Clinical Significance and Molecular Features of the Serrated Colorectal Cancer Pathway

Abstract

Summary Colorectal cancer (CRC) affects approximately 150,000 Americans and 2716 New Zealanders annually, and approximately one third of Americans and 45 percent of New Zealanders will eventually succumb to the condition. In the past, CRC has been one of the best understood malignancies in terms of its molecular alterations. The first step of carcinogenesis involves the development of pre-malignant lesions (polyps) in the colonic mucosa. Certain histological features of the polyp determine its malignant potential, and the risk of cancer will be significantly reduced if all the precursor lesions are removed endoscopically. However, despite close surveillance, many studies have shown that a significant number of CRCs will still develop. This could be because some cancers are missed on prior endoscopy, their precursor lesions are not easily recognised at the time of colonoscopy, or they may be a result of rapid development of cancer. There are two major polyp subtypes which serve as precursors to colorectal cancers (adenoma and serrated polyps). Colorectal cancers mostly arise from adenomas but a lesser proportion can arise from serrated polyps. Once thought to be harmless, the spectrum of serrated colorectal polyps includes some potentially dangerous lesions, such as the sessile serrated adenoma/polyp (SSA/P) and the traditional serrated adenoma (TSA). SSA/Ps develop through a distinct molecular pathway featuring proto-oncogene B-Raf or v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutations, hypermethylation of CpG sites in gene promoters, and loss of expression of mismatch repair gene MLH1. The first part of this thesis focuses on determining the natural history of serrated neoplasms, and the endoscopic detection and treatment of these lesions in Cleveland Clinic, Ohio. The main body of the thesis is based on the hypothesis that hypermethylation of CpG islands results in epigenetic silencing of specific microRNAs (miRNAs) that function as tumour suppressors, and this silencing results in increased cell growth and development of serrated neoplasia. Methods and Results In the first part of thesis I have determeined the natural history of the serrated polyps, compared their detection rate with the adenoma detection rate, and have demonstrated that endoscopic removal of these precursor lesions is feasible. microRNAs are post-transcriptional regulators that may function as tumor suppressors. Previously, with global DNA methylation screening, miR-1247 is found to be hypermethylated in three methylated tumors when compared with three non-methylated tumors and normal colonic mucosa. By TaqMan® quantitative polymerase chain reaction (PCR), this was further confirmed in 20 tumours of each group. Treatment with the DNA de-methylation reagent, 5-Aza-2′-deoxycytidine (5’-Aza), has resulted in upregulation of miR-1247 expression in CRC cell lines. Transiently transfected miR-1247 cells have shown reduced cell growth and less cell migration in vitro. Moreover, stable transfected miR-1247 cells have significantly impaired tumour development when implanted into immunocompromised mice. These results strongly suggest that miR-1247 is a tumor suppressor. Demethylation with 5’-Aza has significantly decreased tumor growth in nude mice. The predicted gene target of miR-1247 is E3 ubiquitin-protein ligase MYCBP2 also known as myc-binding protein 2 or protein associates with myc (PAM), a significant decrease in MYCBP2 protein levels has been seen in cells transfected with miR-1247. Moreover, inhibition of endogenous miR-1247 by a miR inhibitor significantly has increased MYCBP2 expression. Direct targeting of MYCBP2 3’UTR by miR-1247 is confirmed with luciferase assays. Most importantly, with immunohistochemistry staining, a reciprocal relationship between MYCBP2 expression and miR-1247 level in seven methylated versus non-methylated cancers has been demonstrated. Conclusion This thesis has demonstrated that epigenetic regulation plays an important part in the tumorigenesis of serrated CRC. DNA hypermethylation is a key feature in this subtype of colorectal CRC, which in turn suppresses miR-1247 expression and causes an increase in MYCBP2 and its downstream MYC protein. The findings of this thesis open up a whole new area for therapeutic approaches in the treatment of serrated colorectal cancer.