Epigenetic Silience Of RASSF10Promotes Colorectal Cancer Growth By Suppressing P53Signaling

Background: Colorectal cancer is a common malignancy. About1.2million newcases were diagnosed and over600,000patients were died each year. The pathogenesisof colorectal cancer remains unclear. Accumulation of gene mutation and epigeneticchanges are regarded as important factors of colorectal cancer. Epigenetic changes aredrawing attention because it is revisable and epigenetic related therapy becomespossible. RASSF proteins were originally designated on the basis of sequencehomology to domains that associate with Ras-like small GTP-binding proteins. Thesedomains are known as Ras association (RA) domains and are distinct from Ras-bindingdomains (RBD) which bind an alternative set of Ras efectors. There are now10members in the RASSF family (RASSF1-10) subdivided into two distinct subgroups,the classical RASSF proteins (RASSF1-6) and the N-terminal RASSF proteins(RASSF7-10) based on the location of the RA domain. RASSF10gene is located onchromosome11p15.2and contains an over2kb CpG island in the promoter region.RASSF10is frequently methylated in melanoma, thyroid cancer, leukemia, glioma andgastric cancer. However, the epigenetic change and the function of RASSF10incolorectal cancer remain unclear.Objective: To further understand the methylation status, expression, function andmechanism of RASSF10in colorectal cancer.Method: Eight colorectal cancer cell lines,89cases of primary colorectal cancerand5cases of normal colorectal mucosa were involved in this study. Semi-quantitativeRT-PCR was employed to detect the expression of RASSF10before and after thetreatment of5-aza-dc. Methylation specific PCR was employed to examine themethylation status of RASSF10. The function of RASSF10in colorectal cancer wasexplored by MTT, colony formation, flow cytometry and xenograft mice. Gene expression array was employed to explore the possible downstream genes regulated byRASSF10. The results of gene expression array were verified by RT-PCR, western blotand Immunohistochemistry.Results: Loss of RASSF10expression and complete methylation were found inLOVO, RKO and HCT116cells. Expression or reduced expression and partialmethylation were found in LS180, DLD1, HT29, SW480and SW620cells. Restorationor increased expression of RASSF10was found in these colorectal cancer cells by5-aza-2’-deoxycytidine treatment. RASSF10was methylated in60.7%(54/89) ofprimary colorectal cancer. Methylation was associated with tumor stage (p<0.05) andmetastasis (p<0.05). Re-expression of RASSF10inhibited cell proliferation and inducedapoptosis and G2/M phase arrest in RKO and HCT116cells. HCT116cells tumorxenograft was suppressed by RASSF10. Reduced expression of MDM2was revealed bycomparison of gene expression profile in RASSF10expressed and unexpressed RKOcells. RASSF10activated P53signaling in RKO and HCT116cells after UV damage.The expression of MDM2and bcl-2was downregulated and the expression of P53,cleaved caspase-3and bax was upregulated after re-expression of RASSF10. Oppositeresults were found after knockdown of RASSF10. RASSF10sensitized RKO andHCT116cells to docetaxel.Conclusions: RASSF10is frequently methylated in human colorectal cancer andthe expression was regulated by promoter region hypermethylation. Methylation ofRASSF10is associated with late tumor stage and metastasis. RASSF10suppresseshuman colorectal cancer growth by activating P53signaling in colorectal cancer.RASSF10sensitizes colorectal cancer to docetaxel.