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Abstract：
BACKGROUND: Resistance to targeted therapies represents a significant hurdle to successfully treating hepatocellular carcinoma (HCC). While epigenetic abnormalities are critical determinants of HCC relapse and therapeutic resistance, the underlying mechanisms are poorly understood. We aimed to address whether and how dysregulated epigenetic regulators have regulatory and functional communications in establishing and maintaining drug resistance.
METHODS: HCC-resistant cells were characterized by CCK-8, IncuCyte Live-Cell analysis, flow cytometry and wound-healing assays. Target expression was assessed by qPCR and Western blotting. Global and promoter DNA methylation was measured by dotblotting, methylated-DNA immunoprecipitation and enzymatic digestion. Protein interaction and promoter binding of DNMT3a-TET2 were investigated by co-immunoprecipitation, ChIP-qPCR. The regulatory and functional roles of DNMT3a and TET2 were studied by lentivirus infection and puromycin selection. The association of DNMT and TET expression with drug response and survival of HCC patients was assessed by public datasets, spearman correlation coefficients and online tools.
RESULTS: We identified the coordination of DNMT3a and TET2 as an actionable mechanism of drug resistance in HCC. The faster growth and migration of resistant HCC cells were attributed to DNMT3a and TET2 upregulation followed by increased 5mC and 5hmC production. HCC patients with higher DNMT3a and TET2 had a shorter survival time with a less favorable response to sorafenib therapy than those with lower expression. Cancer stem cell-like cells (CSCs) displayed DNMT3a and TET2 overexpression, which were insensitive to sorafenib. Either genetic or pharmacological suppression of DNMT3a or/and TET2 impaired resistant cell growth and oncosphere formation, and restored sorafenib sensitivity. Mechanistically, DNMT3a did not establish a regulatory circuit with TET2, but formed a complex with TET2 and HDAC2. This complex bound the promoters of oncogenes (i.e., CDK1, CCNA2, RASEF), and upregulated them without involving promoter DNA methylation. In contrast, DNMT3a-TET2 crosstalk silences tumor suppressors (i.e., P15, SOCS2) through a corepressor complex with HDAC2 along with increased promoter DNA methylation.
CONCLUSIONS: We demonstrate that DNMT3a and TET2 act coordinately to regulate HCC cell fate in DNA methylation-dependent and -independent manners, representing strong predictors for drug resistance and poor prognosis, and thus are promising therapeutic targets for refractory HCC.
METHODS: HCC-resistant cells were characterized by CCK-8, IncuCyte Live-Cell analysis, flow cytometry and wound-healing assays. Target expression was assessed by qPCR and Western blotting. Global and promoter DNA methylation was measured by dotblotting, methylated-DNA immunoprecipitation and enzymatic digestion. Protein interaction and promoter binding of DNMT3a-TET2 were investigated by co-immunoprecipitation, ChIP-qPCR. The regulatory and functional roles of DNMT3a and TET2 were studied by lentivirus infection and puromycin selection. The association of DNMT and TET expression with drug response and survival of HCC patients was assessed by public datasets, spearman correlation coefficients and online tools.
RESULTS: We identified the coordination of DNMT3a and TET2 as an actionable mechanism of drug resistance in HCC. The faster growth and migration of resistant HCC cells were attributed to DNMT3a and TET2 upregulation followed by increased 5mC and 5hmC production. HCC patients with higher DNMT3a and TET2 had a shorter survival time with a less favorable response to sorafenib therapy than those with lower expression. Cancer stem cell-like cells (CSCs) displayed DNMT3a and TET2 overexpression, which were insensitive to sorafenib. Either genetic or pharmacological suppression of DNMT3a or/and TET2 impaired resistant cell growth and oncosphere formation, and restored sorafenib sensitivity. Mechanistically, DNMT3a did not establish a regulatory circuit with TET2, but formed a complex with TET2 and HDAC2. This complex bound the promoters of oncogenes (i.e., CDK1, CCNA2, RASEF), and upregulated them without involving promoter DNA methylation. In contrast, DNMT3a-TET2 crosstalk silences tumor suppressors (i.e., P15, SOCS2) through a corepressor complex with HDAC2 along with increased promoter DNA methylation.
CONCLUSIONS: We demonstrate that DNMT3a and TET2 act coordinately to regulate HCC cell fate in DNA methylation-dependent and -independent manners, representing strong predictors for drug resistance and poor prognosis, and thus are promising therapeutic targets for refractory HCC.
摘要：
背景：对靶向治疗的抗性代表了成功治疗肝细胞癌（HCC）的重要障碍。虽然表观遗传异常是肝癌复发和治疗耐药的关键决定因素，潜在的机制知之甚少。我们的目的是解决在建立和维持耐药性方面是否以及如何失调的表观遗传调节因子具有调节和功能沟通。
方法：通过CCK-8，IncuCyte活细胞分析，流式细胞术和伤口愈合分析。通过qPCR和Western印迹评估靶表达。通过dotblotting测量全局和启动子DNA甲基化，甲基化DNA免疫沉淀和酶消化。通过共免疫沉淀研究了DNMT3a-TET2的蛋白质相互作用和启动子结合，ChIP-qPCR。通过慢病毒感染和嘌呤霉素选择研究了DNMT3a和TET2的调控和功能作用。DNMT和TET表达与HCC患者的药物反应和生存的关联通过公共数据集进行评估。spearman相关系数和在线工具。
结果：我们确定DNMT3a和TET2的协同作用是HCC耐药的可行机制。抗性HCC细胞的更快生长和迁移归因于DNMT3a和TET2上调，随后5mC和5hmC产量增加。与具有较低表达的人相比，具有较高DNMT3a和TET2的HCC患者的生存时间较短，对索拉菲尼治疗的反应较差。肿瘤干细胞样细胞（CSCs）显示DNMT3a和TET2过表达，对索拉非尼不敏感.DNMT3a或/和TET2的遗传或药理学抑制受损的抗性细胞生长和肿瘤形成，并恢复了索拉非尼的敏感性.机械上,DNMT3a未与TET2建立调节回路，但与TET2和HDAC2形成复合物。该复合物结合了癌基因的启动子(即，CDK1，CCNA2，RASEF），并上调它们，而不涉及启动子DNA甲基化。相比之下,DNMT3a-TET2串扰沉默肿瘤抑制因子(即，P15，SOCS2）通过与HDAC2的共阻遏复合物以及增加的启动子DNA甲基化。
结论：我们证明DNMT3a和TET2以DNA甲基化依赖性和非依赖性方式协同调节HCC细胞命运，代表耐药性和不良预后的强预测因子，因此是难治性HCC的有希望的治疗靶点。
方法：通过CCK-8，IncuCyte活细胞分析，流式细胞术和伤口愈合分析。通过qPCR和Western印迹评估靶表达。通过dotblotting测量全局和启动子DNA甲基化，甲基化DNA免疫沉淀和酶消化。通过共免疫沉淀研究了DNMT3a-TET2的蛋白质相互作用和启动子结合，ChIP-qPCR。通过慢病毒感染和嘌呤霉素选择研究了DNMT3a和TET2的调控和功能作用。DNMT和TET表达与HCC患者的药物反应和生存的关联通过公共数据集进行评估。spearman相关系数和在线工具。
结果：我们确定DNMT3a和TET2的协同作用是HCC耐药的可行机制。抗性HCC细胞的更快生长和迁移归因于DNMT3a和TET2上调，随后5mC和5hmC产量增加。与具有较低表达的人相比，具有较高DNMT3a和TET2的HCC患者的生存时间较短，对索拉菲尼治疗的反应较差。肿瘤干细胞样细胞（CSCs）显示DNMT3a和TET2过表达，对索拉非尼不敏感.DNMT3a或/和TET2的遗传或药理学抑制受损的抗性细胞生长和肿瘤形成，并恢复了索拉非尼的敏感性.机械上,DNMT3a未与TET2建立调节回路，但与TET2和HDAC2形成复合物。该复合物结合了癌基因的启动子(即，CDK1，CCNA2，RASEF），并上调它们，而不涉及启动子DNA甲基化。相比之下,DNMT3a-TET2串扰沉默肿瘤抑制因子(即，P15，SOCS2）通过与HDAC2的共阻遏复合物以及增加的启动子DNA甲基化。
结论：我们证明DNMT3a和TET2以DNA甲基化依赖性和非依赖性方式协同调节HCC细胞命运，代表耐药性和不良预后的强预测因子，因此是难治性HCC的有希望的治疗靶点。
