe ( B C C A ) and A . Siwoski (previously at Brit ish Columbia Cancer Research Centre [BCCRC] ) . 2.1.1 DNA Isolation Tissue was digested in a digestion buffer (10 m M Tris; 1 m M E D T A , p H 8; 0.5 % w/v sodium dodecyl sulphate (SDS); and 50 m M NaCI) at 55°C for 72 hours with 20 pg o f proteinase K spiking every 24 hours. D N A was extracted twice with phenol/chloroform/isoamyl alcohol followed by precipitation using 10 pg of glycogen as a D N A carrier. D N A was resuspended in 10 pL of d tbO. D N A material was extracted from blood in the same manner. 2.1.2 DNA Quantification Five percent of the total D N A isolated from the samples was run out on a 1% w/v agarose gel along with known amounts of D N A . The gel was placed in 1.5 M NaCI and 0.5 M N a O H solution for 30 minutes to denature the D N A . D N A was transferred to HyBond 33 nylon membrane (Amersham Biosciences Corp, Piscataway, N J , U S A ) and allowed to dry for 30 minutes at 80°C. Random Priming Ki t (Invitrogen Life Technologies, Carlsbad, C A , U S A ) was used as suggested by the manufacturer to create a radioactive probe from 200 ng of human genomic D N A . Briefly, 200 ng of denatured D N A was labeled with: 14 m M of each dCTP , dGTP and dTTP, I X Random Priming Buffer and 25 pC i o f [<x32P] d A T P and 20 U of Klenow Fragment (Invitrogen) for 1 hour at room temperature. The probe was placed in Church's Buffer (1 % w/v B S A ; 0.5 m M E D T A ; 0.5 M N a H P 0 4 , pH 7.2; and 7 % w/v SDS) along with the nylon membrane and hybrizided overnight at 65°C. The membrane was washed in 2 X SSC for 5 minutes, in 2 X SSC and 1 % w/v SDS for 15 minutes three times and finally in 0.2X SSC and 0.1 % w/v SDS for 30 minutes two times. The membrane was exposed on Kodak X - O M A T A R autoradiography film and on a phosphoimager cassette for further analysis. The cassette was analyzed by the S T O R M (Amersham) phosphoimager and using ImageQuant 5.0 software (Amersham) to determine the concentration o f D N A . 2.2 Identification of Genomic Alterations 2.2.1 SMALL-PCR Samples were chosen for analysis from the above described selection of archival formalin fixed paraffin embedded lung blocks in which the D N A was isolated. There was no biased patient selection based on smoking, race, gender or age. Samples were chosen for S M A L L - P C R analysis that contained enough D N A to perform the technique as well at least one normal and tumour sample from the same patient were required. Oligonucleotides were purchased from Alpha D N A Inc. (http://www.alphadna.com). Primers used were a-28 and a-36 and their sequences are listed in Table 5. For each reaction, 20 picomoles (pmol) o f each primer were 34 Table 5: List of All Primers and Their Sequence Used In This Thesis Primers Reverse Sequence (5'- 3') Forward Sequence (5'- 3') a-28 gca tac ggc a n/a a-36 cct aga ccg a n/a T7 taa tac gac tea eta tag gga ga n/a T3 att aac cct cac taa agg ga n/a 9p INFA gta agg tgg aaa ccc cca ct tgc gcg tta agt taa ttg gtt D11S1780 gta tgc cac taa aca act gta gtc a ggg ate tgc age aat tac t D11S1887 cct gac att gta tct aaa cct c cct ctg tat tec cac aaa ac D11S4082 aaa tgc cat tat ttc att cc tag atg ccc ate aac taa cc D11S896 agt tea tat cca cct cac aca ate tec cct age tgt ttt gga H D A P K 1 cgc tac etc tct gtc cct tg gga aca aaa gca acg gaa a H G A P D H agg ggt eta cat ggc aac tg ggc etc caa gga gta aga cc H G A S 1 ggc gca gat aca aac agt ga ggg ttg gga agg agg aat ta H F Z D 4 aag cag ggt cca ttt cct tt caa tct ggg gga ett tea ga M F Z D 4 ccc get age aac eta tea aa ctg aag cag aca ggc atg aa M H P R T tga caa cga ttt act gaa agt gg tea gga gag aaa gat gtg att ga M G A P D H gec tct ett get cag tgt cc ggc att get etc aat gac aa 35 end-labeled with 2 uC i o f 5' [yi2P] A T P (6000 Ci/mmole; Amersham) using 1 Unit (U) o f T4 polynucleotide kinase (PNK) (MBI Fermentas, Burlington, ON) , I X T4 P N K Buffer which contained 500 m M Tr is -HCl (pH 7.6), 100 m M M g C l 2 , 50 m M D T T , 1 m M E D T A , 1 m M spermidine (MBI Fermentas) at 37°C for 1 hour and heated to 65° for 5 minutes. S M A L L - P C R reactions were then performed in a 10 pL volume containing 4 ng of D N A isolated from formalin-fixed, paraffin-embedded archival samples. The reaction also contained 2.5 U of Recombinant Taq D N A Polymerase (supplied by Dr. K. Lonergan, B C C R C ) ; 200 p M of each dN T P ; 10 m M Tr is -HCl , p H 8.3; 50 m M KC1; 2 m M M g C l 2 and 0.001 % gelatin. A l l P C R reactions were performed in a PTC-100 Thermal Cycler (MJ Research, Waltham, M A , U S A ) or iCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, C A , U S A ) for 45 cycles (94°C for 1 minute, 35°C for 1 minute and 72°C for 2 minutes). A l l reaction products were run on 7 % non-denaturing polyacrylamide gels at 800 V for 3 hours. Gels were dried for 1 hour at 80°C on a Ge l Dryer Mode l 583 (Bio-Rad Laboratories) and exposed to Kodak X - O M A T A R Radiography film. Each S M A L L - P C R gel consisted of at least one normal and one preinvasive lesion from the same patient. In each experiment, positive and negative controls consisted of D N A extracted from a frozen lung tissue and a reaction without D N A template, respectively. P C R bands that were altered between normal and preinvasive samples from the same patient were noted. P C R alterations that occurred in multiple patients were chosen for further analysis. 2.2.1 Cloning SMALL-PCR Alterations Alterations of interest were cut from the dried polyacrylamide gel and boiled in 20 pL of deionized water (dH 20) for 10 minutes to elute the D N A from the gel. A n aliquot o f the eluted sample was amplified using the same primer(s) that were used in the original 36 S M A L L - P C R reaction but were modified to contain BamH\ or Pstl restriction endonuclease sites to facilitate cloning into a plasmid vector. Two primers were used in the original S M A L L - P C R reaction and it was important to determine which combination of primers amplified the bands corresponding to the alterations chosen for further analysis. There were three possible combinations of primers that amplified the bands thus the re-amplification was done in three separate P C R reactions using each primer independently or in combination with the other. The P C R reaction was performed in a 20 uL final volume containing I X c P C R Buffer (50 m M KC1; 10 m M Tr is -HCl , p H 8.3; 2 m M M g C l 2 ; 20 pg/mL B S A ; 0.1 % v/v Triton X-100; 200 p M of each dNTP) , 10 picomoles o f each primer (or 20 pmol of one primer), 2 U of recombinant Taq D N A polymerase. A l l reactions were performed in a thermal cycler. One cycle o f 94°C for 3 minutes; three cycles of94°C for 1 minute, 35°C for 1 minute, 72°C for 2 minutes; twenty-five cycles of 94°C for 40 seconds, 62°C for 40 seconds, 72°C for 40 seconds; one cycle o f 72°C for 10 minutes followed by a hold at 4°C until the reactions were removed from the thermal cycler. P C R products were digested with the appropriate restriction endonuclease (BamHl or Pstl or both depending the terminal sequence of the fragment) and purified by excising the fragments from a 6 % polyacrylamide gel. D N A was eluted from the gel fragment by placing it in enough d H 2 0 to cover the fragment and placed at 4°C overnight. Plasmid vector, pBluescript S K + , (Stratagene, L a Jolla, C A , U S A ) was digested with either BamHl or Pstl or both and dephosphorylated and combined with the eluted fragment for subsequent ligation at 16°C overnight. Fifty percent o f each ligation was transformed into competent E. coli strain D H 5 a using the standard heat shock method (Bergmans et al. 1981). Cells were plated onto L B agar plates containing 100 pg/mL ampicil l in, I P T G and X - G a l and grown 37 overnight at 37°C. Colonies containing an insert were selected by a blue and white screening procedure. Nine white colonies and one blue colony per transformation were subjected to colony P C R reaction to verify that each clone contained the appropriate sized insert that was seen in the S M A L L - P C R gel. A l l reactions were performed in a 20 pL final volume containing a I X c P C R Buffer, 2 U of recombinant Taq D N A Polymerase, 2 pg of Ribonuclease (RNase) A , 5 pmol o f each T7 and T3 primers (see Table 5 for primer sequence) in a thermal cycler (one cycle o f 94°C for 3 minutes; thirty cycles o f 94°C for 25 seconds, 45°C for 45 seconds and 72°C for 45 seconds). Colonies that contained an insert o f appropriate size were then selected for colony fingerprinting to verify that the inserts all had the same sequence pattern. Colony fingerprinting reactions were carried out in a 10 pL volume containing a I X fingerprinting nucleotide stock (0.05 m M of each dNTP ; 1 m M ddGTP), I X fingerprinting buffer (10 m M Tr is -HCl , pH 8; 50 m M KC1, 1.5 m M M g C l 2 ) , 2.5 U recombinant Taq D N A polymerase, 1 pg RNase A , 0.5 pmol o f 5 ' [y- 3 2P] A T P labeled T7 primers (6000 Ci /mmol; Amersham). T7 primers were end-labeled with 1 U T4 P N K , I X T4 P N K Buffer, 0.33 pmol y - 3 2 P A T P at 37°C for 1 hour followed by 5 minutes at 65°C. Each colony was placed in d t ^ O and heated at 95°C for 5 minutes to lyse the cells. Colony lysate and the colony fingerprinting mixture were combined and placed in a thermal cycler for an initial cycle o f 95°C for 4 minutes; followed by 30 cycles at 95°C for 1 minute, 40°C for 2 minutes and 72°C for 2 minutes. Reaction products were then run on a 5-6 % denaturing polyacrylamide gel with 8 M urea for 50 W for 2 hours. Gels were dried on a Ge l Dryer (Bio-Rad Laboratories) for 1 hour at 80°C. Dried gels were exposed on Kodak X - O M A T A R autoradiography film. Plasmid preparations of the clones containing the 38 appropriate sequence pattern were done using the NucleoBond Plasmid M i n i Columns (BD Biosciences, Mississauga, ON) . 2.2.2 Sequencing SMALL-PCR Alterations Plasmid inserts were sequenced at the B C C R C Sequencing Service or the Nucleotide Ac id Protein Services (NAPS) at the University of British Columbia. Samples that were sequenced at the B C C R C with the Appl ied Biosystems (ABI , Foster City, C A , U S A ) Model 310 Sequencer were done using 3.2 pmol o f T7 primer, 300 - 500 ng of plasmid D N A and 4 pL of BigDye (ABI) v3.0 Terminator Chemistry in a 10 pL volume. Cycl ing conditions consisted of an initial denaturation at 95 °C for 2 minutes followed by 28 cycles at 96° C for 10 seconds, 50°C for 5 seconds, 60°C for 3 minutes and were performed in a thermal cycler. The product was then cleaned by isopropanol precipitation. Sequencing that was performed at N A P S was done with the A B I Appl ied Biosystems P R I S M 377 Sequencer using 3.7 pmol o f T7 primer, 200 ng of plasmid D N A and 4 pL of BigDye (ABI) v3.1 Terminator Chemistry in a 20 uL volume. Cycl ing conditions consisted of an initial denaturation at 95°C for 5 minutes followed by 25 cycles at 96°C for 30 seconds, 50°C for 15 seconds, and 60°C for 4 minutes performed in a thermal cycler. The product was again cleaned by isopropanol precipitation. 2.2.3 Localization of SMALL-PCR Alterations to Chromosomal Regions Sequenced clones were matched to known regions of the human genome project using bioinformatics websites such as U C S C Genome Bioinformatics B L A T at www.genonie.ucsc.edu and N C B I nucleotide-nucleotide B L A S T (blastn) at www.ncbi.nlm.nih.gov/BLAST. Chromosomal location was identified by matched results. 39 2.3 Fluorescent in situ Hybridization (FISH) 2.3.1 Probe Preparation A bacterial artificial chromosome ( B A C ) from 1 l q 14.2 was selected using the bioinformatics websites at U C S C and N C B I which was covering the region o f the fzd4 gene. The B A C was selected from the RPCI-11 library a commonly used human B A C library produced at the Roswell Park Cancer Institute. RP11-736K20 was selected from the library stored at the Genome Sequencing Centre at the B C C A . Five pg of purified B A C was nick-translated with lOpL of N ick Translation Enzyme (Vysis Inc., Downers Grove, IL, U S A ) which contained D N A polymerase I; DNase I in 50 % glycerol; 50 m M Tr is -HCl , p H 7.2; 10 m M MgSCv, 0.1 m M D T T , 0.5 mg/mL nuclease-free B S A . The reaction was in a 50 pL volume with I X N ick Translation Buffer (Vysis) which contained 50 m M Tr is -HCl , pH 7.2; 10 m M MgSC*4; 0.1 m M D T T . The reaction also contained 0.5 nmol o f Spectrum Red dUTP (Vysis), 0.5 nmol dTTP, 1 nmol of each d A T P , dGTP and dCTP and was carried out at 15°C for 2 hours. 20 pL of probe was precipitated with 2 pg human Cot-1 D N A (Invitrogen) and 4.9 mg of human placental D N A (Invitrogen) using ethanol precipitation and resuspended in d H 2 0 . 2.3.2 Slide Preparation 2.3.2.1 Blood Metaphase Slides Normal blood metaphase and interphase cells placed onto glass slides were kindly supplied by O. Ludgovski ( B C C A ) . The slides were dehydrated in a series (70%, 80%), 90%, and 100%>) of ethanol at room temperature for 2 minutes then air dried. They were treated 40 with 70 % formamide and 2 X SSC at 70°C for 2 minutes and then dehydrated in a series o f ethanol and air dried again. 2.3.2.2 Paraffin-Embedded Tissue Sections Unstained formalin-fixed, paraffin-embedded tissue was cut at 5pm sections and placed onto glass slides. Slides were placed in xylene to remove the paraffin and then dehydrated in ethanol. They were treated with 0.2 N HC1 for 20 minutes at room temperature, followed by treatment in 1 M N a S C N (Sigma, St. Louis, M O , U S A ) at 80°C. A protease treatment ensued by placing the slides in 75,000 U of pepsin (Sigma) at 37°C. Slides were visualized with propidium iodide to ensure digestion and background signals were accurate. 2.3.3 Hybridization The prepared B A C probe (RP11-736K20) as well as C E P 11 Spectrum Green (Vysis), specific for the centromeric region o f chromosome 11, was placed on the slide with Hybridization Solution (Vysis), denatured at 95°C in Slide Moat 240000 (Boekel Scientific, Feasterville, P A , U S A ) and then hybridized at 37°C overnight. 2.3.4 Post-Hybridization Washes 2.3.4.1 Blood Metaphase Slides After the overnight hybridization, slides were placed in 0.4X SSC and 0.3 % v/v NP-40 at 73°C then placed in 2 X SSC and 0.1 % v/v NP-40 at room temperature. 41 2.3.4.2 Paraffin-Embedded Tissue Sections Slides were washed in 2 X SSC and 0.3% v/v NP-40 at room temperature followed by 72 °C incubation. In both cases slides were air dried and mounted in DAPI/Anti fade (Vysis) before visualizing on the microscope (Axioscope2, Carl Zeiss, North York, ON). 2.3.5 Imaging Imaging was performed using a fluorescent lamp and 3 sets o f manual filters, DAP I (31000, Chroma Technology Corporation, Rockingham, V T , U S A ) , Texas Red (41004, Chroma Technology Corporation) and F ITC (41001, Chroma Technology Corporation). Software used for the imaging was Northern Eclipse (Empix Imaging, ON) . 2.4 Loss of Heterozygosity Analysis Microsatellite markers were identified in the 1 lq l4 .2 chromosomal region that contained the S M A L L - P C R alteration and fzd4 gene. Primer sequences were obtained from Genome Database at http://www.gdb.org. Table 5 contains a complete list o f primers and their sequences that was used in this thesis. A l l primers were ordered from Alpha D N A Inc. through orders@alphadna.com. Amplif ication o f the loci was performed in a 20 pL volume with 50 pmol o f each primer specific for the region, 2 U recombinant Taq D N A polymerase and I X c P C R Buffer. Depending on the primers used for amplification either 10 ng or 40 ng of template D N A was used in the reaction. Cycl ing began with an initial denaturation at 94° C for 3 minutes followed by 2 cycles o f 94°C for 30 seconds, 52°C for 30 seconds and 72°C for 30 seconds; 11 cycles o f 94°C for 30 seconds, 52°C decreasing by 0.5°C each cycle for 30 seconds and 42 72°C for 30 seconds; finally 27 cycles o f 94°C for 30 seconds, 46°C for 30 seconds and 72°C for 30 seconds performed in a thermal cycler. The P C R products were ran on 7 %, 8 %, 10 % or 12 % denaturing polyacrylamide gels with 8 M urea at D11S1887, D11S1780, D11S896 or D11S4082 respectively. Gels were stained with SybrGold (Molecular Probes, Eugene, OR, U S A ) and visualized on the S T O R M (Amersham) imager. 2.5 Expression Analysis of Selected Genes 2.5.1 mRNA Isolation Frozen tissue blocks containing lung squamous cell carcinomas and corresponding normal blocks from the same patient were trimmed to remove unwanted cell populations (See Table 6). R N A was isolated from the blocks using TRIzol (Invitrogen) following the manufacturers recommendations. R N A quantity and quality were evaluated and from these evaluations thirteen patients with both tumour and normal R N A of good quality and quantity were available for further analysis. 2.5.2 Reverse Transcription (RT)-PCR c D N A was created from the panel o f thirteen patients using 1 pg of total R N A , 0.5 ng of oligo dT (18) primers, I X First Strand Synthesis Buffer (Invitrogen) which contained 50 m M Tr is -HCl (pH 8.3), 75 m M KC1 and 3 m M M g C l 2 ; 0.2 pmol D T T ; 10 nmol o f each dNTP; 36.4 U RNase inhibitor (Amersham) and 200 U of Superscript II Reverse Transcriptase (Invitrogen) in a 20 pL reaction volume. The reaction was initially incubated at room temperature for 10 minutes then incubated at 45°C for 50 minutes followed by and inactivating incubation at 70° C for 15 minutes as recommended by the manufacturer. 43 Table 6: Panel of Frozen Squamous Cell Lung Carcinoma Samples Used For LOH and Expression Analysis *Patient Sample Number % Tumour Cells 1 3380 65 2 3481 30 3 3491 70 4 5853 70 5 5882 50 6 5883 60 7 5968 50 8 6034 70 9 6041 50 10 6042 50 11 6503 60 12 6515 30 13 6756 50 * Number is reference for all L O H and expression data, including figures and tables. 44 2.5.3 Gene Expression Analysis using Semi-Quantitative RT-PCR To evaluate gene expression levels P C R was performed on the panel o f thirteen patients, gapdh and B-actin were used as normalizing genes to evaluate total c D N A levels. A l l reactions were performed in a thermal cycler with an initial incubation of 2 minutes at 95°C was followed by 30 cycles of95°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute. A l l reactions contained I X c P C R Buffer, 10 pmol o f each primer 2 U recombinant Taq D N A polymerase, and 2.5 pL of c D N A from the reverse transcription reaction in a 25 pL volume. Each gene had a negative control which was P C R amplified without template D N A . A l l reactions were run on a 12 % polyacrylamide gel for at least 2 hours at 150 V . D N A was visualized by staining with SybrGreen (Molecular Probes) and imaged on the S T O R M (Amersham). P C R band intensities were calculated using ImageQuant (Amersham) software. Each patient was expression analysis was normalized with gapdh and B-actin for all genes analyzed. 2.5.4 Statistical Analysis of Gene Expression Statistical analysis involved a two tailed paired student's t-test evaluation for paired normal and tumour samples for all genes. A two-tailed homoscedastic or heteroscedastic Student's t-test was used to evaluate brushing sample means to the paired normal and tumour samples with fzd4 evaluation using both gapdh and B-actin. The F-test was used to determine i f variances were equal or unequal among the populations tested. Ninety-five percent confidence intervals were calculated for all genes. 45 2.6 Expression Analysis of fzd4 from Embryonic Lung Tissue Lungs were dissected from CD1 mice embryos at different embryonic time points (Figure 6). Once the lung was dissected it was placed immediately in TRIzo l reagent (Invitrogen). The R N A was then extracted according to manufacturer's directions along with 3 acidic phenol extractions and stored at -20°C. Reverse Transcription (RT) was performed in a 20 pL volume as previously described with the following modifications: 200 ng of random hexamers and 500 ng o f total R N A were used in the reaction for all time points. 500 ng of oligo dT primers along with 500 ng of total R N A were used on one time only, day 0.5 (19.5 dpc). P C R was performed in a 20 pL volume with 20 pmol o f gene specific primers, I X c P C R Buffer, 2 U recombinant Taq D N A polymerase and 1 pL of template from the R T reaction. A negative control for the P C R reaction was R N A that had not been reverse transcribed. Samples were run on a 12 % v/v non-denaturing polyacrylamide gel for 2 hours at 150 V . 2.7 Immunohistochemistry Immunohistochemistry (IHC) was performed using an affinity purified polyclonal antibody for mouse FZD4 (AF194) from R & D Systems Inc. (Minneapolis, M N , U S A ) . Mouse embryos, 11.5 dpc, were formalin fixed paraffin embedded at St. Paul's Hospital (Vancouver, B C ) . The slides were then sectioned at 4 p M and placed onto glass slides. The slides were dewaxed and dehydrated in preparation for staining. Optimization o f the IHC was performed with Retrievit Target Retrieval Solutions (InnoGenex, San Ramon, C A , U S A ) using al l p H solutions (2, 4, 6, 8 and 10) in the sampler kit for antigen/epitope retrieval. The slides were steamed for 30 minutes with Retrievit Solutions (InnoGenex) and then rinsed 46 Figure 5: Representative Examples of the Lungs Dissected from CD1 Mice Embryos at Different Time Points. Different magnification settings are used to display the developing lungs from the mice embryos. (A) 11.5 dpc. (B) 13.5 dpc. (C) 15.5 dpc. (D) 19.5 dpc. 47 with H2O and phosphate buffered saline (PBS). Blocking was performed using 1.5 % v/v normal horse serum (NHS) in P B S for 30 minutes and then incubated with the primary antibody, 15 pg/mL diluted in 1.5 % v/v N H S , for 60 minutes. The slides were washed in PBS and incubated with a biotinylated secondary antibody (BA1300, Vector Laboratories, Burlingame, C A , U S A ) diluted in N H S for 30 minutes. Once this incubation was complete they were washed in P B S and stained with Vectastain A B C - A P Ki t (AK5000, Vector Laboratories) for 30 minutes following the manufacturer's directions. The slides were incubated with substrate (Vector Red Substrate Ki t , 51000, Vector Laboratories) and counterstained with hematoxylin, dehydrated and mounted in Cytoseal. Slides were visualized on an Axioscope2 (Carl Zeiss). Once optimization was performed as described above the selected pH Retrievit Target Solution (InnoGenex) was used to determine i f lower concentrations of the primary antibody could be used and to test the specificity o f the primary antibody binding. As a negative control, the same concentration o f normal goat IgG (AB108-C, R & D Systems Inc.) was applied to the slides instead of the FZD4 antibody while everything else remained constant. 48 Chapter 3: Results 3.1 SMALL-PCR Identifies Recurrent Chromosomal Alterations In order to identify early chromosomal alterations such as translocations, deletions, amplifications and point mutations it is important to have a complete patient set that contains both normal as well as preneoplastic lesions and invasive tumours. Using S M A L L - P C R 64 microdissected samples were analyzed. The analysis comprised of 16 patient sets which all contained at least one normal sample and one progression stage. The majority o f the patients contained 2 or more progression stages. P C R products that were run on polyacrylamide gels were analyzed for a loss o f P C R signal seen either in the normal or cancer stages. A n alteration was deemed potentially important i f it was recurrent in more than one patient set. There were 18 recurrent alterations that were identified from the patient sets. Table 7 displays the details of all alterations including P C R fragment, alteration size from the P A G E , sample number, sample grade, whether it was a gain or loss o f signal in the cancer progression stage compared to normal, primers used to amplify the fragment, frequency o f the alteration, and chromosomal location. 3.1.2 Cloning Sixty-four recurrent alterations originating from the S M A L L - P C R gels were cloned as outlined in the Materials and Methods. Alteration G , involving 4 S M A L L - P C R fragments, is used as an example to demonstrate the cloning procedure. As estimated from the S M A L L gel, alterations G should be approximately 130 bp. Figure 6 shows the reamplification o f the D N A eluted from the fragment excised from the S M A L L gel. The reamplification was done using the original S M A L L primers but modified to contain 49 Table 7: Summary of SMALL-PCR Alterations SMALL Band *Case **Sample Grade Fragment Size Patient Frequency Gain or Loss Cloning Primers Chromosomal Location A-1 126 6.1 310 3/7 Gain 28 13q33.1 A-2 127 6.1 A-3 131 6.1 A-4 143 " 6.1 A-5 145 6.1 A-6 146 6.1 A-7 120 8.1 B-l 75 8.1 400 2/7 Gain Reamplified with different primers B-2 143 6.1 C- l 71 6.1 280 4/7 Gain 28-36 Repeat Element C-2 76 6.1 C-3 143 6.1 C-4 144 6.1 C-5 145 6.1 C-6 120 8.1 D-l 160 3.1 220 3/7 Loss 28-36 Repeat Element D-2 85 3.1 D-3 121 1 E-l 84 6.1 170 3/7 Gain 28-36 l lq l4 .2 E-2 20 6.1 E-3 120 8.1 F-l 81 1 160 2/7 Loss 28-36 No match in database F-2 92 3.2 F-3 162 3.2 G- l 71 6.1 130 4/7 Gain 28-36 14q24.2 G-2 84 6.1 G-3 75 8.1 G-4 146 6.1 H-l 85 6.1 120 4/7 Gain 28-36 2p25.3 H-2 131 6.1 H-3 75 8.1 H-4 145 6.1 1-1 69 6.1 100 4/7 Gain 28-36 Repeat Element 1-2 84 6.1 1-3 146 6.1 1-4 128 6.1 J- l 6 6.1 320 4/9 Gain 28 13q33.1 J-2 51 8.5 J-3 14 6.1 J-4 15 6.1 J-5 16 6.1 J-6 18 6.1 J-7 62 8.1 Continued... 50 Table 7: Summary of SMALL-PCR Alterations SMALL Band *Case **Sample Grade Fragmen tSize Patient Frequency Gain or Loss Cloning Primers Chromosomal Location K-1 4 6.1 K-2 8 6.1 K-3 10 6.1 250 2/9 Gain 28-36 20p13 K-4 28 6.1 K-5 3 6.1 L-1 10 6.1 220 2/9 Gain Reamplified with L-2 87 6.1 different primers M-1 8 6.1 M-2 50 3.1 120 4/9 Gain 28-36 Ligation Failed M-3 15 6.1 M-4 62 8.1 N-1 6 6.1 N-2 10 6.1 N-3 28 6.1 110 4/9 Gain 28-36 No match in N-4 153 8.1 database N-5 87 6.1 N-6 18 6.1 0-1 151 1 0-2 161 1 150 3/9 Loss 28-36 9q21.33 0-3 64 3.1 0-4 86 1 P-1 160 3.1 770 2/7 Loss 28-36 Ligation failed P-2 85 3.1 Q-1 132 1 Q-2 147 1 530 3/7 Loss 28-28 E. coli match Q-3 92 3.2 R-1 143 6.1 460 2/7 Gain 28-28 Ligation failed R-2 120 8.1 *Case number refers to Appendix II and III. A description of the patient history as well as tumour grade is present in these appendices. Appendix VI displays another summary o f the S M A L L - P C R alterations in terms of biopsied site, sample alteration frequency and grade o f the lesion. * *See Appendix IV for lung S C C grading. 51 Figure 6: ^amplification of Alteration G Using Different Primer Combinations. Different primer set combinations determine if bands excised from the SMALL-PCR polyacrylamide gel were amplified using the same primer combination. G1-G4 represents the fragments seen in four different samples. 28 is the a-28 primer that was used and 36 is the a-36 primer sequence that was used. Combinations of the primers are used to determine which primer set amplified the fragment alteration (G) in each case. Here it is evident that 28:36 reamplified all G fragments thus the cloning procedure is continued. Arrow is at the expected size of the fragment. 100 bp ladder was used as a size marker (L). 52 restriction endonuclease recognition sites (BamHI and PstI) at the 5' end to facilitate cloning of the fragment into the vector. If all fragments are reamplified with the same primers in all cases then the cloning procedure continues. The fragments are then digested with the appropriate restriction enzymes (BamHI or PstI or a combination o f the two) and then purified on a polyacrylamide gel. The P C R products matching the appropriate size are excised from the gel and the D N A is eluted. The fragments are ligated into an appropriately digested and dephosphorylated pBluescript p S K + vector containing a p-galactosidase reporter gene and ampicil l in resistance gene. Ligated products are then transformed into competent E. coli strain D H 5 a cells by heat shock. At least 7 white and 1 blue colonies were selected for colony P C R to ensure that the appropriate size fragment is cloned into each colony. Figure 7 shows an example o f the colony P C R for alteration G . The primers are specific for the vector thus an addition o f 168 bp of vector length plus the expected size of the fragment, in this case is 130 bp. Together a fragment o f 298 bp is expected for the colony P C R from alteration G . Notice in Figure 7 that not all colonies selected give the appropriate size fragments so those colonies would not be selected for further evaluation. A l l colonies that were confirmed to have the appropriate size fragments were then tested for correct sequence pattern using colony fingerprinting (FP) technique described in the Materials and Methods. Figure 8 displays an example o f al l G fragments clones that were selected to have the appropriate size inserts as determined by the colony P C R . For this fragment all clones display the same pattern on the colony FP denaturing acrylamide gel therefore all colonies wi l l contain the same sequence information. A control vector without insert is run as a guide to determine where the vector sequence ends and where the clone insert begins. 53 L G l G2 L G3 G4 2<ww &emm sea • e e s s —* I •a W it l Figure 7: Colony-PCR of Alteration G to Determine if the Correct Size Fragment was Cloned. At least 8 clones are chosen to evaluate the insert fragment size. 100 bp ladder is used as a size maker (L). Primers used for colony PCR produce a fragment that is larger than the insert. Negative controls (-ve) represent the size of the fragment produce from the vector alone without insert which is 168 bp. The expected size of the cloned G fragment was 300 bp (including vector). It is evident that some of the clones from G l and G2 either contain no insert or contain the inappropriate sized insert. These clones would be discarded and no longer evaluated. 54 Figure 8: Colony Fingerprint to Illustrate Appropriate Sequence Pattern of Cloned Inserts from Alteration G. A positive control (+ve) of vector alone is run to determine where the vector sequence ends and thus the insert sequence begins. A l l clones from fragments G1-G4 have the same sequence pattern. Any one clone can then be chosen for sequencing analysis. This technique reduces the cost of sequencing multiple clones. 55 One or two representative colonies that contain the appropriate size insert as well as the same sequence pattern are grown for plasmid purification. Purified plasmids are then sequenced at Nucleic Acids Proteins Unit (NAPS) at the University of British Columbia or at the BCCRC Sequencing Service. Figure 9 shows the sequence output from NAPS of fragment G. The restriction endonuclease sites are located in the sequence and between them is the sequence from the S M A L L - P C R alteration. 3.1.3 Localizing to Chromosomal Regions A l l cloned sequences were matched to known regions of the human genome project using bioinformatics websites such as B L A S T at NCBI or at the Human Genome Browser at the University of Southern California (UCSC). There is no advantage of using one database over the other as they both use the same high throughput sequenced draft series. Figure 10 shows a display screen from UCSC which aligns the cloned query sequence with known sequences of the human genome project. Table 8 is a summary of the S M A L L - P C R fragment that localized to known regions of the human genome. Of interest is that 2 S M A L L - P C R fragments sets from alterations A and J localized to the exact same chromosomal region at 13q33.1. These fragments were identified from 2 separate gels indicating that S M A L L - P C R is a reproducible technique when the same primers sequences are used. Appendix IV displays a summary of the S M A L L alterations in terms of sample frequency of alteration, biopsied site, grade of lesion as well as localization to chromosomal regions. Known genes 1 Mb on either side of the S M A L L - P C R were evaluated for their oncogenic or tumour suppressor potential from the literature. The genes selected to be of interest due to their tumourogenic potential and the actual number of genes within 1 Mb on either side of the S M A L L - P C R sequence is displayed in Table 8. 56 T N C T 1 / A G G N N C C G G T C C G G N N T C C C C T C G A G G C C G A C G G T A T C G A T A A G C T T G A T A T C G A A T T 1 C T G C A G ; C A T A C G G C A T A G C A C A T A G G G G A T C A T T T A G T C A A A G C T G T C T G A G G A T C A C A A T G A T T G G G C « B 9 B I B B 1 1 B 1 ? 8 1 3 B A G G G G C T C C C A T C C T T C C T C C T G A C A G G A T G G A G C C T G A C A G T C A A G G T C A A G C A T C 1 « B 1 S B 1 6 8 1 7 B 1 A B 1 < M A C C T C C G A G G T G A G A G A A G G C A C G G G T T A G A C A C A G C A T C G G T C T A G G | G G A T C C | A Figure 9: Sequence Analysis of a SMALL-PCR Alteration as determined by NAPS. Restriction endonuclease recognition sites used initially to clone the fragments are located (marked by bold boxes). Between these regions is the sequence of the alteration identified by SMALL-PCR. 57 UCSC Genome Browser on Human May 2004 Assembly move zoom m f ^ W W f ^ l z o o m o u t fl^|17|flQ^ position chrl 1.85,700.000-87,700.000 ; 2,000,001 bp image width; 900 U"™P aesaeeeel S7eae9«9l Chromosome Bands L o c a l i z e d FISH Mapping Clones 11Q14.2 RefSeq Genes Base P o s i t i o n Chromosome Band KSPC138 FLJ23514 HC3 PRSS23 rzw FT—1221 M WW3S CTSC CT5C move start Click on a feature for details Click on base position to zoom in around cursor. Click on left mini-buttons for track- move end [*j 2.0 Q specific options BfclT 0 reset all 11 hide all | Chromosome 0 Guidelines 0 Labels: left 3 center E 1 r e f r e s h Use drop down controls below and press refresh to alter tracks displayed. Tracks with lots of items will automatically be displayed in more compact modes Figure 10: Display Screen from UCSC Genome Browser. A web based genome bioinformatics site that contains reference sequences and working draft assemblies for a large collection of genomes. Database facilitates the localization a query sequence of unknown chromosomal location to a specific region of the genome. 58 Table 8: Successfully Cloned and Mapped Recurrent Chromosomal Alterations Discovered by SMALL-PCR Chromosome Frequency # Genes within 1 Mb Candidate Genes 2p25.3 4/7 Patients 4 None identified 9q21.33 3/9 Patients 4 gasl dapk l l q l 4 . 2 3/7 Patients 5 fzd4 13q33.1 7/16 Patients 7 ercc5 14q24.2 4/7 Patients 10 psenl numb 20pl3 2/9 Patients 18 angpt4 csnklal tcfl5 sox 12 59 3.2 Fluorescent in situ Hybridization Verif ication o f S M A L L - P C R alteration using 1 lq l4 .2 as an example was initially done by F ISH to illustrate that S M A L L - P C R as a useful tool in identifying chromosomal abnormalities. F ISH was performed on normal metaphase nuclei as previously described in the Materials and Methods section to verify probe specificity and localization. It is important to localize the probe to metaphase chromosomes to ensure the background signals are low and the designed probe localizes to the correct chromosome at the appropriate region. A commercial centromeric probe for chromosome 11 (CEP11) ensures that the probes is localizing to the appropriate chromosome. As seen in Figure 11 normal blood metaphase cells are hybridized with nick translated B A C (RP11-736K20) and CEP11 . Both probes are localized to the same chromosome and gave distinct signals specific to chromosome 11. Once the verification o f the probe is completed the next question was whether there was an observable gain or loss o f the B A C probe in squamous cell tumours o f the lung to verify the alteration initially seen in with the S M A L L - P C R . Both probes were then hybridized to multiple formalin fixed, paraffin embedded tumour sections from multiple patients. The results o f the tissue hybridization were inconclusive. With all sections hybridized the red probe (RP11-736K20) showed very weak signal i f at all. The treatment and hybridization were working wel l because in all cases the green probe (CEP11) gave strong signals in most of the cells analyzed. It was expected that some cells would give no signals due to tissue sectioning but for most cells analyzed the red probe was questionable. Different concentrations of nick translated B A C resulted in the same inconclusive results. Figures 12 and 13 illustrate common examples o f the hybridization on tissue sections. 60 Figure 11: Ver i fy ing F I S H Probe on a Normal Blood Metaphase Spread. The red probe is nick translated RP11-736K20 B A C and the green probe is CEP11 centromeric probe for chromosome 11. This image displays the specificity and localization for the test B A C RP11-736K20 in red to the appropriate chromosomal location. Nuclei and metaphase chromosomes are counterstained with DAPI . 61 Figure 12: FISH Analysis of Formalin Fixed Lung Squamous Cell Carcinoma. Green probe is CEP11 centromeric probe and the red probe is a nick translated B A C , RP11-736K20. Strong signal in most nuclei for green cetromeric probe but absence of red probe indicates hybridization is functional but a problem with signal intensity of the red probe. 62 Figure 13: FISH Performed Using an Increased Concentration of Nick Translated BAC on Formalin Fixed Lung Squamous Cell Carcinoma. Both the CEP11 centromeric probe (green) and nick translated B A C RP11-736K20 (red) are hybridized to the tissue. There is evidence of strong signal with the green probe but absence of the red probe. 63 3.3 Map Description of llql4.2 Short nucleotide repeats are scattered throughout the genome, STS markers or microsatellite markers take advantage of this fact. Unique D N A on either side of the repeat is used as primer landing sites. Microsatellite analysis requires that the repeat segment from each homologous chromosome be of different length or heterozygous to be informative in order to detect L O H . For example, i f both maternal and paternal chromosomes have the same length o f repeats, the loci being tested wi l l be uninformative as both chromosomal fragments wi l l resolve at the same distance on the gel. If both alleles are o f different size then they wi l l resolve at different positions on the gel and a conclusive analysis can be performed. The analysis wi l l either result in retention or allelic imbalance. Retention results when both copies o f the homologous chromosome are present in the test D N A . Al le l ic imbalance occurs when one allele is represented at different intensities when comparing the test D N A to the control D N A . Al le l ic imbalance can be either allelic amplification or deletion in the test D N A compared to the control D N A . Another possible result is a homozygous deletion whereby both copies of the allele at the test loci are lost compared to the control D N A and no band is resolved on the gel. STS markers at 1 l q l 4 .2 were used to validate the genomic alterations seen through S M A L L - P C R . Figure 14 displays a detailed map of the region from the U C S C Genome Browser to illustrate the genomic position o f the STS markers used for L O H analysis. Figure 14 also displays the S M A L L - P C R alteration, the genes at 1 lq l4 .2 as well as the assembly o f B A C s covering the region. The B A C coverage displays the genomic regions of the B A C chosen for F ISH analysis. Table 9 illustrates the numerical genomic position, the expected size and percentage of heterozygosity within the human population o f the STS markers. 64 • - -in m i • ajfc. Dl 131887 D11S896 D13S4082 D11S1780 SMALL-PCR A P 9 9 2 4 9 2 . 3 ftF991331.5 MP993959.2 HP 989654 . 4 RP11 -736K23 O P 492967 2 ftF989-31 1 . 4 RP993497.2 n p e * i 7 & 4 5 ftpaee75e.6 flP99333S 2 RP995436 . 1 ftP M l 642 4 ftC 9 119SS .Q ME3H-fH~ 865889991 8 7 9 « 9 9 8 9 | Chromosome Banas L o c a l i z e d Py F I S H mapping C l o n e s l l q l 4 , 2 STS M a r k e r s on G e n e t i c <biue> and R a d i a t i o n H y & r i d <Biacn> Maps Vour Sequence f r o m BLflT s e a r c h • Known Genes B a s e d on SHISS-PROT, TrCMBL, BRMft, and R e f S e a -4 S F W E i * F2D+ 4 ACSMfelly f rom F r a-sd^ei-.ts 0.5 Mb Figure 14: Map Description of llql4.2 to Illustrate STS Marker Positions and BAC Assembly. The map displays the genes in the region (light blue) relative to the genomic position of the S M A L L - P C R alteration (red). The B A C assembly (brown) that covers the region displays the location of the B A C chosen for FISH analysis. The STS markers chosen for the microsatellite analysis (green) were on either side o f the S M A L L - P C R alteration. 65 Table 9: Numerical Description of the STS markers used for LOH analysis at llql4.2 STS Marker Genomic Position (bp) *Expected Size (bp) Heterozygosity %in population D11S1887 86,117,329 253-279 75 % D11S896 86,393,981 169-183 7 4 % D11S4082 87,006,674 125-131 6 4 % D11S1780 87,376,476 173-191 74 % * Expected size refers to fragment length of polymorphism in the human population 66 3.4 Loss of Heterozygosity Analysis Microsatellite analysis is a well established method of assessing allelic imbalance on a locus-specific basis. Many studies have identified regions of chromosomal instability in lung cancer using microsatellite markers distributed throughout the genome. Due to the quantity limitations of the microdissected material that was originally used in the S M A L L - P C R analysis, another panel o f D N A was extracted from fresh frozen tissue. The panel consisted of 13 squamous cell carcinomas and paired normal samples from the same patient. The frozen blocks were trimmed to remove unwanted cell populations from the perimeter o f the sample but were not microdissected to isolate specific cell populations. Table 6 from the Materials and Methods section describes the percentage of tumour in each case. Al le l ic imbalance is defined as a difference in the P C R signal ratio o f the paternal and maternal alleles o f the normal D N A compared to the tumour D N A . Table 10 describes the summary of results for all loci that were tested. The summary includes an STS marker at 9p, INFA, which was used as a control for D N A quality. As seen in Table 10, only one patient showed allelic imbalance at D11S1887. There were 7 patients that showed a potential homozygous deletion at one or more loci. A homozygous deletion was defined as showing no P C R amplification at a locus but did show P C R amplification at the control locus. Eight patients demonstrated retention o f both alleles at one or more loci and 1 patient was non-informative at all loci tested. See appendix V for a representation o f a denaturing polyacrylamide gel. 67 Table 10: Summary of LOH Analysis Performed at llql4.2 and Control at 9p. Patient % Tumour D11S1887 D11S896 D11S4082 D11S1780 INFA (control) 1 65 N l HD? HD? GOOD 2 30 N l HD? R N FAINT 3 70 N l R HD? R GOOD 4 70 Al N l N l GOOD 5 50 GOOD 6 60 HD? N l NI HD? GOOD 7 50 HD? HD? HD? HD? GOOD 8 70 NI N l NI NI T FAINT 9 50 Nl R NI HD? GOOD 10 50 N l N l HD? HD? GOOD 11 60 NI R GOOD 12 30 N FAIL N FAIL NI " N FAIL 13 50 N FAIL N FAIL NI NI N FAIL TABLE KEY AI= Allelic imbalance HD? = Potential homozygous deletion R = Retention NI = Non-informative N = Normal T = Tumour Color code: Blank background indicates non-informative alleles. Green background indicates that both copies of the chromosome are still present (retention). Red background indicates allelic imbalance. Orange background indicates a potential homozygous deletion. 68 3.5 Gene Expression Analysis, Semi-Quantitative RT-PCR S M A L L - P C R identified chromosomal regions altered in squamous cell lesions. Potential oncogenes or tumour suppressor genes in those regions were identified. To test whether there was altered expression o f messenger R N A (mRNA) of selected genes, R N A was isolated from a panel (see Table 6) o f tumour and corresponding normal frozen tissue. The R N A was reverse transcribed using oligo dT primers to isolated m R N A with poly A tails from the remainder o f the R N A , transfer R N A ( tRNA) and ribosomal R N A ( rRNA). Expression analysis was performed using two reference genes, glyceraldehyde-3-phosphate dehydrogenase (gapdh) and fi-actin, for normalization of c D N A quantity. 3.5.1 Frizzled 4 (fzd4) at llql4.2 Expression analysis was performed as described in the Materials and Methods section. The first S M A L L - P C R region that was selected to test altered expression o f the genes in the region was 1 lq l4 .2 . This region was selected first was because it contained the most upstream gene in the Wnt pathway, the transmembrane receptor Frizzled 4. To identify changes in m R N A expression in tumour samples P C R band intensities were measured using ImageQuant Software (Amersham Biosciences). Table 11 displays the output signal intensities from ImageQuant that were used in the analysis. The signal intensities for each case were normalized by dividingfzd4 intensity by either gapdh or $-actin intensity. Figure 15A and Figure 15B display the normalized expression for both the normal and tumour samples for each patient normalized with gapdh and $-actin used as reference genes. 69 Table 11: Output Intensities from ImageQuant Software for fzd4 and gapdh RT-PCR Analysis. Sample* Intensity Area** gapdh N l 1317143 625 fzd4 N l 627952 625 gapdh T l 3796735 625 fzd4T\ 610357 625 gapdh N 2 3898811 625 fzd4 N 2 2708101 625 gapdh T2 5090973 625 fzd4 T2 1134536 625 gapdh N3 3523901 625 fzd4 N3 1977545 625 gapdh T3 5550593 625 /z<#T3 694717 625 gapdh N 4 3858926 625 ./zc# N 4 3270375 625 gapdh T4 6628239 625 T4 1262814 625 gapdh N5 3776845 625 N5 2439097 625 gapdh T5 3457196 625 ./zaW T5 1142349 625 gapdh N 6 3398577 625 /zfiW N 6 4156920 625 gapdh T6 5261994 625 /z<# T6 1761585 625 gapdh N 7 3677512 625 / z J 4 N 7 2627520 625 gapdh Tl 4787196 625 fzd4 T7 1437121 625 Sample* Intensity Area** gapdh N8 4054041 625 fzd4 N8 2405045 625 gapdh T8 5585845 625 fzd4 T8 1935189 625 gapdh N 9 3436043 625 fzd4 N 9 2774688 625 gapdh T9 4360986 625 fzd4 T9 898954 625 gapdh m o 2813812 625 fzd4 N10 1499098 625 gapdh T10 4799216 625 fzd4 T10 1495276 625 gapdh N i l 2647981 625 fzd4 N i l 2138073 625 gapdh T l 1 4095403 625 fzd41\\ 2146249 625 gapdh N12 1570827 625 fzd4 m 2 1249212 625 gapdh T12 4672711 625 fzd4T\2 1197040 625 gapdh N13 3137214 625 fzd4m3 1380276 625 gapdh T13 3540485 625 / z t#T13 750753 625 * Sample number refers to Table 6 from the Materials and Methods section * * Area refers to the total area on the polyacrylamide gel that was calculated for pixel intensity. For analysis it is important that all areas are equal. 70 A. 1.4 1.2 i 0.8 I 0.6 • E 0.4 0.2 B. [ • Normal i I Tumour n : i 1 1 . i i L . 1 1 1 111 I i l l I i i i i i i i i 1 2 3 4 5 6 7 8 Sample 1 1! 1 1! 9 10 11 12 13 > 1.4 1.2 1 4^0.8 • Normal • Tumour 1 2 3 4 5 6 7 8 9 10 11 12 13 Sample Figure 15: Normalized fzd4 Expression Levels Evaluated by RT-PCR. (A) Expression levels are normalized with gapdh as a reference gene. (B) Expression levels are normalized with fi-actin as a reference gene. Blue bars represent the normal tissue expression and the red bars represent the tumors tissue expression offzd4. Sample number refers to Table 6 for all expression charts. 71 respectively. Expression of each gene is normalized by dividing the intensity o f the gene o f interest by the reference gene for both normal and tumour samples. Figure 15A and Figure 15B illustrate that using two different genes, gapdh and $-actin respectively, to normalize expression levels gives different expression ratios. In order to display differences between the normal and tumour sample expression the tumour normalized expression is then divided by normal expression which has been normalized as well , see Figure 16A and Figure 16B for gapdh and fi-actin respectively. Because the samples were not microdissected, the tissue heterogeneity is considerable for some samples (see Table 6). Even with the degree of tissue heterogeneity present in most samples, Figure 16A displays a decrease offzd4 expression in all patients' tumour samples when gapdh was used to normalize the data. Nine of the 13 samples had a less than half the relative expression of fzd4 in the tumour samples compared to the normal samples. When using fi-actin as a reference gQX\s,fzd4 expression is reduced in 11 of the 13 tumours evaluated (Figure 16B). Four o f the 13 samples had a less that 0.5 relative expression offzd4 in the tumour samples compared to the normal samples. Both reference genes displayed that fzd4 expression is decreased in tumour samples which was statistically significant with a p-value o f less than 0.001 using a paired t-test with and alpha value of 0.05, Figure 17. In order to determine whether the normal samples that were used, lung parenchyma, were an appropriate normal control for an epithelial solid tumour, lung brushings from Dr. S. Lam ( B C C A ) were provided as an epithelial normal control, gapdh and fi-actin were again used as reference genes to evaluate expression levels offzd4 in these samples. Figure 18 displays the average of the lung bmshings and lung parenchyma 72 A. 1.4 1 2 3 4 5 6 7 8 9 10 11 12 13 Sample Figure 16: Relative Expression Levels offzd4 Using Paired Lung SCC and Normal Tissue from the Same Patient. (A) Normalized fzd4 expression levels with gapdh as a reference gene. A l l patients show a strong decrease of expression in tumour tissue compared to normal. ( B ) Normalized fzd4 expression levels with $-actin as a reference gene. 11 /13 patients show a decrease of expression in tumour compared to normal. 73 Normal Tumour Normal Tumour gapdh gapdh actin actin Sample Groups Figure 17: Mean Relative Expression Levels offzd4 in Lung SCCs Compared to Normal Tissue using gapdh or P-actin as Reference Genes. Bars represent 9 5 % confidence intervals. Both are statistically significant with p-values of O.OOOl for gapdh normalization and 0 .001 for /3-actin using a student's paired t-test. Purple bars represent the gapdh analysis whereby dark purple is normal expression levels and the light pruple is the tumour expression levels of\fzd4. Green bars are the $-actin analysis, dark green is the tumour levels and light green is the normal tissue levels. 74 gapdh gapdh gapdh actin actin actin Sample Groups Figure 18: Mean Relative Expression Levels offzd4 in Lung SCCs compared to Normal Lung Parenchyma and Normal Lung Brushings Using gapdh or R-actin as Reference Genes. Bars represent 95% confidence internals. Both gapdh and p-actin are statistically significant, p-values of O.OOOl for gapdh normalization with both parenchyma and brushings. p-value = 0.001 for /3-actin normalization with both parenchyma and brushings. A student's paired and unpaired t-test was used for the statistical analysis. Bars are the same colors are previously described with the addition of maroon as the gapdh brushing and metallic green as the $-actin brushing evaluation. 75 expression offzd4 normalized with gapdh and $-actin. Using gapdh and $-actin as reference genes, the lung brushings had statistically equivalent means of that o f the lung parenchyma. The lung brushings also displayed that the tumour samples had a decreased expression which was again statistically significant (p-value <0.001) with an unpaired t-test and an alpha value of 0.05. From this we can deduce that the lung parenchyma can be used as a normal control. 3.5.2 Growth Arrest Specific 1 (gasl) at 9q21.33 The next region, 9q21.33, that was selected for analysis contained two genes potentially associated with squamous cell carcinogenesis o f the lung, gasl and dapk both reside within 0.5 M b from the S M A L L - P C R alteration (Figure 19). The same panel o f 13 squamous cell carcinomas and paired normal samples that were used for fzd4 expression analysis were also used for gasl analysis (Table 6). Figure 20A displays the normalized expression of gasl using gapdh as a reference gene in tumour compared to normal expression levels. Almost all tumours (12/13) displayed a decreased expression o f gasl compared to the normal parenchyma expressions levels. O f those tumours showing a decrease in expression, 4 samples displayed a less than 0.5 relative expression level. Figure 20B represents the normalized expression levels o f gasl in tumours compared to normal expression levels using R-actin as a reference gene. Ten of the 13 tumours evaluated showed a decreased expression level compared to normal parenchyma levels. When using R-actin to normalize the data only 2 tumours displayed a less than 0.5 relative expression level. Figure 21 illustrates the mean expression values of both tumour and normal parenchyma using both gapdh and R-actin as reference genes. Statistically, gapdh revealed a significant 76 Figure 19: Map Description of 9q21.33. The red bar and arrow indicate the location of the S M A L L - P C R alteration. The genes in the region are blue in color. Both genes evaluated reside within 0.5 Mb on either side of the S M A L L - P C R alteration. 77 A. 1.2 * l l 1 0 g-1 i O) 3 o 0.8 O Q) ? « £ > » 5 04 ro Sf £ o ° - o £ g 8 0.2 0.0 6 7 8 Sample 10 11 12 13 Figure 20: Relative Expression Levels of gasl Using Paired Lung SCCs and Normal Tissue from the Same Patient. (A) Normalized gasl expression using gapdh as a reference gene. Twelve of 13 tumours show a decrease o f expression compared to normal levels (B) Normalized gasl expression levels using $-actin as a reference gene. Nine of 13 tumours show a decrease of expression compared to normal levels. 7 8 Normal gapdh Tumour gapdh Normal Tumour actin actin Sample Groups Figure 21: Mean Relative Expression Levels of gasl in Lung SCCs Compared to Normal Tissue Using gapdh or $-actin as Reference Genes. Bars represent 95% confidence intervals, p-value using gapdh as a control is significant (0.002) whereas p-value using B-actin as a control is not significant (0.181). Statistical analysis was done using the student's paired t-test. 79 difference amongst tumour and normal parenchyma populations (p=0.002) in terms of gasl expression levels. While using $-actin as a normalizing gene did not reveal a statistically significant difference in expression levels amongst tumour and normal populations' (p=0.181). 3.5.3 Death Associated Protein Kinase (dapk) at 9q21.33 Figures 22A and 22B illustrate the expression levels of dapk in lung SCCs using gapdh and B-actin as reference genes respectively. A l l 13 tumours evaluated had a decreased expression level o f dapk using gapdh as a normalizing gene (Figure 22A). And in fact 8 of the 13 tumours had a less than 0.5 relative expression level in tumour compared to normal. When using B-actin as a normalizing gene, 11 tumours had decreased expression of dapk compared to normal parenchyma and 4 tumours had a less than 0.6 relative expression level (Figure 22B). From Figure 22B it is evident that patient 13 has an overexpression o f dapk in tumour compared to normal which wi l l affect the mean expression levels. Calculating the mean expression levels indicated that normal and tumour populations have significantly different expression levels o f dapk when using gapdh as a normalizing gene (Figure 23). When using B-actin as a reference gene, the significance is less than gapdh although still apparent among the tumour and normal populations in terms o f dapk expression levels. A students' paired t-test was performed using both gapdh and B-actin normalized populations. Both tumour populations were significantly different from the normal populations with p-values o f 0.002 and 0.035 for gapdh and B-actin, respectively. 80 A. B. Figure 2 2 : Relative Expression Levels of dapk Using Paired Lung SCCs and Normal Tissue from the Same Patient. (A) Normalized dapk expression using gapdh as a reference gene. A l l 13 tumours show a decrease in dapk expression when using gapdh as a control. (B) Normalized dapk expression using $-actin as a reference gene. Eleven of 13 tumours display a decrease in expression using ft-actin as a control. 81 1.2 0 — Normal Tumour Normal Tumour gapdh gapdh actin actin Sample Groups Figure 23: Mean Relative Expression Levels of dapk in Lung SCCs Compared to Normal Tissue using gapdh or $-actin as Reference Genes. Bars represent 95% confidence intervals, p-values for gapdh (0.002) are significant as well as the p-values for R-actin (0.035) normalization using a student's paired t-test. 82 3.6 Embryonic Expression of fzd4 Many Wnt signaling molecules are associated with embryogenesis. The expression offzd4 mXenopus had been evaluated in the developing embryo using R T - P C R as well as in situ hybridization (Shi and Boucaut 2000). One year earlier Borello et al. (1999) examined the entire fzd family expression in mouse embryos but analysis offzd4 was questionable. Favre et al. (2003) evaluated the expression analysis of genes from endothelial cells o f the lung of an adult mouse and identified fzd4 as having a 2 fold higher expression level as compared to unpurified cells o f the lung. The expression of fzd4 in development has not been wel l documented and has not been described at all in the developing lung. Figure 24 illustrates fzd4 expression occurring within the embryo as early as 6.5 dpc although the expression levels are somewhat weaker than the neighboring analysis at 7.5 dpc which continues onto 9.5 dpc. In order to determine whether expression offzd4 is involved in lung development, lungs were dissected from CD1 mice embryos at different time points, 11.5 dpc, 15.5 dpc and 19.5 dpc. Expression analysis offzd4 was performed using R T - P C R and m R N A levels were based on the reference genes mgapdh and mhprt. Embryonic lung buds start to form at 9.5 dpc from the ventral surface of the forgut, described in more detail in section 1.4.2.1 from the Introduction. Many Wnt signaling genes and associated genes such as shh,fgf, and ptch have been implicated in the formation o f the lung during embryogenesis. There have been no reports as to whether fzd4 is expressed or involved in early lung development. Figure 25 illustrates fzd4 expression present as early as 11.5 dpc and continues until postnatum in the lung. 83 mgapdh mfzd4 mhprt 6.5 7.5 8.5 9.5 dH20 -RT L 6.5 7.5 8.5 9.5 dH,0 -RT L 6.5 7.5 8.5 9.5 dH20-RT Figure 24: Embryonic Expression of mfzd4 from Different Time Points of a Developing Mouse Embryo. Numbers refer to dpc. mfzd4 expression occurs as early as 6.5 dpc and continues through all time points measured. A size marker (L) is a 100 bp ladder. Numbers indicate the embryonic dpc. A negative RT control (-RT) is included to determine if the primers are amplifying RNA. As well, a water control (dH20) is included to ensure a clean PCR reaction, mgapdh and mhprt are used to evaluate total RNA levels. A l l time points using mgapdh appear to be the same but the first 2 time points with mhprt are at lower levels. 84 mfzd4 mgapdh mhprt Random primers dT Random primers dT Random primers dT L H,0 11.5 15.5 19.5 19.5 -RT H,0 11.5 15.5 19.5 19.5-RT L H20 11.5 15.5 19.5 19.5-RT Figure 25: Embryonic Expression of mfzd4 from Different Time Points of Dissected Lungs from CD1 Mice Embryos. Numbers refer to dpc. mfzd4 expression occurs as early as 11.5 dpc in a developing lung. The size marker (L) is a 100 bp ladder. Numbers indicate the embryonic dpc. A negative RT control (-RT) is included to determine if the primers are amplifying RNA. As well, a water control (dH20) is included to ensure a clean PCR reaction, mgapdh and mhprt are used to evaluate total RNA levels. A test to determine if random primers or oligo dT (dT) primers would yield the most or the best quality R N A was performed. At 19.5 dpc the same amount of RNA was used to create cDNA with both random primers and with oligo dT primers. Random primers performed better with mfzd4 and mgapdh but oligo dT primers worked best with mhprt. 85 3.7 Immunohistochemistry Immunohistochemistry (IHC) was performed on 11.5 dpc mouse embryos as suggested by the manufacturer using a polyclonal antibody (Ab) specific for FZD4 ( R & D Systems Inc.). The initial optimization using antigen retrieval solutions appeared to indicate that pH 2 Retrievit Target Solutions (Vector Laboratories) gave the most intense, specific staining using this primary Ab. Figure 26 illustrates the staining of the embryos using the different pH solutions as well as a negative control using no primary antibody. While p H 2 seemed to give the most intense, specific staining, pH 10 also gave an intense staining but the entire embryo was stained using this solution. This seemed to indicate that the p H 10 antigen retrieval was non-specific. The reason the p H 2 was selected was because using no antigen retrieval solutions seemed to stain the embryos in a similar pattern only far less intense. The next step was to alter the primary antibody concentration and to use a true negative control, normal goat IgG. Figure 27 displays the varying concentrations (3, 5, 7.5 and 15 pg/mL) o f primary A b that were used on the embryos. The manufacturer recommended 15 pg/mL but often a lower concentration can be used. A l l concentrations that were used stained with the same intensity as the recommended concentration. The next optimization was the use of a true negative control, affinity purified normal IgG from goat. This control was used because the FZD4 primary antibody was also produced in the goat. From Figure 28 it is evident that the IgG control is binding the same areas as the antibody that was supposed to be specific for FZD4. Therefore this antibody was not specific for mouse FZD4 and should not be used in any further evaluations. The company was contacted with the results but could offer no further support. 86 A . B . Figure 26: IHC Optimization Using Target Antigen Retrieval Solutions. (A) No primary antibody. (B) No Retrievit Solution. (C) pH 2. (D) pH 4. (E) pH 6. (F) pH 8. (G) pH 10 Retrievit Solution. pH 2 (panel C) was chosen for further evaluation as it produced the most specific and strongest signal. 87 Figure 27: IHC Antibody Concentration Optimization. (A) 3 pg/mL. (B) 5 pg/mL. (C) 7.5 pg/mL. (D) 15 pg/mL of primary antibody. A l l concentrations show an equal amount of staining similar to the first optimization experiment. 88 Figure 28: IHC Negative Control Using Normal IgG Purified from Goat. (A) 1 ug/mL IgG (B) 1 ug/mL FZD4 A b (C) 3 ug/mL IgG (D) 3 ng/mL FZD4 A b (E) 5 ug/mL IgG (F) 5 ug/mL FZD4 Ab. A l l concentrations show similar staining of both FZD4 A b and IgG. This experiment concludes that FZD4 A b is not specific. 89 Chapter 4: Discussion 4.1 Summary of Results The focus of this thesis was to identify and characterize genomic alterations occurring in premalignant squamous cell lung lesions. S M A L L - P C R was used as a genome wide scan to identify recurrent alterations occurring in multiple patients using minute amounts o f archival D N A . 16 patients were screened by S M A L L - P C R which identified 18 recurrent alterations within the 64 microdissected samples analyzed. A l l 18 recurrent alterations occurring in at least 2 patients were cloned. O f those, 7 alterations were mapped to chromosomal regions at 2p25.3, 9q21.33, l l q l 4 . 2 , 13q33.1, 14q24.2 and 20pl3. Verif ication o f the chromosomal alterations was performed at 1 lq l4 .2 using F ISH and L O H analysis. The verification was done using a different sample set than was previously used for the S M A L L - P C R screen due to the limitations of the premalignant material. Expression analysis was performed on selected genes at 2 loci identified by S M A L L , 1 lq l4 .2 and 9q21.33. Three genes, fzd4, gasl and dapk, were analysed for m R N A expression levels in a paired patient set o f normal and lung squamous cell carcinomas. The analysis identified two genes that were statistically different in expression patterns in the tumour cell populations when compared to normal expression levels. A temporal pattern offzd4 gene expression was identified in the developing embryo and more specifically in the developing lung of a mouse embryo. A spatial pattern o f F Z D 4 protein expression was attempted in the developing mouse embryo using IHC. 90 4.2 Validation for the SMALL-PCR Approach to Identify Recurrent Alterations in SCC S M A L L - P C R has identified 18 recurrent genetic alterations in premalignant lesions which could be critical to the progression of lung cancer. This genome-wide analysis required the use of microdissected material due to the heterogeneity o f the lesions. Very little material was available for this assay due to the small size o f the premalignant lesions. Four ng of D N A was used to screen the genome using D N A that was extracted from formalin fixed, paraffin embedded tissue. Conventional methods of genome-wide assays such as C G H or multicolour F ISH require the use of much more D N A and the resolution is poor, allowing for 1-5 M b of visualized area which can contain dozens of genes. Other techniques such as L O H which only look at one regions of the genome at one time would require the use too much D N A i f a full genome-wide scan were done and this would be very labour intensive. One problem with the alterations obtained from S M A L L - P C R is the inability to determine whether it is a gain or a loss o f the genomic region of interest. The mechanism underlying the change in the tumour sample is unknown. This problem can be resolved by other follow-up methods such as expression analysis or F ISH. Eighteen recurrent regions were discovered by S M A L L - P C R and were described in section 3.1 and Table 7. The significance of these findings is described below. 4.3 Significance of Regions Discovered by SMALL-PCR O f interest, 4 o f 6 regions listed in Table 12 contain genes that belong to important development pathways. In particular F Z D 4 , C S K N 2 A 1 , TCF15 and PSEN1 have potential to be directly involved in Wnt signaling cascade. GAS1 has been shown to be activated by W N T signaling molecules due to its inhibitory role in the Shh signaling pathway (Lee, 2001). Details concerning the involvement of these genes are to follow in the next few sections. 91 Table 12: Successfully Cloned and Mapped Recurrent Chromosomal Alterations Discovered by SMALL-PCR Chromosome Frequency Genes 2p25.3 4/7 Patients None identified 9q21.33 3/9 Patients gasl dapk l l q l 4 . 2 3/7 Patients fzd4 13q33.1 7/16 Patients erccS 14q24.2 4/7 Patients psenl numb 20pl3 2/9 Patients angpt4 csnk2al tcfl5 sox 12 92 4.3.1 Novel llql4.2 Alteration in SCC of the Lung Alterations at 1 l q l 4 have not been wel l characterized in lung cancer to date. Cycl in D l , a cell cycle regulator, genomic information resides at chromosomal position 1 lq l3 .3 and most studies focus on this gene in the region o f 1 l q . C G H analysis o f N S C L C often report regions of alteration at 1 l q both amplifications and deletions. The resolution o f this technique is inappropriate to implicate specific regions that contain an identifiable number of genes therefore, further follow-up is required to fine map the regions identified by C G H (Berrieman et al. 2004; Luk et al. 2001; Walch et al. 1998; Michelland et al. 1999; Chan et al. 1996). Using S M A L L - P C R 43% (3/7) o f the patients that were screened contained an alteration at 1 lq l4 .2 . The alteration is 0.4 M b from the fzd4 gene. FZD4 is a member of the Frizzled family o f G-coupled receptors which are specific for secreted Wnt signaling molecules. The Wnt signaling pathway and its implications in cancer and other aspects have been discussed in the introduction in detail. This thesis initially focused on determining i f FZD4 had implications in lung cancer because of the number of developmental genes residing in the area of the alterations, in particular those of the Wnt signaling pathway. fzd4 was the first gene chosen for evaluation as it begins the intracellular signaling cascade as it is a transmembrane receptor. Secondly, Wnt signaling has been shown to have significant effects on other tumour types such as those previously described in particular colorectal cancer which is another epithelial type o f cancer. Lastly, the vast majority o f therapeutic targets are receptor molecules because they offer target accessibility due to their location on the cell surface. 93 4.3.1.1 Description of the DNA Analysis of llql 4.2 Alteration Two methods of D N A verification were performed on 1 lq l4 .2 , F ISH and L O H , as described in the Materials and Methods section. 4.3.1.1.1 FISH Analysis at llql4.2 The B A C that was selected for genomic analysis, RPT1-736K20, was completely sequenced and its genomic position is stable as it doesn't reside within or near a region with gaps in the genomic coverage. Metaphase F ISH verified the genomic position o f the probe to chromosome 1 l q . Unfortunately the formalin fixed tissue sections that were used to analyze frequency o f alteration in N S C L C were incapable o f producing a strong enough fluorescent signal for visualization from the probe that was synthesized. On the other hand the synthetically labeled centromeric probe produced an intense signal that was seen in virtually all nuclei. Other groups have had difficulty visualizing with nicktranslated material and the suggestion was made to use more than one B A C in the reaction to cover a broader area. As previously stated with metaphase chromosomes there is the limitation of resolution but with interphase nuclei there is less intense signaling due to the signal being spread out because o f the uncoiling o f the D N A . Another method for D N A verification was sought. 4.3.1.1.2 LOH Analysis at llql4.2 Microsatellite analysis was used to determine i f the region at 1 lq l4 .2 displayed any genomic instability in S C C of the lung. The panel used for L O H analysis was not microdissected (Table 6). This limitation was not recognized until after the experiment was completed. Some of the samples contained as little as 30% tumour cell population which is not suitable for P C R conditions. Regardless, instability was detected within these samples at 94 multiple loci with multiple tumours. One tumour displayed L O H at D11S1887 while there were seven tumours that displayed a potential homozygous deletion in the 1 lq l4 .2 region. Further follow-up is required to determine i f this is a true homozygous deletion or an artifact of the sample. Homozygous deletion is more probable as all the samples that contained the homozygous deletion were tested at another locus which detected strong signal at both alleles. Therefore it can be determined from this L O H analysis that 1 lq l4 .2 is a frequent target o f genomic instability for tumour success. 4.3.1.2 Description of the Expression Analysis of fzd4 at llql4.2 FZD4 homodimerization occurs in the endoplasmic reticulum (ER) (Kaykas et al. 2004). A mutant truncated version of the receptor wi l l still bind wi ld type FZD4 and retain it in the E R impairing its signaling acting in a dominant negative fashion (Kaykas et al. 2004). Wnt-5A activates signaling through FZD4 (Umbhauer et al. 2000). Endocytosis o f the receptor required D v l 2 and p-arrestin 2, an adaptor molecule responsible for desensitizing second messenger signaling (Chen et al. 2003). Expression patterning in the mouse embryo has been performed at 9.5 dpc and 10.5 dpc in which the authors propose the telencephalon to the telencephalic vesicles are e x p r e s s i n g ( B o r e l l o et al. 1999). Another expression study was performed with the Xenopus embryo in which expression offzd4 was constant in the fertilized egg until at least the larval stage (Shi and Boucaut 2000). fzd4 inhibition was common to undifferentiated malignant embryonal carcinomas and that with treatment to differentiate these cells upregulation offzd4 was seen (Walsh and Andrews 2003). Expression analysis offzd4 ensued the genomic verification o f 1 lq l4 .2 in this thesis to evaluate genes in the S M A L L - P C R region. A statistically evident decrease in expression of fzd4 was observed with both gapdh and R-actin used as reference genes. This could imply 95 that a decrease offzd4 is necessary for primary lung squamous cell carcinomas to be successful. The expression pattern seen in mouse embryos indicates fzd4 is important for embryonic development and as well fzd4 has a role in the development o f the lung. This data suggests that genes involved in development are differentially expressed in tumour cells. More specifically, because developmental genes regulate the maturation o f cells it would be advantageous for a tumour cell to inhibit these genes in order to become less differentiated. Thus the decrease offzd4 seen in lung SCCs could be an escape the tumour cell requires in order to avoid the normal constraints of cell maturation. It was shown that fzd4 is expressed in the developing lung but the role it plays still needs to be investigated. 4.3.2 Candidate genes at 9q21.33: gasl and dapk S M A L L - P C R detected an alteration in 3 of 7 patients screened at 9q21.33. The S M A L L alteration was a loss of P C R signal seen in multiple tumours compared to a band consistently present in normal samples. From the literature, 40% of N C S L C s have alterations detected by L O H or C G H at 9q where as only 25% of S C L C s harbor alterations at this chromosome arm (Sanchez-Cespedes 2003). 9q21.33 contains candidate genes that have a potential role in lung carcinogenesis. This thesis chose to evaluate expression levels o f gasl and dapk in S C C of the lung to address the second hypothesis. It states that i f the regions altered by S M A L L - P C R are important then they should contain genes that are differentially expressed between normal and tumour samples. A n investigation into what is currently known about these genes and the results obtained in this thesis may help understand the observations. 96 4.3.2.1 GAS1 GAS1 is an integral plasma membrane protein that has an inverse U shape found on the surface of the cell (Del Sal et al. 1994). The gasl gene was isolated due to its induction in serum deprived and high density fibroblast cells (Schneider et a l l 988) . Overexpression of gasl in quiescent cells inhibited the transition from Go to the S phase of the cell cycle (Del Sal et al. 1992). GAS1 induced cell cycle arrest required endogenous P53 (Del Sal et al. 1995). Ruaro et al. (1997) discovered a proline rich region on murine P53 that is required for GAS1 inhibition o f cell cycle. Induction of c - M Y C transcription factor is able to repress gasl expression through transcriptional control (Lee et al. 1997). The opposite is also possible whereby expression of gasl is able to repress c-myc expression (Lee et al. 2001). Wnt signaling can induce the expression of gasl (Lee et al. 2001). Wnt signaling molecules are known to activate c-myc expression through P-catenin (He et al. 1998). Perhaps indirectly through c - M Y C , Wnt signaling controls the expression of gasl. In the developing embryo Wnt secreted factors are antagonists to Shh signaling due to the opposing roles they have in development (Lee et al. 2001). Lee et al. (2001) also demonstrated GAS1 as a binding repressor o f Shh presumably limiting its ability to induce cell growth. Lee et al. (2001) were the first to report gasl involvement in apoptosis. They found that gasl induction of interdigital cell death in the mouse embryo was P53 independent. Contrary to the above arguments it was found that GAS1 functions to strongly protect cells from apoptosis and not significantly inhibit cell growth in endothelial cells (Spagnuolo et al. 2004). Vascular endothelial (VE)-cadherin as well as vascular endothelial growth factor 97 ( V E G F ) up-regulated gasl consistently with their anti-apoptotic activity but independent from their mitogenic effect (Spagnuolo et al. 2004). 4.3.2.1.1 GAS1 a Role in Cancer The above data suggests that GAS1 may have a role in the tumourogenic process as its activity is controlled by or controls a variety of important signaling elements that have a well established role in cancer. The confusion lies in the activity o f G A S 1 , whether it has oncogenic or tumour suppressive abilities. There is a large body of evidence suggesting that GAS1 behaves as a tumour suppressor. High levels o f gasl expression in transfected NIH3T3 cells and tumour cells greatly inhibited growth rate and when anti-sense m R N A was used the cells grew to a much higher saturation and were not contact inhibited as they previously were (Evdokiou and Cowled 1998, Evdokiou and Cowled 1998). Consistent with the above arguments, gasl overexpression prevents the transition from Go to S phase of the cell cycle (Del Sal et al. 1992). Recently, Spagnuolo et al. (2004) demonstrated a novel role for G A S L They demonstrated that gasl overexpression in endothelial cells has an anti-apoptotic role by promoting cells stabilization and resistance to cells death by toxin induction (Spagnuolo et al. 2004). This thesis demonstrated that gasl expression levels in tumour cells were significant different from the expression levels in normal parenchyma cell populations in the lung when using gapdh as a normalizing gene. On the other hand, when using R-actin as a normalizing gene a significant difference between normal and tumour cell expression o f gasl was not seen. A difference in expression is clearly dependent on which reference gene is used for normalization. The use of reference genes to evaluate gene expression level wi l l be discussed in more detail in section 4.3.2.2.3. Because there were 2 genes in the 98 S M A L L - P C R region that had tumour suppressor potential it would be more definitive to evaluate the expression levels o f dapk along with gasl. 4.3.2.2 DAPk Death-associated protein kinase (DAPk) is a relatively new addition to the apoptotic family o f signaling molecules. It was discovered in the mid 1990s in a genetic screen to identify genes necessary for interferon (IFN)-y induced cell death (Deiss et al. 1995). In subsequent years the identity o f family members ( D R P - l / D A P k 2 , Dlk/ZIP-kinase, D R A K 1 and D R A K 2 ) that contain a high degree of catalytic domain homology were cloned and identified (Kogel, Prehn, and Scheidtmann 2001). A discussion about the structural domains of D A P k followed by its mode of action and finally its role in cancer wi l l be discussed in the following paragraphs. 4.3.2.2.1 DAPk Structure Figure 29 illustrates the known functional domains o f D A P k . The catalytic domain o f D A P k is critical to its death inducing functions and in fact mutation o f one amino acid (Lys42) causes a loss o f catalytic activity (Cohen et al. 1997). This region is thought to be the A T P binding site similar to other kinases. The kinase domain is adjacent to a Ca 2 + /calmodul in (CaM) regulatory domain. It is proposed that the C a M domain acts as a pseudo-substrate that blocks the catalytic cleft from substrate interaction. Upon activation o f C a M with C a 2 + binding, changes in the conformation of the protein exposes the active site thus enabling the catalytic functionality o f the protein (Bialik et al. 2004). 99 aa: 200 400 600 800 1000 1200 1426 t Ser rich tail Figure 29: DAPk Structural Protein Domains. The kinase domain (red box) of D A P k is critical to its death inducing catalytic action. This region is thought to be the A T P binding site similar to other kinases. Ca2+/calmodulin (CaM) (blue) regulatory domain acts as a pseudo-substrate that blocks the catalytic cleft from substrate interaction. Upon activation o f C a M with Ca2+ binding changes in the conformation o f the protein expose the active site and thus enabling the catalytic functionality of the protein. The ankyrin repeats (orange boxes) are protein-protein interaction motifs common to cytoskeletal proteins. The two P-loops (green) have an unknown function as of yet. The cytoskeletal binding domain (purple) is responsible for D A P k localization to the actin filament network. The death domain (gold) is responsible for D A P k kil l ing ability. The serine rich region (baby blue) in the C-terminus is responsible for negative regulation of the death domain. The position of the protein domains are indicated by amino acid (aa) position above the diagram. 100 The remainder o f the protein is involved in regulation and localization. The ankyrin repeats are protein-protein interaction motifs common to cytoskeletal proteins. The cytoskeletal binding domain is responsible for D A P k localization to the actin filament network. Mutation analysis o f both domains revealed their importance for D A P k localization (Bialik and K imch i 2004). The two P-loops have an unknown function as o f yet. The C-terminal o f D A P k harbors a death domain followed by a serine rich region which is common to other death domain containing proteins including death receptors and their adaptor proteins (Feinstein et al. 1995). Deletion of the death domain eliminates D A P k ability to induce ki l l ing but an adaptor for this domain has not yet been identified (Bialik et al. 2004). The serine rich region in the C-terminus is responsible for negative regulation of the death domain. It was shown through deletion analysis o f this region that enhanced ki l l ing by D A P k was evident, as well through enhanced expression of this region the kil l ing abilities of D A P k were abolished (Raveh et al. 2000). 4.3.2.2.2 DAPk Function The studies on D A P k over the past few years have revealed that D A P k has multiple functions and that it lies at a critical junction o f cell death signaling (Bialik et al. 2004). Several groups have shown that suppression o f D A P k function can slow death induction by various stimuli such as T N F a , IFNy, Fas, T G F p , detachment from the extra cellular matrix and C6 ceramide (Cohen et al. 1997; Inbal et al. 1997; Jang et al. 2002; Pelled et al. 2002; Deiss et al. 1995). Once D A P k is activated it can trigger a range of death responses leading to multiple phenotypes. These responses wi l l be discussed in the following paragraph. D A P k has been shown to induce characteristics o f a programmed cell death called autophagic cell death (Inbal et al. 2002). This type of cell death has the appearance of 101 lysosomal bodies as intracellular compartments referred to as autophagic vacuoles (Assuncao Guimaraes and Linden 2004). These vacuoles are a means of recycling organelles and other proteins for normal cellular nourishment. Other cellular features of this type of cell death are the enlargement o f the mitochondria, endoplasmic reticulum and Golg i apparatus and most noticeable the blebbing of the plasma membrane (Assuncao Guimaraes and Linden 2004). Autophagy was induced by D A P k through IFN-y mediated kil l ing o f HeLa cells (Inbal et al. 2002). It was shown that the autophagy death pathway is a caspase-independent cell death pathway (Inbal et al. 2002). Furthermore, Wang et al. (2002) discovered that D A P k can induce pro-apoptotic activity by suppressing integrin mediated cell survival signals. This occured in anchorage-dependent growth conditions whereby the loss o f adhesion in certain cell types subsequently activated a caspase-dependent type of apoptosis called anoikis (Wang et al. 2002). Other groups also identified D A P k as activating caspase-dependent cell death via the presence of active p53 in response to hyperproliferative signals or D A P k mediates T G F p induced apoptosis signaling through S M A D s (Raveh et al. 2001; Jang et al. 2002). The multiple pathways involved in D A P k ki l l ing ability indicates that D A P k plays a central role in programmed cell death and also implicates cross talk between pathways and the complexity of cellular signaling. 4.3.2.2.3 DAPk a Role in Cancer Evidence from the literature of aberrant D A P k activity in a vast array o f tumour types is overwhelming. A highlight of the main evidence from animal models and cell culture as well as human tumour screens wi l l be summarized. 102 As previously stated, D A P k is responsible for inducing cell death by means of P53 activation o f apoptosis. Raveh et al. (2001) showed that D A P k protein is upregulated in response to oncogenes activation ( c - M Y C and E2F-1) and that loss of D A P k resulted in a decreased apoptotic response to oncogene activation. This implicates D A P k as an early tumour suppressor which acts in conjunction with P53 to induce apoptosis as a safeguard against mitogenic signaling. D A P k was also shown in animal modeling to influence metastasis o f tumour migration. A highly metastatic Lewis lung cell carcinoma was shown to be D A P k negative whereas a less metastatic counterpart had normal levels o f D A P k expression (Inbal et al. 1997). The highly metastatic cells were restored to normal levels o f D A P k expression and injected into mice. This suppressed their ability to form lung metastases in mice. Whereas, injection of the less metastatic cells into mice infrequently resulted in lung metastases and when analyzed they demonstrated loss o f D A P k protein expression at high frequency (Inbal et al. 1997). This implicates an advantage of D A P k loss to metastatic tumours. dapk hypermethylation has been extensively studied in a variety o f tumour types ranging from lymphoma/leukemia to a vast number of solid tumours. The percentage o f hypermethylation ranges from 85 % in lymphoma to 15 % in colon and breast cancers (Raveh and K imch i 2001). dapk hypermethylation has also been reported in some premalignant stages of gastric cancer and oral S C C (To et al. 2002; Kulkarni and Saranath 2004). Hypermethylation o f dapk in non-small cell lung carcinomas have been reported at 44 % (59/135) (Tang et al. 2000), 25 % (47/185) (K im et al. 2001) and 28 % (21/75) (Yanagawa et al. 2003). Yanagawa et al. (2003) also reported the status o f dapk hypermethylation in non-neoplastic lung tissue at 13 % (10/75) which the other groups did 103 not report. Homozygous deletion and L O H have also been reported on dapk but infrequently compared to hypermethylation (Simpson et al. 2002). There is clear evidence from the literature that dapk gene expression is altered in tumours. The common mechanism for dapk alteration is epigenetic hypermethylation o f the promoter region. The results from this thesis support what has been documented in the literature. A decrease in dapk expression was apparent in lung SCCs. A 52.2 % decrease of expression was seen in the tumour samples with gapdh as a normalizing gene and a 19.3 % decrease was seen with B-actin as a normalizing gene, both were statistically significant. These are the two most common normalizing genes reported in the literature but the different results that both present are disturbing. The difference between normalizing genes reflects a need for better control elements in determining expression levels o f selected genes. Because the samples were not microdissected this may reflect a difference in expression of the surrounding cell populations. A look into the mechanism o f how dapk is being decreased in these tumour samples would be advantageous. This would determine i f S C C of the lung follows the standard mechanism o f deactivating dapk, hypermethylation, as previous groups have reported. Methylation is a reversible process, agents capable o f demethylating D N A are ideal for cancer therapy. Although caution should be taken as the current demethylating drugs are not gene specific as they affect the entire genome. One must consider the implications o f demethylating the entire genome. Hopefully in the near future there wi l l be a possibility o f gene specific demethylation as a valuable tool for cancer therapy. 104 4.3.3 Candidate genes at 20pl3: angpt4, tcfl5, soxl2 and csnk2al S M A L L - P C R identified an alteration at 20pl3 in 2 o f 9 patients screened. The chromosomal regions at 20p has not been wel l documented in lung cancer but a few cytogenetic profiles have described alterations in this region in other types of epithelial cancers (Hauptmann et al. 2002; Weber et al. 1998; Nakao et al. 1998). The alteration resides less than 1 M b from the telomeric most region of 20p. This region harbors a large number of known genes (18) many open reading frames (10) and hypothetical genes (17). O f the known genes in the region angpt4, csnk2al, tcfl5 and sox!2 have tumourogenic potential and these wi l l be described in the following paragraphs. 4.3.3.1 ANGPT4 Angiopoietin-4 (ANGPT4) is a member o f the vascular endothelial growth factor ( V E G F ) family. A N G P T 4 is an agonist o f the Tie2 receptor like its family member A N G P T 1 (Valenzuela et al. 1999). A N G P T 1 in involved in the later stages of vascular development to promote remodeling, maturation and stabilization o f the vessels (Sato et al. 1995; Suri et al. 1996). Overexpression o f A N G P T 1 in transgenic mice leads to hypervascularization (Suri et al. 1998). Tumour vascularization is a well established fundamental (Hanahan and Weinberg 2000). Upregulation o f A N G P T 1 and A N G P T 2 have been described in N S C L C (Xing et al. 2003; Tanaka et al. 2002; Takahama et al. 1999) but A N G P T 4 has not been assessed in lung cancer. angpt4 expression has been evaluated in a few types of cancers with positive correlation o f tumourogenic involvement (Brown et al. 2000; Currie et al. 2001). 105 4.3.3.2 TCF15 TCF15 is a member of the T-cell factor (TCF) family o f basic helix-loop-helix (bHLH) transcription factors (Hidai et al. 1995). The function o f TCF15 has not been established but other family members' functions have been wel l characterized. In the absence of Wnt signaling, T C F acts as a target repressor of wnt genes (Brannon et al. 1997; Bienz 1998). Upon activation o f the Wnt signaling pathway, p-catenin can convert T C F into a transcriptional activator of the same genes that it was repressing alone (Nusse 1999). T C F is involved in many cell fate decisions and other roles in development as the targeted activation o f Wnt signaling (Verbeek et al. 1995; Galceran et al. 1999; Korinek et al. 1998). The function of T C F 15 is not currently known but there is substantial evidence that other members o f the T C F family are involved in carcinogenesis (Roose and Clevers 1999). 4.3.3.3 SOX12 T C F family members contain a D N A binding domain called high-mobility group ( H M G ) domain (Brantjes et al. 2002; Clevers and van de Wetering 1997). Although distantly related (30%), S O X transcription factors also contain H M G domains. S O X proteins interact with a 6-7 base pair D N A sequences through a 79 amino acid protein motif to control transcription of target genes (Kamachi et al. 2000). S O X proteins along with co-transcription factors are responsible for many events in embryogenesis (Kamachi et al. 2000). SOX12 is most closely related to S O X 4 and SOX11 by sequence homology and has wide spread expression in adult and fetal tissue suggesting a role embryogenesis and cell fate decisions (Jay et al. 1997). Overexpression of sox4 has been established in both S C L C and N S C L C primary tumours as well as cell lines (Friedman et al. 2004). Due to sequence similarity o f 106 S 0 X 4 and S O X 12 there is potential for S O X 12 to have a role in lung tumourogenesis and further evaluation should be done. 4.3.3.4 CSNK2A1 Casein Kinase (CK) 2 is a protein serine/threonine kinase composed of heterotetromeric subunits. The catalytic subunits (a and a') and the regulatory subunit ( P ) exist as either (X2P2, a ' c ^ or cfifa configuration. 20pl3 harbors the alpha (a) subunit o f C K 2 . C K 2 is one of the most conserved protein kinases in evolution and the deletion o f both catalytic subunits is lethal (Padmanabha et al. 1990). C K 2 is found in the cytoplasm and nucleus and has been implicated in a broad array of cellular functions (Litchfield 2003). Most findings have suggested that C K 2 has a functional role in the regulation o f cellular growth, protein synthesis, division of cells and protein traffic (Litchfield 2003). Despite the fact that this enzyme was discovered 48 years ago, the mechanisms that regulate its activity under physiological conditions are still a puzzle (Litchfield 2003). There are more than 300 genes that C K 2 phosphorylates among them A P C , Dv l -1 , Dvl-2 and TCF-4 belong the Wnt signaling pathway (Pinna 2002). In contrast to other kinases C K 2 mutations causing constitutive activation are not found in cancer probably due to its wide spread role. On the other hand, over representation o f C K 2 has been found in the vast majority of tumours in many studies (Guerra and Issinger 1999; Tawfic et al. 2001). C K 2 was elevated in lung tumours when compared to the corresponding non-neoplastic tissue of the same patient (Yayl im and Isbir 2002; Daya-Makin et al. 1994). These have been the only studies to examine this protein in lung cancer and more investigation is require to determine i f this protein has an intricate role in the development o f lung tumourogenesis. 107 4.3.4 Candidate gene at 13q33.1: ercc5 S M A L L - P C R identified an alteration in 7 of 16 patients screened at 13q33.1. The reproducibility o f S M A L L - P C R was confirmed with the identification o f two alterations (fragments A and J , see Table 7) from separate experiments being amplified with the same primer sequence which resolved at the same region of the S M A L L gel. Chromosome arm 13q is often associated with lung cancer as approximately 60 % of N S C L C and 90 % of S C L C display L O H or cytogenetic alterations (Sanchez-Cespedes 2003). Most often these alterations are associated with other known tumour suppressor genes on this arm, R B at 13ql4.2 and B R C A 2 at 13ql3.1. The involvement o f R B in lung cancer has previously been discussed in the introduction. S M A L L - P C R discovered the alteration at 13q33.1, a remote distance from the other known tumour suppressor genes. C G H studies implicating 13q involvement most often assume the gene targeted for deletion to be rb but these results could implicate another chromosomal region on 13q associated with lung cancer. Tobacco carcinogens cause D N A damage and a reduced D N A repair capacity could lead to multiple regions o f genomic instability. A s previously discussed, the LUCA region at 3p21.3 is a region of target for B P D E damage in lung cancer cases (Wu et al. 1998). There are 2 main pathways involved in D N A repair: base excision repair (BER) which removes a single damaged nucleotide and nucleotide excision repair (NER) which removes damaged oligomers 25-32 nucleotides long (Wood 1996). Excision repair cross-complementing (ercc) genes are involved in the N E R pathway and one of the members, ercc5, resides at 13q33.1. This gene is approximately 1 M b from the S M A L L - P C R alteration but it is an obvious candidate to be altered in lung cancer. In fact, Cheng (2000) found a significant 108 decrease in ercc5 expression in lung cancer patients compared to normal controls. ercc5 has also been described in other types of cancer such as head and neck squamous cell carcinomas (Cheng et al. 2002) and prostate cancer (Hyytinen et al. 1999). Further studies should identify the ercc genes as key targets in lung cancer therapeutics. 4.3.5 Candidate genes at 14q24.2: psenl and numb Approximately 30% of N S C L C s and S C L C s have L O H or chromosomal copy number changes at 14q (Sanchez-Cespedes 2003). S M A L L - P C R identified 4 of 7 patients with alterations at chromosome 14q24.2. This region harbors many genes most o f which are hypothetical genes but a few known genes in the region possess tumourogenic potential. 4.3.5.1 PSEN1 The extensive research on presenilin 1 (psenl) resulted from the genetic linkage o f this gene to early onset Alzeimer's disease. In the last few years researchers have discovered that PSEN1 contains the active site o f y-secretase activity that cleaves type I single pass membrane receptors within the membrane (Kimberly and Wolfe 2003). y-secretase is a high molecular weight complex formed at the plasma membrane that involves the activities PSEN1 and other co-factors such as presenilin enhancer 2 (PEN2), nicastrin and APH1 (Takasugi et al. 2003). The most thoroughly described role for PSEN1 y-secretase activity is the cleavage o f Notch, P-ameloid precursor protein (PAPP) and ErbB-4. A generalized model for y-secretase activity involves receptor (Notch, PAPP or ErbB-4) stimulation upon binding to its ligand. The ligands are also type I transmembrane proteins, thus receptors are limited to regulating interactions between physically proximal cells (Gridley 2001). Once the receptor binds its ligand it permits a disintegrin 109 metalloprotease ( A D A M family) to clip the extracellular domain o f the receptor near the plasma membrane. The remainder o f the receptor then serves as a substrate for y-secretase, which cuts within the transmembrane domain. The released intracellular domain is then able to translocate to the nucleus and alter gene transcription (Selkoe and Kopan 2003). Notch is a conserved cell signaling system that regulates cell fate specification, stem cell maintenance and initiation of differentiation in embryonic and postnatal tissues (Grego-Bessa et al. 2004). PSEN1 is not only implicated in y-secretase activity but has been shown to bind P-catenin in vivo (Cox et al. 2000) but that the y-secretase activity is not responsible for PSEN1 interaction with p-catenin (Meredith et al. 2002). As previous described, P-catenin is a critical mediator o f the canonical Wnt signaling pathway and an important regulator o f cadherin-based cell adhesion complexes. The interaction between PSEN1 and P-catenin targets p-catenin phosphorylation and subsequent destabilization resulting in its degradation (Kang et al. 2002). From this new found involvement in Wnt signaling regulation, it is possible that loss o f psenl genetic information would enable the success of a tumour cell by increasing its mitogenic signaling capabilities through p-catenin. 4.3.5.2 NUMB Located directly telomeric to the genetic information ofpsenl is the genetic information of the numb gene on chromosome 14. The N U M B protein functions as a cell fate determinant in neuronal precursors cells where it influences cell fate by antagonizing signaling from the Notch receptor (Frise et al. 1996). N U M B co-localizes with endocytic vesicles and was shown to directly bind the intracellular domain o f Notch as well as a clathrin adaptor, a-adaptin (Berdnik et al. 2002; Santolini et al. 2000). These observations 110 suggest that N U M B acts as an adaptor between a-adaptin and its Notch cargo and promotes the down-regulation of Notch by endocytosis (Berdnik et al. 2002). Due to the characteristics o f the normal function of Notch, regulation of cell fate, stem cell maintenance and initiation of differentiation, there is potential for tumourogenic applications of this protein i f alterations arise (Grego-Bessa et al. 2004). Recent studies have implicated Notch as an oncogenic potential in Pre-T cells but others have implicated Notch in suppressing tumour development in keratinocytes (Weng et al. 2003). Notch has also been implicated in epithelial-mesenchymal transitions for multiple metaplasias which are precursors for tumour formation (Timmerman et al. 2004). The Notch pathway is a complicated pathway and its involvement in carcinogenesis is being unfolded. Any molecule such as N U M B that has an established role in the regulation o f Notch could also be involved in carcinogenesis process. I l l Chapter 5: Conclusions 5.1 Conclusions The following key points summarize the data collected in this thesis: 1. Sixty-four samples from 16 patients were screened for alterations by S M A L L - P C R 2. S M A L L - P C R identified 18 recurrent regions that were altered in squamous lung lesions. O f those, 7 were cloned and mapped to known human chromosomal regions. 3. Four regions contain genes involved in or interacting with the Wnt signaling pathway. 4. Expression analysis offzd4 by R T - P C R revealed a statically significant decrease in expression in lung S C C compared to normal tissue expression with p-values of <0.0001 and 0.001 for gapdh and B-actin normalization respectively. 5. Lung parenchyma is statistically similar in expression o f selected genes to lung brushings and can be used as a normal control for epithelial tumours. 6. Expression analysis o f gasl by R T - P C R revealed a statistically significant decrease in expression in lung SCCs compared to normal tissue expression when using gapdh as a normalizing gene but not when using B-actin as a normalizing gene, p-values were 0.002 and 0.181 for gapdh and B-actin respectively. 7. Expression analysis o f dapk by R T - P C R revealed a statically significant decrease in expression in lung S C C compared to normal tissue expression with p-values of 0.002 and 0.035 for gapdh and B-actin respectively. 8. A clear difference in expression analysis can be seen with different genes used to normalize data. A need for a better expression control is required. 9. fzd4 is expressed early in mouse development and was seen at all time points tested in whole embryos from 6.5 dpc to 9.5 dpc. 112 10. Dissected lungs from mice embryos revealed that fzd4 is expressed from 11.5 dpc to 19.5 dpc, the first postnatal day. 11. Immunohistochemistry studies revealed that purchased antibodies are not always specific to the antigen they are intended to target. 5.2 Significance of Findings The above conclusions address the hypotheses of this thesis. In the following paragraphs the significance of these findings wi l l be examined. S M A L L - P C R is capable of detecting alterations from minute quantities of premalignant lung lesions. It is important to evaluate these lesions for genetic alterations as these alterations may be the driving force behind tumourgenesis. By the time a lesion is invasive it has acquired numerous genetic alteration and deciphering the key genetic events becomes extremely difficult. Working with cell lines to uncover these key events is even more trivial as they no longer represent a biologically normal state. Thus determining the early genetic events leading to the progression of lung cancer has a higher probability of directing the future in lung cancer prevention. S M A L L - P C R identified 18 recurrent regions of alteration occurring in multiple patients. O f the 6 regions described in Table 8, 4 regions contain genes that are involved in development processes. More specifically, these genes interact with or belong to the Wnt signaling pathway. The frequency at which these genes were detected implicates developmental gene irregularity in lung cancer progression. These genes are responsible for the maturation and differentiation o f cells as they progress through development. A tumour may become more prolific i f it is capable o f down regulating or shutting off genes that would normally signal a cell to mature. 113 This thesis demonstrated the decrease in expression of selected genes in lung SCCs which are normally involved in developmental processes. Specially, fzd4 was shown to have a statistically significant decrease in expression in lung tumours. fzd4 was also shown to be expressed in a developing lung from a mouse embryo. Clearly FZD4 has a role in lung development and this role needs to be better defined. A more defined role may help in the understanding of why this gene is down regulated in lung cancer progression. 5.3 Future Directions The purpose of this thesis was to identify genetic alterations occurring in early stage lung cancer. The use of genetic markers to predict disease progression and patient outcome are o f beneficial use to the clinician in order to treat patients appropriately. Ideally one would hope to find a genetic alteration that i f reverted could change a cancerous phenotype back to normal. Protein expression of the genes underlying the genetic alterations is the key to identifying the functional unit o f the cell. A mechanistic role of this protein is also important as this w i l l lead to a better understanding of the molecular workings inside the cell. The work of this thesis has established a few genetic alterations involved in early lung carcinogenesis but much work must be done to better understand the role o f these alterations in a tumour setting in order for them to be of use in a clinical practice. Understanding how fzd4 is being down regulated within a tumour cell could help researchers identify the important aspects o f the genetic make-up of this gene. Gene silencing may be occurring through epigenetic factors such as D N A hypermethylation or histone deacetylation. There could be mutations in the promoter region of the gene which could disable transcription factors from binding thus causing a decrease or complete inactivation o f the gene. Other mutations that could occur are mutations in the gene 114 sequence leading to either a faulty protein sequence or a truncated protein. The protein being produced may not be able to get to or leave the endoplasmic reticulum because it is being recognized as a defective protein. Other mutations could be in the 3' untranslated region (UTR) region of the gene. Therefore, discovering the mutations and/or the transcription factors associated with the expression of fzd4 could lead to a potential prognostic marker or a better understanding of the molecular features which could aid in treatment strategies. Iffzd4 is a strong tumour suppressor gene then perhaps patients may benefit the most, due to time constraints, i f understanding its role in tumour progression occurred first. Because there is evidence that this gene is down regulated in tumourogenesis perhaps by expression studies one could over express fzd4 in a tumour cell line and determine i f it has the potential to revert the cell line back to a normal phenotype. Or vice versa, by introducing a dominant negative FZD4 protein into a normal cell does it have the potential to convert a normal cell into a cancerous one? Frizzled 4 has been shown to have altered m R N A expression in lung carcinomas. The next step would be to look at the protein expression and pattern o f FZD4. Using a proteomic approach it would allow for sub-cellular localization o f F Z D 4 , association with Wnt ligands or others, any modifications that occur to the protein, the rate at which these modifications occur, and any protein interactions. Understanding the native role of FZD4 and perhaps its role in lung development would be a great benefit. As pointed out in this thesis it is expressed in early lung development but what role does it have? In vivo work is necessary as quite often working in vitro is an artificial scenario. There are many cellular layers and many interacting features that are present in vivo which is impossible to replicate in a Petri dish. Selecting the appropriate animal model could help 115 determine i f there are any benefits to the treatment. If the treatment passes all expectations up to this point it would now be possible to take it to a clinical trial and apply the treatment to humans to determine i f there are any toxic effects and curative potential. S M A L L - P C R has identified multiple regions of interest involved in squamous cell carcinoma of the lung. In the other regions identified, 2p25.3, 13q33.1, 14q24.2 and 20p l3 , genes have not yet been tested for altered m R N A expression. A greater cohort o f patient samples to test altered expression would further validate any findings. Genes that were tested at 9q21.33, gasl and dapk, should be followed-up using the same methodology as described above. The most important thing researchers must do is follow-up and determine a role for each gene individually to determine its role and potential in carcinogenesis. 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Cell 106 (2):781-92. 133 Appendix I*: Non-small Cell Lung Cancer TNM Staging Guide T= Primary Tumour N = Lymph Nodes M = Distant Metastasis T X : Primary tumour cannot be assessed N X : Lymph nodes cannot be assessed M X : Metastasis cannot be assessed TO: No evidence of primary tumour NO: No nodal invasion MO: No metastasis present Tis: Carcinoma in situ (local non-invasive tumour) N l : Nodes found in lung on the same side as primary tumour M l : Disease has spread to distant organs T L A tumour that is 3 cm or less in greatest dimension, no proximal invasion N 2 : The tumour has spread to lymph nodes in lung and chest on same side T2: A tumour more than 3 cm in diameter or invading pleura or involves the main bronchus N 3 : The tumour has spread to lymph nodes in chest on opposite side of primary tumour or lymph nodes in neck on either sides T3: Tumour o f any size with extension into the chest wall, diaphragm, mediastinal T4: Tumour of any size with invasion of mediastinal organs or vertebral body *Derived from Greene et al.(2002) 134 Appendix II: List of Patient Profiles that were Available from the Archival Samples Patient Sex Birthdate Smoking Status Pack Years 1 F 8/17/28 Current Smoker 42 2 F 3/18/28 Current Smoker 96 3 F 7/19/38 Current Smoker 23 4 F 10/18/32 Current Smoker 36 5 F 1/18/37 Current Smoker 50 6 M 1/24/38 Current Smoker 66 7 M 1/14/23 Current Smoker 67.5 8 M 7/10/40 Current Smoker 35 9 M 7/17/13 Current Smoker 64 10 M 1/10/23 Current Smoker 61 11 M 8/21/30 Current Smoker 70.83 12 M 5/21/27 Current Smoker 70.5 13 M 11/7/46 Current Smoker 34 14 M 10/7/38 Current Smoker 31.25 15 F 2/5/38 Previous Smoker 50 16 F 6/23/28 Previous Smoker 45 17 F 10/6/18 Previous Smoker 108 18 F 9/9/22 Previous Smoker 75 19 M 10/14/18 Previous Smoker 32 20 M 6/11/44 Previous Smoker 47 21 M 1/23/24 Previous Smoker 100 22 M 1/19/37 Previous Smoker 80 23 M 5/16/08 Previous Smoker 10 24 M 7/12/31 Previous Smoker 48 25 M 3/19/19 Previous Smoker 75 26 M 6/15/29 Previous Smoker 123 27 M 11/14/32 Previous Smoker 64 28 M 7/13/38 Previous Smoker 107.3 29 M 3/26/15 Previous Smoker 43 30 M 10/20/43 Previous Smoker 30 31 M 7/8/21 Previous Smoker 47.25 32 M 2/11/20 Previous Smoker 50 33 M 10/6/46 Previous Smoker 72 34 M 11/2/32 Previous Smoker 33 35 M 5/13/34 Previous Smoker 45 36 F 8/3/52 Never Smoker 0 37 F 8/27/30 Never Smoker 0 38 F 1/29/30 Never Smoker 0 39 F 10/22/58 Never Smoker 0 40 F 6/7/60 ? ? 41 M ? ? ? 135 Appendix III: Patient Cohort Available for SMALL-PCR Patient Case Grade 1 44 8.2 45 3.1 *2 1 6.1 2 7.1 3 6.1 4 6.1 5 6.1 6 6.1 7 6.1 8 6.1 9 6.1 10 6.1 113 1 3 24 1 25 6.1 26 6.1 89 6.1 90 6.1 154 5.4 *4 27 2.2 28 6.1 88 6.2 163 9.4 5 135 8.1 136 3.2 137 4.2 138 3.2 6 46 8.1 114 1 7 47 8.1 48 3.1 *8 149 6.1 150 5.2 151 1 152 8.1 153 8.1 *9 86 1 87 6.1 161 1 10 11 1 12 6.2 13 6.1 *11 51 8.5 49 8.5 50 3.1 Patient Case Grade 12 52 8.2 53 8.2 115 1 *13 63 8.1 64 3.1 **14 54 3.1 55 8.3 15 35 1 36 6.1 37 6.1 38 6.1 39 6.1 40 6.1 16 56 8.5 116 1 17 57 8.1 58 1 18 59 8.4 60 8.4 93 3.1 *19 14 6.1 15 6.1 18 6.1 16 3.1 17 1 *20 61 1 62 8.1 *21 68 1 69 6.1 70 7.1 160 3.1 71 6.1 22 79 6.2 80 3.1 *23 82 6.1 83 8.1 84 6.1 85 3.1 24 102 6.1 103 6.1 104 6.1 105 1 25 95 6.1 96 7.1 97 8.1 Patient Case Grade 25 98 3.1 99 6.1 100 6.1 101 6.1 26 155 5.2 156 6.1 157 6.1 158 6.1 *27 126 6.1 127 6.1 128 6.1 129 6.1 130 6.1 131 6.1 132 1 28 106 6.1 107 5.4 108 6.1 109 1 110 6.1 111 8.1 112 6.1 139 4.2 140 5.2 141 4.2 142 5.2 29 133 7.1 134 3.1 30 122 6.1 123 6.1 124 1.0 125 8.3 31 118 6.1 119 1.0 32 41 7.1 42 7.1 164 5.4 165 5.4 166 4.2 167 5.2 168 5.4 169 5.1 172 5.1 173 4.2 174 4.2 Patient Case Grade 32 177 6.1 178 6.1 179 6.1 180 6.1 43 1.0 170 4.2 171 4.1 175 4.2 176 4.2 *33 75 8.1 76 6.1 77 6.1 78 6.1 74 6.1 81 1 *34 143 6.1 144 6.1 145 6.1 146 6.1 147 1 148 5.4 159 3.1 35 31 8.2 32 1 29 8.2 30 1 *36 19 6.1 20 6.1 21 6.1 92 3.2 162 3.2 37 22 6.1 94 9.4 23 1 38 72 6.1 73 1 117 1 *39 120 8.1 121 1 40 65 6.1 66 3.1 67 5.4 41 33 7.1 34 6.1 91 9.4 *Patients in which a SMALL-PCR alteration was detected **Patients that were screened by SMALL-PCR but no alteration was cloned 136 Appendix IV: Lung SCC Lesion Grading System Grade Phenotype 1 Normal 2 Inflammation 2.1 Polys 2.2 Eosinophils 2.3 Plasma cells 2.4 N / A 2.5 Lymphocytes 2.6 Granulomatous 3 Hyperplasia 3.1 No mataplasia 3.2 Metaplasia 4 Mild Atypia 4.1 No mataplasia .4.2 Metaplasia 5 Moderate Atypia 5.1 No mataplasia 5.2 Metaplasia 5 Severe Atypia 5.3 No mataplasia 5.4 Metaplasia 6 Carcinoma in situ 6.1 Squamous 6.2 Glandular 6.3 Other 137 Grade Phenotype 7 Microinvasive 7.1. Squamous 7.2 Glandular 7.3 Other 8 Carcinoma 8.1 Squamous 8.2 Glandular 8.3 Large Cel l 8.4 Small Ce l l 8.5 Other 9 Unsatisfactory 9.1 Too small 9.2 Wrongly oriented 9.3 Epithelium lost 9.4 Epithelium incomplete 9.5 Other 9.6 Not a deep cough 9.7 Too few cells 9.8 Poorly preserved Obscured by 9.9 inflammation Appendix V*: Representation of Microsatellite Analysis performed at D11S1780 1 2 3 4 5 6 7 8 N T N T N T N T L N T N T N T N T ^ 175 bp 9 10 11 12 13 N T L N T N T N T N T * Fragment length of polymorphism in the human population for this marker is between 173-191 bp. The 25 bp ladder (L) used as a size marker is emphasized at the 175 bp fragment with a red arrow. Normal (N) and tumour (T) samples for each patient (number) are displayed side by side. 138 Appendix VI: Case Alteration Frequency Identified by SMALL-PCR *Patient Case Biopsy Site **Grade ***SMALL Alteration Chromosome 2 3 L M B 6.1 K-5 20pl3 2 4 R U L 6.1 K - l 20pl3 2 6 Trachea 6.1 J-1 13q33.1 2 8 6.1 K-2 20pl3 2 10 6.1 K-3 20pl3 4 28 R L L 6.1 K-4 20pl3 8 153 R U L 8.1 0-1 9q21.33 9 87 L B 1+2 6.1 0 - 2 , 0 - 3 9q21.33 11 51 8.5 J-2 13q33.1 13 63 L U L E N 8.1 0-4 9q21.33 19 14 R B 2 6.1 J-3 13q33.1 19 15 R B 3 A 6.1 J-4 13q33.1 19 16 R B 2 6.1 J-5 13q33.1 19 18 R B 3 A 6.1 J-6 13q3.1 20 62 8.1 J-7 13q33.1 21 71 6.1 G - l 14q24.2 23 84 R U L E 6.1 E - l , G-2 l l q l 4 . 2 , 14q24.2 23 85 L B 1+2 6.1 H - l 2p25.3 27 126 L U L 6.1 A - l 13q33.1 27 127 L U L E 6.1 A-2 13q33.1 27 131 M C 6.1 A - 3 , H-2 13q33.1,2p25.3 33 75 R B 7 8.1 G-3, H-3 14q24.2, 2p25.3 34 143 P O S T R M B 6.1 A-4 13q33.1 34 145 R M L 6.1 A-5, H-4 13q33.1,2p25.3 34 146 R U L B 6.1 A-6 , G-4 13q33.1, 14q24.2 36 20 6.1 E-2 l l q l 4 . 2 39 120 L B 9 8.1 A-7 , E-3 13q33.1, l l q l 4 . 2 * Number is reference for Appendix II and III * * See Appendix IV for lung S C C grading * * * See Table 7, p.51 for S M A L L - P C R alteration descriptions Table Key L M B = Left middle bronchus R U L = Right upper lobe R L L = Right lower lobe L B = Left bronchus R B = Right bronchus 139 

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