Department of Medical Oncology, Cancer Research Center Groningen, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.
Department of Gynecologic Oncology, Cancer Research Center Groningen, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.
J Transl Med. 2024 Jun 10;22(1):556. doi: 10.1186/s12967-024-05311-7.
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 ARID1A loss and genome-wide DNA methylation are co-regulated in OCCC and identify putative therapeutic targets epigenetically regulated by ARID1A.
DNA methylation of ARID1Amt/ko and ARID1Awt 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 ARID1Amt/ko vs ARID1Awt 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.
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 did not cause global DNA methylation changes but 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. 13 Leading-edge DM genes extracted from the enriched gene-sets 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 ARID1Amt compared to ARID1Awt OCCC cells, which was reversed by 5-aza-2'-deoxycytidine treatment.
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.
卵巢透明细胞癌（OCCC）的化疗反应差和高 DNA 甲基化引起了广泛关注。最近，我们揭示了人类激酶组和其他癌症相关基因的突变景观，发现在 46%的 OCCC 患者中，SWI/SNF 染色质重塑复合物的组成部分 ARID1A 存在有害突变。本研究旨在全面研究 OCCC 中 ARID1A 缺失和全基因组 DNA 甲基化是否受到共同调控，并确定 ARID1A 表观遗传调控的潜在治疗靶点。
采用 Infinium MethylationEPIC BeadChip 分析 ARID1Amt/ko 和 ARID1Awt OCCC 肿瘤和细胞系的 DNA 甲基化。采用全基因组甲基化的层次聚类分析，分析 OCCC 肿瘤与临床和肿瘤突变状态的聚类关系。利用 GEO 表达谱鉴定 DM 基因及其在 ARID1Amt/ko 与 ARID1Awt OCCC 中的表达水平。结合三个预先排列的 GSEA，揭示了 ARID1A 表观遗传调控的途径和前沿基因。通过计算机模拟验证并在肿瘤和细胞系中显示一致的 ARID1A 相关甲基化变化的前沿基因被视为候选基因，并最终通过亚硫酸氢盐测序和 RT-qPCR 进行验证。
全基因组甲基化的层次聚类分析显示 OCCC 肿瘤有两个聚类。肿瘤分期、ARID1A/PIK3CA 突变和 TP53 突变在两个聚类之间有显著差异。OCCC 中的 ARID1A 突变并未导致全基因组 DNA 甲基化改变，但与 2004 个基因的 DM 启动子或基因体 CpG 岛有关。三个预先排列的 GSEA 共同揭示了 ARID1A 相关 DM 基因富集的 EZH2 和 H3K27me3 相关基因集。从富集基因集中提取的 13 个前沿 DM 基因通过基于表达的计算机模拟验证，并在肿瘤和细胞系中显示出与 ARID1A 相关的一致甲基化变化。亚硫酸氢盐测序和 RT-qPCR 分析显示，与 ARID1Awt OCCC 细胞相比，ARID1Amt 中的 IRX1、TMEM101 和 TRIP6 启动子呈超甲基化，表达水平降低，5-氮杂-2'-脱氧胞苷处理可逆转这种情况。
本研究表明，ARID1A 缺失与 OCCC 中许多基因的差异甲基化有关。已经确定了 ARID1A 依赖性 DM 基因是许多癌症相关途径的关键基因，这可能为 OCCC 的靶向治疗提供新的候选药物。
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