It is well known that epithelial-mesenchymal transition (EMT) can confer cancer cells with invasive and migratory capabilities associated with distant metastasis. As a key upstream factor in the Hippo pathway, Kibra (wwc1 gene) has been shown to suppress EMT in breast cancer cells, and we have found that its expression is reduced or lost completely in both human breast cancer cell lines and clinical tissue samples, particularly in triple negative breast cancer (TNBC). Unfortunately, the molecular mechanisms underlying this progression-associated event remain to be elucidated. Epigenetic gene silencing is one of the most common causes of suppressed expression of tumor suppressor genes. Furthermore, recent studies have demonstrated that EZH2 can recruit DNA methyltransferases, resulting in DNA methylation and subsequent gene silencing in certain circumstances. Thus, we hypothesized that there may exist a link between EZH2 and DNA methylation in association with wwc1 silencing in breast cancer. To test this hypothesis, we performed bisulfite sequencing, shRNA, co-IP, ChIP, MeDIP and ChIP-qPCR. As expected, RG108 or 5-Aza treatment improved the wwc1 gene transcription and Kibra protein expression. Both bisulfite sequencing and MeDIP demonstrated higher CpG methylation of the wwc1 promoter the TNBC cells (MDA-MB-231) than in luminal breast cancer cells (MCF7). It is noteworthy that ChIP and co-IP assays showed that EZH2, H3K27me3 and DNMT1 are enriched at the wwc1 promoter, and there exist physiologically relevant protein-protein interactions between them. We also found that EZH2 knockdown leads to a partial increase in Kibra expression and a considerable reduction in H3K27 and DNMT1 trimethylation. Moreover, ChIP-qPCR revealed more DNA fragments containing the wwc1 promoter in MDA-MB-231 than in MCF7 cells after immunoprecipitation with EZH2, DNMT1 and H3K27me3 antibodies. Collectively, our results reveal crosstalk between H3K27me3 inhibition catalyzed by EZH2 and CpG island methylation mediated by DNMT1 within the wwc1 promoter, which synergistically silence wwc1 gene expression in TNBC. Based on these results, we conclude that EZH2 shows promise as a potential anti-tumor target.

众所周知，上皮-间质转化（EMT）可使癌细胞具有与远处转移相关的侵袭和迁移能力。作为河马途径中的关键上游因素，Kibra（wwc1已经证明该基因可抑制乳腺癌细胞中的EMT，并且我们发现其表达在人乳腺癌细胞系和临床组织样品中，特别是在三阴性乳腺癌（TNBC）中均被完全降低或丧失。不幸的是，这种进展相关事件的分子机制仍有待阐明。表观遗传基因沉默是抑制肿瘤抑制基因表达的最常见原因之一。此外，最近的研究表明，EZH2可以募集DNA甲基转移酶，从而在某些情况下导致DNA甲基化和随后的基因沉默。因此，我们假设EZH2和与wwc1相关的DNA甲基化之间可能存在联系在乳腺癌中沉默。为了验证该假设，我们进行了亚硫酸氢盐测序，shRNA，co-IP，ChIP，MeDIP和ChIP-qPCR。如预期的那样，RG108或5-Aza处理改善了wwc1基因转录和Kibra蛋白表达。亚硫酸氢盐测序和MeDIP均显示TNBC细胞（MDA-MB-231）的wwc1启动子的CpG甲基化程度高于腔内乳腺癌细胞（MCF7）。值得注意的是，ChIP和co-IP分析显示EZH2，H3K27me3和DNMT1在wwc1处富集启动子，并且它们之间存在生理相关的蛋白质-蛋白质相互作用。我们还发现，EZH2敲低导致Kibra表达的部分增加以及H3K27和DNMT1三甲基化的显着降低。此外，用EZH2，DNMT1和H3K27me3抗体免疫沉淀后，ChIP-qPCR在MDA-MB-231中显示的MCw7细胞中含有wwc1启动子的DNA片段更多。总的来说，我们的研究结果揭示了EZH2催化的H3K27me3抑制与DNMT1介导的wpc1启动子内DNMT1介导的CpG岛甲基化之间的串扰，这使TNBC中的wwc1基因表达协同沉默。基于这些结果，我们得出结论，EZH2显示出有望作为潜在的抗肿瘤靶标。