Epigenetics Of Colorectal Cancer: Multiple Gene Promoter Methylation Profile And Its Clinicopathological Significance

Epigenetics of colorectal cancer: multiple gene promoter methylation profile and its clinicopathological significancePh.D Candidate: Xu Xiao LiDirector: Prof. Shi Da RenObjectives: To study the epigenetics of colorectal cancer. 1. To investigate the multiple gene promoter methylation profile of both sporadic colorectal cancer (SCRC) and HNPCC (hereditary non-polypsis colorectal cancer). 2. To analyze the relationship between multiple gene promoter methylation profile and the clinicopathological features of colorectal cancer. 3. To investigate the effects of promoter methylation on protein expression of multiple genes in colorectal cancer. 4. To study the relationship between mismatch repair (MMR) gene hMLH1 promoter methylation status and the mismatch repair deficiency phenotype--microsatellite instability high (MSI-H) and loss of mismatch repair gene hMLH1 protein expression in colorectal cancer.Methods: Methylation specific PCR (MSP) was applied to detect 32 gene promoter methylation profile in 65 cases of colorectal cancers (including 39 sporadic colorectal cancer and 26 HNPCC), 8 colorectal adenomas, 16 non-cancerous counterpart mucosas of colorectal cancer, and 1 normal colorectal mucosa. Immunohistochemical test of 11 genes with distinct promoter methylation status was performed on 58 colorectal cancer using home-made tissue-microarray. Microsatellite instability of 62 colorectal cancers taken from the total 65 cases was assessed using GeneScan on ABI 310 Genetic Analyzer. Results: Out of the total 32 loci detected, 22 were methylated. At least 7 loci were methylated in each colorectal cancer, adenoma and non-cancerous mucosa, but only 4 loci N33, SFRP1, MAGEA1 and COX2 were methylated in the one normal mucosa. The number of methylated loci tends to increase from non-cancerous mucosa and adenoma to cancer. Patients older than 65 years and left site tumor have more loci methylated than other groups (p<0.05). Methylation of 9 loci ER, MGMT, hMLH1, P16, P14, APC, RASSF1a, MINT1 and MINT31 were only found in colorectal cancers and adenomas, and MGMT, p14, RASSF1a and MINT31 mathylation was not found in adenomas. ER methylated in all cancerous and adenomatous samples while no methylation was detected in non-cancerour mucosas. 61.5% detected CRC samples showed at least one of the 7 loci of MGMT, hMLH1, p16, p14, APC, RASSF1a, MINT1 and MINT31, and 33.8% showed at least one of MGMT, p14, RASSF1a and MINT31 was methylated. It was associated with patients' age older than 65 years (both p<0.05). The methylation frequency of MGMT (26%) and hMLH1 (23%) was higher than that of HNPCC (0 and 10% respectively), while MINT31 (10%) was lower than HNPCC (30%). p16 promoter methylation was associated with left tumor site and poor differetiation of CRC (p<0.05). 15.4% SCRC have 3 of loci MGMT, hMLH1, p16, p14, APC, MINT1 and MINT31 methylated synchronous (defined as CpG island methylator phenotype-CIMP+), and these were all lymph node metastasis cases, and also was related to poor differentiation. CDH13 un-methylation and poor differentiation of CRC associated with short survival time after surgical treatment (p<0.05). Both hMLH1 low expression and MSI-H showed trends of good prognosis of colorectal cancer. Reduced CDH13 and hMLH1 protein expression was related to their promoter methylation (p<0.05). 16% CRC was identified as MSI-H including 2 of SCRC and 8 of HNPCC. Out of the 2 sporadic MSI-H CRC, 1 had hMLH1 promoter methylation. One of the 5 HNPCC without hMLH1 mutation had promoter methylation. MSI-H was not associtated with overall multiple gene promoter methylation pattern.Conclusion: We concluded that MGMT, hMLH1, p16INK4a, p14ARF, APC, RASSF1a, MINT1 and MINT31 methylation is relatively specific in CRC and colorectal adenoma. MGMT, p14ARF , RASSF1a, and MINT31 were methylated only in colorectal cancer. Multiple gene promoter methylation profile of SCRC may be different to that of HNPCC. Aging, left tumor site and poor differentiation were the closest factors related to promoter methylation.