ROLE OF CC3 IN COLORECTAL CANCER PROGRESSION PEK LI TING, SHARON (BSc (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF Ph.D DEPARTMENT OF PATHOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgement The first person that comes to my mind is definitely my supervisor, Dr Robert Hewitt. I sincerely thank Robert for his consistent understanding and encouragement over the last four years. Although I was given a framework to follow in the beginning of my PhD, Robert has given me lots of freedom in the course of my research, allowing me to explore areas of my interest. I was also provided opportunities to be involved in writing research grants and choosing conferences I was interested in. Even though my research work revolved around cancer studies and molecular biology, that has not stopped Robert from getting me involved in tissue-banking conferences. Being an avid reader himself, Robert has always shared his thoughts from reading books and lets us think beyond doing bench work and writing publications. I would also like to thank Dr Eng Chon Boon, molecular biologist of NUS-NUH Tissue Repository (currently the Director). He is the one with the most creative ideas in troubleshooting whenever I ran into trouble with cloning and PCR. He was always happy to give me a lift to work or back home at my convenience and that saved me lots of hassle and time. I would like to thank Dr Rajeev Singh, for his help in scoring immuno-histochemistry and teaching me basic histology. I appreciate the help from Wentong and Thiri, from NUH Cancer Registry, who have provided me with deidentified patient data for immunohistochemical studies. My labmates: Yibing, Kelly, Chiou Huey, Fiona, friends from neighboring labs, Lee Lee, Mary and Li Kian have been a constant source of encouragement and has given me lots of scientific input that sometimes helped in my experiments. I appreciate all the help from the administrative i staff from Pathology office, especially Rohana, who has given me lots of assistance from handling scholarship matters all the way to thesis submission. Lastly, I would like to thank my family for their support, even though they had no clue as to what I was really doing. Whenever the stress from research gets piling, it was always the comfort of home and the trust from my family, which gives me faith in what I do. With the wide exposure that I was given, I am confident that the skills I have learnt from this lab will continue to benefit me in the future. ii Table of Contents Acknowledgement i Table of contents iii Summary vii List of Tables x List of Figures xi Abbreviations xiii Chapter Introduction 1.1 Colorectal cancer statistics …………………………… .……….…… … … 1.2 The Anatomy of Normal Colonic Crypts ………………………….… .…… .1 1.3 Cancer is a disease of deregulated cell proliferation ………………………… .2 1.4 Prognostic Indicators in Colorectal Cancer ……………………….… 1.5 Apoptosis ………………….………………… 1.5.1 Intrinsic and Extrinsic apoptotic pathways ………………., .8 1.5.2 Measurement of Apoptosis and In Vitro Chemo-sensitivity and Resistance Assays ……………… ………….…… 13 1.6 Chemotherapeutic treatments 1.6.1 Therapeutic targeting of cell proliferation and apoptosis……………… 14 1.6.2 Determinants of responses to 5-FU .15 1.7 Epigenetics …………………………………… .19 1.7.1 DNA methylation …………… 19 1.7.2 Reactivating silenced genes ………………………… … .…… .21 1.7.3 Methods to study methylation ……………………….………………23 1.7.4 Early detection of cancer …………………… ….…… ……………26 1.8 CC3 ……………………………………………………… … .……….…… .26 iii 1.8.1 Functional Studies in CC3 …………………………… .… ………27 1.8.2 Studies of CC3 expression in tissues ……………….….….….…….29 1.8.3 Methylation of CC3 …………………………………………….… 31 1.9 Hypothesis, objectives and significance ………………….………………….32 Chapter Materials and Methods 2.1 CC3 monoclonal antibody production ……………………….…….…… ….34 2.1.1 Antigen selection ……………………………………… ….………34 2.1.2 Validation of CC3 monoclonal antibodies … .……… .… .… 35 2.2 Clinical Specimens ……………………………………………….… …… 36 2.3 Tissue microarray (TMA) ………………………………… ….….….… .…36 2.4 Immunohistochemistry in tissue sections ……………… ….….… .… … 37 2.5 Laser Capture Microdissection (LCM) …………………….……………… 38 2.6 Functional study of CC3 in colorectal cell lines 2.6.1 Cell lines ……………………………………… .…… ….…… .39 2.6.2 Generation of Stable Transfectants Overexpressing CC3 .… … .39 2.6.3 siRNA transfection …………………………………… ….………40 2.6.4 Cell viability (Trypan Blue exclusion and Cell Titer Blue) ….……40 2.6.5 Proliferation Assay ……………………………… ……….………41 2.6.6 Detection of apoptosis 2.6.6.1 Flow cytometric DNA content assessment assay using PI/RNAse A ………………………… … ……….….… 42 2.6.6.2 Flow Cytometric Apoptosis Assays with AnnexinV-FITC and 7AAD ……………… … …… …… .… 43 2.6.6.3 TUNEL ……………… … …………………… ….……43 2.6.6.4 DNA laddering … ………………… .……… .….……44 2.6.6.5 Immunofluorescence of anti-Bax in cell lines .………45 2.6.7 Caspase 3/7 activity ………………………………… .… ………45 2.6.8 Anchorage independence in ―soft agar‖ assay …… .…………….46 2.6.9 In vitro invasion assay …… .…………………………… ………46 2.6.10 Wound Healing assay …… .……………………… ….………47 2.7 RNA Isolation iv 2.7.1 RNA Isolation from Laser captured cells ……………….… ……47 2.7.2 RNA Isolation from cell lines ………………………… ….………48 2.8 cDNA synthesis ………………………………………………… .….………48 2.9 Real time PCR amplification …………………………………… .….………49 2.10 Semi-quantitative PCR amplification ………………………… … ……….50 2.11 DNA Isolation 2.11.1 DNA Isolation from Laser captured cells ………… …………….53 2.11.2 DNA Isolation from cell lines …………………… …………….53 2.12 Protein extraction 2.12.1 Total proteins ……………………….…………… ….………….54 2.12.2 Subcellular fractionation ………….………….… …………….54 2.13 Western Blotting and Immunodetection …….………….… ……………55 2.14 Sequencing of CC3 Exon …….……………………….… ……………57 2.15 Drug preparation …….……………………….… ….………58 2.16 Methylation studies on CC3 promoter in colorectal cell lines 2.16.1 CpG island and promoter analysis .… …………… …….……58 2.16.2 Bisulfite Treatment .… ………………… .……….…… ……59 2.16.3 TA cloning and sequencing …………… .…………….… ……59 2.16.4 Colony PCR screening …………… .…………………… ……60 2.16.5 Methylation-Specific PCR (MSP) …… ………………………61 Chapter Results 3.1 Novel monoclonal CC3 antibody is specific …………… .………… … 62 3.2 CC3 is expressed on the tips of normal colon mucosa ……………… 64 3.3 Mucinous carcinomas show decreased CC3 expression ……….……… 65 3.4 Downregulation of CC3 expression is associated with colon cancer progression ……………………………………… ….… 68 v 3.4 Immunohistochemical analysis of CC3 in human normal and tumor tissues ….70 3.5 Selection of Cells with LCM in Tumor Tissues ………………… ….79 3.6 Sequencing of CC3 exon in colorectal cancer tissues, cell lines and breast cancer cell lines …………… ………………………………………………… 80 3.7 Expression CC3 in 13 colorectal cell lines …………………… .….… … …81 3.8 Generation of CC3 over-expressing Colo320 and HCT116 cell line …… …. 83 3.9 Over-expression of CC3 inhibited cell growth ………………………… .….85 3.10 Reduction of cell growth was due to increased apoptosis ……………….… 88 3.11 Over-expression of CC3 reduced anti-apoptotic genes ………………….… 94 3.12 Induction of apoptosis by CC3 was in part due to Bcl-2 and Bcl-xl …….… 99 3.13 CC3 is up-regulated in colorectal cell lines upon 5-FU treatment …….….…101 3.14 Over-expression of CC3 sensitized cells to drug-induced cell death .…… .105 3.15 Caspase activity was sustained for a longer time course in CC3-transfected Colo320………………………………………………………….………… .…108 3.16 Sustained caspase activity in CC3 vector cells is, in part due, to increased active Bax …………………………………………………………………… …….…111 3.17 Hypermethylation of CC3 promoter is associated with transcriptional repression ……………………………………………… ….115 3.18 Inhibition of DNMT enzymes restored CC3 expression in Colo320 .118 3.19 Hypermethylation of CC3 promoter contributes to survival of Colo320 … .122 3.20 Over-expression of CC3 in HCT116 cell line suppressed cell invasion through extracellular matrix (ECM) …………………… ……………………………… 125 4. Discussion .128 5. Concluding remarks and significance of study . 144 6. References 146 vi Summary The CC3 gene was first identified as a novel tumor suppressor gene of variant small cell lung carcinoma (v-SCLC) as its over-expression was found to suppress the metastatic potential of v-SCLC cells. Consistent with its proposed function as a tumor suppressor gene, the CC3 gene is expressed at low levels in some highly aggressive tumor cell lines derived from gastric, SCLC and human hepatocellular carcinoma. While the tumor suppressive effects of CC3 had been extensively studied, a recent study in prostate cancer showed that increased CC3 expression was associated with invasion and metastasis. These findings suggest that CC3 may have bi-functional roles and its mechanism of action may be highly cell-type specific. Hence, it is necessary to investigate the role of CC3 in other cancer types in order to understand its function in human carcinogenesis. Colorectal cancer is the third most common cancer expected to occur and the third highest expected number of deaths from cancer. To this end, we developed a novel CC3 monoclonal antibody for immunohistochemistry. We characterized that CC3 acts as a tumor suppressor in colorectal cancer, where significant loss of CC3 was found in advanced colorectal cancer tissues, more aggressive mucinous sub-types and tumors which metastasized to the liver. In normal colon tissues, CC3 was stained strongly at the luminal surface of the crypt and negatively stained at the base of the crypts. This differential staining is consistent with its hypothesized function as a pro- vii apoptotic protein, where cells are the surface epithelium are committed to cell death and cells at base of crypts are rapidly proliferating. Over-expression of CC3 in colorectal cancer cell lines resulted in reduced cell growth and increased apoptosis as shown by various techniques. We show that these were, in part, due to a reduction in Bcl-2, Bcl-xl as well as an increase in caspases-3, -8 and gene products. Increased CC3 reduced invasiveness of cells as compared to cells transfected with empty vector. This reduction could be due to a reduction in matrix metalloproteinase-1. 5-Fluorouracil (5-FU)-based adjuvant chemotherapy has been efficacious in reducing mortality for lymph node positive colon. We demonstrated that CC3 also contributes to resistance to 5-FU. Work on a pair of isogenic cell lines showed that the resistant cell line was low in endogenous CC3 and upon 5-FU treatment, CC3-induces apoptosis via the mitochondrial pathway and causes cytochrome c release followed by activation of the caspases. In this dissertation, we also define possible mechanism of CC3 silencing by DNA hypermethylation. We demonstrated that the promoter of CC3 is hypermethylated, leading to a loss of CC3 expression. Upon treatment with 5-Aza-2'deoxycytidine (5Aza), a demethylating drug, CC3 expression was up-regulated. A combination drug regimen of both 5-Aza and 5-FU, further showed that CC3 was first up-regulated viii followed by increased cell death. This is consistent with our previous finding, where exogenous expression of CC3 and 5-Fluorouracil leads to increased cell death. Hence, induction of CC3 might be exploited as a therapeutic strategy, along with 5-FU-based combinatorial chemotherapy for colorectal cancer. ix Our studies of CC3 in clinical samples, its putative mechanism of action and its role upon 5-FU treatment have shown that CC3 acts as a tumor suppressor in colorectal cancer and has prognostic significance as well as predictive significance in 5-FU response. Due to its potential translational relevance, it was also beneficial to elucidate how CC3 was regulated. Since we and others have found no mutation in CC3 clinical samples and cell lines, we focused our research in epigenetic modulation of CC3. Even though our methylation studies did not include tissue specimens, it has been reported that the tumor-type specific CpG island hypermethylation profile is still maintained in the long-established cell lines(Smiraglia, Rush et al. 2001; Paz, Fraga et al. 2003). This shows that colon cancer cell lines are in general relevant in vitro models, comparable with the in vivo situation(Lind, Thorstensen et al. 2004). Using bioinformatics tools, a CpG island was found in the CC3 promoter. In both bisulfite sequencing and methylation specific PCR, we observe a correlation between high promoter methylation with a low CC3 expression. The presence methylation in CC3 CpG island in our work, as well as in hepatocellular carcinoma(Lu, Ma et al. 2008), together with its putative tumor suppressive role in cancer makes it an even more attractive candidate to evaluate its putative clinical usefulness as a diagnostic marker of disease. Furthermore, we used a pharmacological reactivation approach to show that CC3 was upregulated following 5-Aza-dC treatment in Colo320 cell lines. We recognize that 5Aza-dC, a nucleoside anti-metabolite and a potent inhibitor of DNA methyltransferase (Dnmt1), will have a global effect on other methylated genes(Soengas, Capodieci et al. 139 2001; Scott, Dong et al. 2006) and does not specifically target CC3. However, the results presented in this study demonstrate that exposure of Colo320 cell lines to 5-Aza-dC effectively demethylated the CpG regions within the CC3 promoter (Fig 3.22), leading to re-expression of CC3gene expression. We were also able to show that, upon silencing of CC3, the effect of 5-Aza-dC in enhancing chemotherapy sensitivity could be abolished (Fig 3.23), thus indicating that, as a consequence of CC3 promoter demethylation by 5Aza-dC, higher CC3 levels led to greater chemosensitivity and improvements in chemotherapy response. Since over-expression of CC3, in combination with 5-FU, appears to be efficacious in contributing to significantly increased apoptosis, our methylation work lead us to believe that the use of a demethylating agent which restores CC3 expression can also be an effective adjunct to chemotherapy in colorectal cancer and possibly other tumors with CC3 promoter hypermethylation. The clinical utility of 5-Aza-dC has been restricted to the treatment of leukemias and lymphomas(Karon, Sieger et al. 1973; Rivard, Momparler et al. 1981; Garcia-Manero, Kantarjian et al. 2006) but no significant effect has been demonstrated with solid tumors because of two major reasons: Firstly, its short half life prevents adequate accumulation deep within solid tumors, and secondly, high doses required to achieve clinical efficacy are associated with toxicity, like myelosuppression(Appleton, Mackay et al. 2007). However, our results suggest that a demethylating agent would function as a chemosensitizer, in part due to increased CC3 expression, and therefore, lower doses could be administered in combination with 5-FU to achieve the desired clinical response. 140 The observation that CC3 was reduced in metastasized colorectal tumors (Fig 3.4) implies that CC3 may be involved in invasion and metastasis. HCT116 cells stably expressing CC3 had significantly reduced invasive capacity through ECM with a concomitant reduction in MMP1 (Fig 3.24), as compared to empty vector cells. The ECM is regarded as a barrier to tumor invasion and increased MMP activity enables greater ECM degradation, thereby facilitating tumor invasion and poor prognosis in colorectal cancers(Wagenaar-Miller, Gorden et al. 2004). MMPs are normally induced at the level of transcriptional activation(Ye, Eriksson et al. 1996) hence the regulation of MMP1 by CC3 may be a novel pathway in invasion. This finding where CC3 acts upstream of MMPs, is supported by two recent reports in hepatocellular carcinoma(Jiang, Pecha et al. 2007) and lung carcinoma(Tong, Li et al. 2009). However, contrary to their findings, we did not see a significant difference in the motility of cells upon CC3 overexpression (Fig 3.25). We had mainly focused our work on elucidating the role of CC3 as a tumor suppressor and its function in response to 5FU treatment. Another possible function of CC3 is its indirect anti-angiogenic role(NicAmhlaoibh and Shtivelman 2001), which may contribute to metastasis. Using a PCR array to investigate possible molecular actions of CC3, we found a number of angiogenic factors that were up-regulated. For example, Col18a1(also known as endostatin) is a known endothelial specific angiogenic inhibitor(Sim 1998), which can block the VEGF-VEGFR/Flk-1 pathway to inhibit the angiogenesis of colon tumor in nude mice(Jia, Dong et al. 2004). CC3 also down-regulated FGFR2, a receptor 141 which works with FGF (Fibroblast Growth Factor) to induce angiogenesis in tumors(Seghezzi, Patel et al. 1998). Hence, CC3 may work both as a tumor suppressor and a metastasis suppressor. 142 Concluding remarks and significance of study This is the first report where the role of CC3 in colorectal cancer progression has been studied. Using a novel CC3 monoclonal antibody, we have shown that CC3 is downregulated in more advanced, aggressive and metastatic colorectal cancer. Furthermore, our pilot study in a panel of normal tissue shows that CC3 is ubiquitously expressed. This is also the first CC3 immunohistochemistry to be performed in a number of cancer subtypes compared with its normal tissues. More mechanistic work could focus on the regulation of CC3 in normal tissues and elucidate its physiological role. For example, it has been shown that TGF-β1 upregulates CC3(Lindfors, Halttunen et al. 2001) in small intestines. TGFβ is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs)(Di Sabatino, Jackson et al. 2009). Prior to this dissertation, much of the studies on CC3 have been focused on liver and lung cancers. In both cancers, CC3 was shown to act as a tumor suppressor by apoptotic and indirect anti-angiogenic mechanisms. It was proposed that CC3 may be involved in the mitochondrial pathway. Data obtained in our work have expanded the paradigm to colorectal cancer, where CC3 acts as a tumor suppressor to induce apoptosis, mainly by its regulation of Bcl-2 family members. In addition, it was shown previously in other cancers, that CC3 could sensitize cancer cells to apoptosis upon drug treatments. 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Methylation of cytosines in the 5’-position of the pyrimidine ring is the most important epigenetic alteration In animals, methylcytosine is mainly found in cytosine-guanine (CpG) 19 dinuleotides(Bird 1986) The presence of 5-methylcytosine in the promoter of specific genes prevents binding of transcription factors In addition, the presence of 5methyl group attracts methyl-DNA-binding proteins and histone... LCM of the Tumor Colorectal Samples 3.6 Expression profile of CC3 in colorectal cell lines 3.7 Over-expression of CC3 in HCT116 and Colo320 colon cancer cell lines 3.8 Over-expression of CC3 in Colo320 cells is associated with reduced cell viability while in HCT116, reduced anchorage-independent growth 3.9 Cell cycle analyses of empty vector and CC3 transfected Colo320 cell lines 3.10 Apoptosis of empty... association with cancer subtype 3.4 Cell cycle analyses of CC3 over-expression in Colo320 and HCT116 cells after low and high doses of 5-FU drug treatment x List of Figures 1.1 Normal epithelium of murine, showing colonic crypts and surface epithelium 1.2 Intrinsic and extrinsic pathway of apoptosis 1.3 Classification of Bcl-2 family proteins 1.4 Metabolism of 5-FU 1.5 Methylation of Cytosine in the Mammalian... common cancer expected to occur and the third highest expected number of deaths from cancer, accounting for 9% of all cancer deaths, projected for 2008 in both men and women(Jemal, Siegel et al 2008) In Asia, the epidemiology of colorectal cancer has changed and has a rapidly rising trend Data from the CancerBase of the International Agency for Research on Cancer (IARC) show that the incidence in many... proteins, except the most N-terminal, BH4 domain Group 3 consists of a large and diverse collection of proteins whose only common feature is the presence of the BH3 domain Antiapoptotic proteins (Bcl-2 and Bcl-xl) sequester the pro-apoptotic BH-3 domain only proteins in stable mitochondrial complexes, and thus prevent activation and translocation of Bax or Bak to mitochondria Initiation of the intrinsic... proteins inactivate the IAP (inhibitors of apoptosis proteins) protein family, which consists of XIAP, IAP1/2 and survivin XIAP is a direct caspase 13 inhibitor(Du, Fang et al 2000) Other IAPs including survivin, which is the one most differentially expressed between malignant and healthy tissue The tumor suppressor gene p53 not only mediates G1growth arrest by inducing the cyclin-dependent kinase inhibitor... the increase in proliferation due to CC3 silencing 3.16 Time-course treatment with 5-FU in colorectal cell lines 3.17 CC3 sensitizes Colo320 and HCT116 cells to drug-induced cell death 3.18 Caspase activities in 5-FU treated Colo320 empty and CC3- transfected cells 3.19 CC3 vector cells induced active Bax at earlier time points upon 5-FU treatment 3.20 CC3 promoter is methylated in Colo320 cell lines... decades in Singapore The rising trend in incidence and mortality highlights the importance of understanding the mechanism and molecular pathogenesis underlying colorectal cancer 1.2 The Anatomy of Normal Colonic Crypts In order to understand how tumorigenesis of the colon occurs, we have to appreciate the architecture of normal colonic crypt organization The colon epithelium is a single layer of highly... benign tumor, which can transform into a malignant cancer through a step-wise progression known as the Adenoma-Carcinoma Sequence About 2.5% of polyps will transition into cancer over a five-year period(Stryker, Wolff et al 1987) 1.4 Prognostic Indicators in Colorectal Cancer The most widely used prognostic indicator for colorectal cancer is tumor staging The original classification developed and subsequently... Genome and Inhibition of methylation with 5-Azacytidine 1.6 Methylation in normal and cancer cells 3.1 Validation of CC3 monoclonal antibody 3.2 Immunostaining of normal colon tissue Staining is exclusive to the tips 3.3 Immunohistochemistry on TMAs of colorectal cancer tissues 3.4 Matched primary tumor, metastases to lymph node and metastases to liver 3.4.1 Immunohistochemistry on TMAs of normal and . of the Tumor Colorectal Samples 3.6 Expression profile of CC3 in colorectal cell lines 3.7 Over-expression of CC3 in HCT116 and Colo320 colon cancer cell lines 3.8 Over-expression of CC3 in. ….79 3.6 Sequencing of CC3 exon 3 in colorectal cancer tissues, cell lines and breast cancer cell lines …………… ………………………………………………… 80 3.7 Expression CC3 in 13 colorectal cell lines ……………………. is necessary to investigate the role of CC3 in other cancer types in order to understand its function in human carcinogenesis. Colorectal cancer is the third most common cancer expected to