Epigenetic Alterations Of A Novel Tumor Susceptibility Gene SLC22A3 In Familial Esophageal Cancer

Objective: Esophageal squamous cell carcinoma(ESCC)occurs frequently among the Chinese population,especially the high-risk Northern Chinese.Our previous studies have reported that SLC22A3 is significantly down-regulated in non-tumor(NT)esophageal tissues from familial ESCC patients compared with those from sporadic ESCCs.However,the mechanism of how SLC22A3 regulates familial ESCC remains unknown.SLC22A3(or organic cation transporter 3,OCT3)is located on human 6q,is a part of IGF2R-SLC22A2-SLC22A3 gene cluster.The SLC22A3 gene encodes for a cation transport protein belonging to the SLC22 A family(SLC22A1-3 or OCT1-3),which is critical for drug transportation and cellular detoxification.Gene structure analysis found that the promoter region of SLC22A3 is located within a Cp G island,suggesting that regulation of this gene may largely depend on methylation marks.Therefore,we aim to explore whether SLC22A3 promoter methylation is associated with its transcription and confers susceptibility to ESCC.Considering the majority of ESCC patients have eating habits of hot food and drink,we will also explore the effect of reduced SLC22A3 expression in response to heat-stress in normal human esophageal epithelial cells.Methods: To test and verify the association between SLC22A3 and ESCC in familial ESCC high-risk individuals,we performed a post hoc GWAS analysis to investigate the influence of common genetic variants around the SLC22A3 gene region on ESCC risk.As SLC22A3 promoter region contains a Cp G island,we speculated that SLC22A3 down-regulation in ESCC is associated with its promoter methylation.Two ESCC cell lines(KYSE30 and KYSE140)without SLC22A3 expression were treated with a DNA methyltransferase inhibitor(5’-Aza).The m RNA expression level of SLC22A3 after the Aza treatment was examined and the relationship between SLC22A3 expression and DNA methylation was also investigated.The methylation pattern of SLC22A3 was further analyzed in more detail in ESCC cell lines,peripheral blood and tissues by using bisulfite genomic sequencing(BGS)and real-time methylation-specific PCR(q-MSP).Furthermore,we looked into the antioxidant function of SLC22A3 by knocking down the endogenous SLC22A3 in normal esophageal cell lines NE1 and NE3.We then performed heat-shock treatment and detected the ROS level and DNA damage status in normal esophageal cells with or without SLC22A3 expression.Results: We demonstrated that SLC22A3 is a susceptibility gene for ESCC;SLC22A3 promoter methylation is one of the regulatory mechanism of ESCC;SLC22A3 methylation confers susceptibility to ESCC in a high-risk population;SLC22A3 protects normal esophageal cells from heat stress-induced reactive oxygen species(ROS)and DNA damage.Conclusion: Our results show that SLC22A3 is a novel tumor susceptibility gene and anti-oxidant gene in ESCC.Promoter hypermethylation of SLC22A3 may serve as a potential diagnostic and prognostic biomarker for high-risk ESCC population.