Targeting P16INK4A In Uterine Serous Papillary Carcinoma Through Inhibition Of Histone Demethylation

BACKGROUNDEndometrial cancer is one of the most common gynecologic malignancies worldwide.Based on the current pathologic classification,endometrial cancer is mainly categorized into two types:Type â… ,the uterine endometroid carcinoma(UEC),and Type â…¡,mostly the uterine serous papillary carcinoma(USPC).The UEC,which is the most common type,is moderately or well differentiated,and usually has favorable prognosis.While USPC,accounting for 5 to 10 percent of the whole endometrial cancers,is highly aggressive and associated with poor outcomes.Compared to its counterpart UEC,USPC has tendency to develop radioresistance and chemoresistance after primary surgeries,especially in the advanced stage.As most USPC is hormone-receptor negative,USPC patients rarely benefit from the available hormone agents.Additionally,the current limited targeted therapies are principally designed and indicated for UEC.Therefore,there is an urgent need to search for potential targets and develop treatment regimens for USPC.P16INK4A,encoded by CDKN2A gene,has been reported as a tumor suppressor,as it can inhibit Cyclin-dependent kinase 4/6(CDK4/6)and arrests the cell cycle.Thus,deficiency of P16INK4A plays an important role in the development of many cancers.However,several other reports indicate that P16INK4A is frequently overexpressed in tissue of USPCs and can serve as one of protein markers for differential diagnosis of USPCs from UECs.Some studies have showed that P16INK4A is required for the cell proliferation and migration in cervical cancer and liver cancer.In addition,P16INK4Aoverexpression has been found in some cancer types,including cervical cancer.Although its biological function in these tissue samples is not well elucidated,researchers have identified P16INK4A might play a crucial role in the survival of cervical cancer cells.Also,in another study,P16INK4A has been reported to promote the migration of liver cancer cell.These aforementioned findings suggest that,P16INK4A might exert distinct biological function either as a tumor suppressor or oncogenic protein in a tissue-specific manner.It has been reported that P16INK4A expression is regulated by the methylation level of histone 3 lysine 27(H3K27).Targeting histone lysine demethylase(KDM)6B by GSK-J4,a KDM6B inhibitor,increases the level of H3K27 trimethylation,which is an effective approach to curtail P16INK4A expression and consequently reduce cell proliferation.Thus,P16INK4 A is worthy of being considered as a therapeutic target for some P16INK4A positive cancers.OBJECTIVESIn this study,we aim to examine the biological functions of P16INK4A in USPC and to explore if targeting KDM6B could be used as a potential therapeutic option for the treatment of endometrial cancer.METHODSWe first screened P16INK4A expression by western blot analysis of 5 endometrial cancer cell lines and selected ETN-1 for further investigation due to its high level of P16INK4A expression.Using shRNA targeting P16INK4A or RB,we downregulated P16INK4A or RB in ETN-1 cells and examined the effects of P16INK4A or RB knockdown on cell viability and migration via 2D/3D clonogenic and wound-healing assays.Finally,we used ex vivo culture of patient derived xenograft system to evaluate the treatment effects of GSK-J4,a histone demethylase inhibitor,on endometrial tumor tissues.RESULTSAfter P16INK4A was knocked down,proliferation and migration of ETN-1 cells declined significantly compared to that of controlled cells.The same phenomenon was also observed after knocking down RB in ETN-1.When exposing to GSK-J4,the levels of P16INK4A were almost completely abrogated and cell viability was significantly reduced in both ETN-1 cells and ex vivo cultured PDX tumor explants.We also demonstrated the correlation between the levels of P16INK4A and the methylation status of histone 3 lysine 27(H3K27)in both ETN-1 cell line and human USPC tumor samples.CONCLUSIONP16INK4A is oncogenic in most endometrial serous cancers.The level of P16INK4A is regulated by methylation status of H3K27.Augmenting methylation of H3K27 is able to downregulate P16INK4A which,in turn,will eventually reduce cell proliferation and invasiveness in endometrial cancer.Our findings may shed light on the development of a potential therapeutic strategy for P16INK4A-positive endometrial cancer,especially the USPC.