基于自噬介导YAP1/STAT3途径探讨PD-L1在肝癌免疫抑制微环境失调进程中的作用及双氢青蒿素的调控效应

Hepatocellular carcinoma (HCC), the fifth most common cancer, is the third most prevalent cause of cancer-related death in the world. Despite continuous improvement in medical technology over the past decade, immunosuppressive microenvironment reduces therapy efficacy in HCC. The overexpression of programmed death ligand-1 (PD-L1), one of the immune checkpoint inhibitors, is a key factor in the development of the microenvironment. STAT3 is usually constitutively activated and promotes PD-L1 expression in HCC. Additionally, Yes-associated protein 1 (YAP1) activates at early HCC stage and increases the expression level of STAT3. Decreasing the expression of YAP1 is a strategy to inhibit its activity. Furthermore, autophagy is a key pathway for the protein degradation. However, the mechanism of autophagy-mediated YAP1/STAT3 pathway on the HCC immunosuppressive microenvironment via PD-L1 is not clear. Artemisia carvifolia has the function of supporting healthy energy to eliminate evils. In our previous research, we found that chemotherapeutic agents, such as Nutlin-3 and Etoposide, activate IFI16 inflammasome, leading to the decreased apoptosis rate in HCC. Meanwhile, dihydroartemisinin (DHA),the main active component of artemisinin after metabolism in vivo, enhances autophagy, inhibits the activity of IFI16 inflammasome, and reduces the expression level of YAP1 in HepG2215 cells. Therefore, we speculate that autophagy-mediated YAP1/STAT3 pathway activates and promotes PD-L1 expression, leading to immunosuppressive microenvironment in HCC. While, DHA may promote YAP1 degradation and inhibit the pathway. To verify this hypothesis, we will detect the influence of autophagy-mediated YAP1/STAT3 pathway on PD-L1 expression and immunosuppressive microenvironment in Yap1flox/flox；albumin-Cre HCC mice in vivo, and HepG2215 and HepG2 cells with Yap1 knockout or overexpressed in vitro. Therefore, we may provide new ideas and therapeutic strategies on preventing immunosuppressive microenvironment in HCC with traditional Chinese medicine.

免疫抑制微环境失调是肝癌治疗药物耐受产生的主要原因，但其确切机制不明。PD-L1高表达是免疫抑制失调的关键环节。持续激活的STAT3导致肝癌生成，促进PD-L1表达；而YAP1活化发生在肝癌早期，可调控STAT3表达。自噬是选择性降解蛋白的关键途径，但由自噬介导的YAP1/STAT3途径在肝癌免疫抑制微环境失调中的作用尚不明确。青蒿可扶正祛邪，预实验发现，化疗药物激活IFI16炎性体导致凋亡降低；而双氢青蒿素(DHA)降低IFI16炎性体活性及YAP1表达。据此提出假说，自噬介导的YAP1/STAT3途径活化，促进PD-L1高表达，导致免疫抑制微环境失调；DHA可能促进YAP1降解改善免疫抑制微环境。为验证假说，本项目拟在YAP1基因条件敲除肝癌小鼠和细胞株模型上，探讨YAP1/STAT3途径调控PD-L1表达的分子机制，探索其防治新靶点和药物，为中医药防治肝癌提供新思路和新策略。

免疫抑制微环境失调是肝癌治疗药物耐受产生的主要原因，PD-L1高表达是免疫抑制失调的关键环节。持续激活的STAT3导致肝癌生成，促进PD-L1表达；而YAP1活化发生在肝癌早期，可调控STAT3表达。自噬是选择性降解蛋白的关键途径，但由自噬介导的YAP1/STAT3途径在肝癌免疫抑制微环境失调中的作用尚不明确。本项目的研究内容是探讨YAP1在肝癌免疫抑制微环境失调进程中的作用；分析自噬介导的YAP1/STAT3途径调控PD-L1表达的机制；验证YAP1与肿瘤自噬活性的相关性；探讨DHA改善肝癌免疫抑制微环境的作用。通过研究开展，本项目取得如下结果：建立了DEN/TCPOBOP诱导的肝细胞特异性Yap1 基因敲除小鼠肝原位肿瘤模型，探讨了YAP1介导的JAK1/STAT1,3调控PD-L1表达的机制；通过肝癌组织芯片，验证了YAP1与肝癌自噬活性的相关性，分析了自噬介导的YAP1调控PD-L1表达的机制；进一步阐明YAP1促进肝癌免疫抑制微环境失调的作用和转录组学及代谢组学机制；证实DHA能抑制YAP1表达，减少肝细胞脂滴沉积、提高肠道AKK菌丰度等改善免疫抑制微环境。本基金标注的成果先后发表在Phytotherapy Research、Phytomedicine、Frontiers of Medicine、 The FASEB Journal、Experimental Cell Research、Molecular and Cellular Biochemistry、Acta Histochemica和中药新药与临床药理等国内外期刊上。本项目的实施，阐明了自噬介导的YAP1/STAT3途径提高PD-L1表达促进肝癌免疫抑制微环境失调的作用和机制，进一步证明自噬对YAP1途径的负向调控作用，为肝癌防治提供新理论依据和作用位点；探讨DHA作为抑制YAP1的中药小分子，改善免疫抑制微环境，提高PD-1靶向抗体治疗肝癌的中医药新策略。