PD-L1-DNMT1信号通路参与肝癌索拉非尼耐药的机制及逆转耐药新策略的研究

Chemotherapy resistance is the major cause of sorafenib treatment failure in hepatocellular carcinoma. It has been reported that aberrant activation of multiple signaling pathway are involved in it. Therefore, multi-molecular targeted therapy has emerged as a crucial treatment direction to overcome resistance. Our previous work revealed that the levels of immune checkpoint molecule (PD-L1) and DNA methyltransferase (DNMT1) mRNA and protein are enhanced in sorafenib-resistant cells, and exhibits a positive correlation. Furthermore, our data indicated that NF-κB activated PD-L1 by transcription regulation, which mediated phosphorylation of STAT3. Then p-STAT3 was found to bind to directly to the promoter of DNMT1 gene, which caused DNMT1 overexpression and hypermethylation of CpG island of tumor suppressor gene (CDH1) and induced gene silencing. Finally, HCC cells achieved resistance to sorafenib. Genetic disruption of PD-L1 or DNMT1 or its inhibitor sensitized sorafenib-resistant cells to sorafenib by blockade the signaling axis. Based on this, we hypothesize a novel PD-L1-DNMT1 signal pathway, which contributes to sorafenib resistance of HCC cells. In this project we will further explore the mechanism underlying resistance to sorafenib. In addition, a promising combination targeted therapy strategy of DNMT1 inhibitor and anti-PD-L1 antibody is designed in vitro and in vivo. This combines epigenetics treatment and immunotherapy, which will help to identify new therapeutic targets to overcome sorafenib resistance of HCC cells.

化疗耐药是导致索拉非尼（sorafenib）治疗肝癌（HCC）失败的主因。目前认为多种信号通路异常激活参与其中，故多靶点协同治疗是克服耐药的重要方向。我们前期发现，在HCC sorafenib耐药细胞中PD-L1和DNA甲基化转移酶（DNMT1）mRNA和蛋白水平均升高，且存在正相关；NF-κB能直接转录激活PD-L1，进而激活磷酸化STAT3作为转录因子和DNMT1的启动子直接结合并调控其高表达，引起抑癌基因CDH1 CpG岛高甲基化，进而基因沉默，导致耐药；基因敲除PD-L1或DNMT1或其抑制剂可使耐药细胞对sorafenib敏感。基于此，本项目提出一个全新的PD-L1-DNMT1信号通路，深入探索其参与耐药的机制，并设计了针对表观遗传治疗和免疫治疗的多靶点协同治疗方案，即联合使用DNMT1抑制剂和PD-L1抗体进行体内外研究，为克服HCC对sorafenib耐药提供新策略。

目前索拉非尼是我国治疗晚期肝癌（HCC）的一线药物，但是先天或获得性耐药导致病人不能获益。本研究发现PD-L1/DNMT1信号通路参与HCC对索拉非尼的耐药。NF-κB能直接转录激活PD-L1，进而激活磷酸化STAT3作为转录因子和DNMT1的启动子直接结合并调控其高表达，引起抑癌基因CDH1 CpG岛高甲基化，进而基因沉默，导致耐药；基因敲除PD-L1或DNMT1或其抑制剂可使耐药细胞对索拉非尼敏感。这为克服HCC对索拉非尼耐药提供了新策略。此外，本研究利用与吉林大学化学学院的化学-医学交叉合作平台，拓展了部分实验内容：①构建载anti-miR221纳米金颗粒，并证实AuNPs-anti-miR221通过抑制miR-221/p27/DNMT1信号通路增强索拉非尼的抗HCC作用；②构建载索拉非尼并用抗VEGFR表面修饰的癌细胞-巨噬细胞杂交膜包被的近红外响应的空心硫化铜纳米颗粒(CuS-SF@CMV NPs)，通过Ras/Raf/MEK/ERK和PI3K/AKT信号通路抑制HCC细胞增殖和血管生成，最终实现了化疗联合光热协同抗HCC的效果，证实CuS-SF@CMV NP具有免疫逃逸、肿瘤靶向和载药能力，以及固有的光热转化能力，使其成为针对HCC的协同光热/化学治疗的理想选择；③合成了载ATM抑制剂并用TGF-β抗体修饰的中空硫化铜纳米颗粒(CuS-ATMi@TGF-β NPs)，具有光稳定性、精准靶向肿瘤释放药物和以近红外形式升高温度的性能，通过下调热休克蛋白表达以低温的光热作用协同化疗抑制HCC生长，同时避免了对正常细胞的损伤。本研究完成了预定的研究内容和目标，在基金的资助下发表SCI论文9篇，作为第一参与人获得吉林省科技厅自然科学奖二等奖1项。