Role of CC3 in colorectal cancer progression

PEK LI TING, SHARON

Publication

Role of CC3 in colorectal cancer progression

PEK LI TING, SHARON

Abstract

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-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 - 9 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 (5-Aza), 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 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.