Genome-wide DNA methylation in relation to ARID1A deficiency in ovarian clear cell carcinoma

Abstract Background: The poor chemo-response and high DNA methylation of ovarian clear cell carcinoma (OCCC) have attracted extensive attentions. Recently, we revealed the mutational landscape of the human kinome and additional cancer-related genes and found deleterious mutations in ARID1A , a component of the SWI/SNF chromatin-remodeling complex, in 46% of OCCC patients. The present study aims to comprehensively investigate whether ARID1Aloss and genome-wide DNA methylation are co-regulated in OCCC and identify putative therapeutic targets epigenetically regulated by ARID1A. Methods: DNA methylation of ARID1A mt/ko and ARID1A wt OCCC tumors and cell lines were analyzed by Infinium MethylationEPIC BeadChip. The clustering of OCCC tumors in relation to clinical and mutational status of tumors were analyzed by hierarchical clustering analysis of genome-wide methylation. GEO expression profiles were used to identify differentially methylated (DM) genes and their expression level in ARID1A mt/ko vs ARID1A wt OCCCs. Combining three pre-ranked GSEAs, pathways and leading-edge genes epigenetically regulated by ARID1A were revealed. The leading-edge genes that passed the in-silico validation and showed consistent ARID1A -related methylation change in tumors and cell lines were regarded as candidate genes and finally verified by bisulfite sequencing and RT-qPCR. Results: Hierarchical clustering analysis of genome-wide methylation showed two clusters of OCCC tumors. Tumor stage, ARID1A/PIK3CA mutations and TP53 mutations were significantly different between the two clusters. ARID1A mutations in OCCC were related to DM promoter or gene-body CpG islands of 2004 genes. Three pre-ranked GSEAs collectively revealed the significant enrichment of EZH2- and H3K27me3-related gene-sets by the ARID1A -related DM genes. Leading-edge DM genes (238) were extracted from the enriched gene-sets, of which 13 passed the expression-based in-silico validation and showed consistent ARID1A -related methylation change in tumors and cell lines. Bisulfite sequencing and RT-qPCR analysis showed promoter hypermethylation and lower expression of IRX1 , TMEM101 and TRIP6 in ARID1A mt compared to ARID1A wt OCCC cells, which was reversed by 5-aza-2′-deoxycytidine treatment. Conclusions: Our study shows that ARID1A loss is related to the differential methylation of a number of genes in OCCC. ARID1A -dependent DM genes have been identified as key genes of many cancer-related pathways that may provide new candidates for OCCC targeted treatment.