Study of Novel Genes Regulated by Aberrant DNA Methylation and Application in Colon Cancer Treatment

Background and Significance Colorectal cancer (CRC) is the most common malignant neoplasm in Taiwan and a leading cause of cancer death in the United States, Taiwan, and worldwide. Colorectal cancer death rates have been decreasing in several Western countries as early detection methods improve. Therefore, biomarkers that enable early detection and intervention are needed to improve patient outcomes. New blood-based biomarkers for early detection of CRC are urgently needed. Aberrant promoter hypermethylation of CpG islands associated with TSGs can lead to transcriptional silencing; abnormal gene body methylation associated with oncogene transcriptional activation, resulting in tumorigenesis. Genomic screening of 98 different primary human tumors indicates that, on average, each tumor has approximately 600 aberrantly methylated CpG islands. Recent studies indicate that several genes are often methylated in the multiple-step process from normal colonic epithelium to adenocarcinoma. The evaluation of therapeutic efficacy is necessary to predict the outcome of patients with predictive chemosensitivity assays and biomarkers to optimize efficacy and minimize toxicity in patients. Genome-wide methylation patterns have been identified by Illuminar 450k methylation array in twenty-six paired tumor and matched normal tissues from Taiwan colon cancer patients. Comparisons of methylation patterns in another 10 paired USC colon cancer patients obtained from the Cancer Genome Atlas (TCGA) website showed substantial differences in heavy methylation loci between Taiwan and U.S.A. colon cancer patients. For example, promoter region of BEND5 gene showed 53.8% (14/26) hyp ermethylation in Taiwan patients, but only 30% (3/10) hypermethylation in U.S.A. patients. The data indicated the need for further comprehensive genome-wide methylation studies in more colorectal cancer patients in Taiwan to develop new biomarkers for early prediction, therapeutic efficacy and clinical outcome prediction, and novel drug targets. The study has found that a candidate tumor suppressor gene, BEND5, showed 82.35% (70 of 85) hypermethylation in colorectal tumors compared to matched normal colorectal tissues. Especially in patients with metastasis in regional lymph nodes, the percentage was 95.2% (20/21) (P = 0.030). In addition, the 71% (24 of 34) of BEND5 mRNA expression was down-regulated in CRCs compared to matched normal colorectal tissues. Low expression of BEND5 protein was associated with poor prognosis (P = 0.018). Transient transfection of BEND5 and/or si-BEND5 found that BEND5 inhibits colon cancer cell proliferation. Specific aims and Study design: Specific aim 1: To discover novel cancer-associated genes that are inhibited by aberrant DNA methylation in colon tumorigenesis by performing Illumina Methylation 450K array-based assay in 50 colon cancerous tissues and in 50 paired normal colon tissues. Comparisons of methylation patterns between Taiwan and U.S.A. colon cancer patients to identify unique aberrantly genome-wide methylation loci of colon cancer in Taiwan. Specific aim 2: a. To verify whether the aberrant DNA methylation levels of novel cancer-associated genes can be detected in another 150 colon cancerous tissues and matched plasma samples but not in matched normal colon tissues and not in plasma samples from healthy persons. b. To determine whether aberrant methylation alters the expression of specific cancer-associated genes, this study analyzed the DNA methylation level, mRNA and protein expression level of cancer-associated genes. c. To evaluate the potential use of the novel cancer-associated genes as biomarkers, this study analyzed the association between the aberrant DNA methylation and clinicopathological parameters, clinical outcome, including disease-free survival, recurrence-free survival, and chemotherapy response. Specific aim 3: To investigate the mechanisms of DNA methylation alterations in cancer-associated genes, the proposed study will investigate whether aberrant DNA methylation is associated with methyltransferases and demethylase-associated enzymes. a. We will also examine mRNA and proteins expression of DNA methyltransferase genes (DNMTs), tet methylcytosine dioxygenase (TETs) and thymine-DNA glycosylase (TDG). b. Finally, we will further clarify the association between aberrant DNA methylation and colon cancer risk factors, including diet, smoking, alcohol, and exercise. Specific aim 4: To study the role of novel cancer-associated genes in colon tumorigenesis and to evaluate whether cancer-specific genes are potential drug targets, we will manipulate the expressions of siRNA, sh-RNA or exogenous genes in cells. We will also examine cellular phenotypic changes including cell proliferation, cell cycle progression, migration and invasion, colony formation and apoptosis. Anticipated results and goals: Identifications of novel cancer-associated genes involved in colon tumorigenesis through aberrant DNA methylation in Taiwan and development of novel therapeutic potential drugs target for anti-cancer treatment. In addition, those cancer-associated genes also could have the opportunities to be provided as the critical targets for early detection, clinical outcome prediction.