内源性PD-1/PD-L1通过mTOR通路调节自噬促进肝癌转移的机制研究

The molecular mechanism and the key regulatory links of hepatocellular carcinoma recurrence and metastasis remain controversial. Our previous study found that liver cancer cells not only express PD-L1, but also express PD-1. Hepatoma cell intrinsic PD-1 can bind to PD-L1 and promote tumor recurrence and metastasis independently of the immune system. On this basis, we used mass spectrometry to determine the different proteins between PD-1-overexpressing hepatoma cells and control cells. Bioinformatics analysis suggested that mTOR and autophagy pathway related proteins had the most significant difference between the two groups. Subsequent Co-immunoprecipitation and pull down initially demonstrated that ATG13, a key protein of autophagy pathway, can physically bind to PD1. Combined with the close relationship between autophagy and prognosis of hepatocellular carcinoma found in our previous research, we proposed the hypothesis that intrinsic PD-1 / PD-L1 regulates autophagy through the mTOR pathway, thus promotes hepatocellular carcinoma recurrence and metastasis. In this study, we will use GST affinity chromatography and laser confocal to confirm the physical correlation between PD-1 and ATG13. Then in vitro and in vivo experiments and molecular biology methods were applicated to explore the mechanism of how PD-1 regulates autophagy through ATG13 to promote tumor recurrence and metastasis. In addition, whether the PD-1 antibody combined with mTOR inhibitors can increase the efficacy of PD-1 antibody also researched. Finally, the above findings were further validated in another expanded clinical cohort and the relationship between sorafenib and drug resistance was initially explored on the basis of autophagy.

深入探讨肝癌复发转移的分子机制及关键调控环节对于提高肝癌患者的预后意义重大。我们前期发现内源性的PD-1可以与肝癌细胞表面的PD-L1结合，独立于免疫系统而促进肝癌细胞复发转移。在此基础上，我们应用质谱分析测定了过表达PD-1与对照组的差异蛋白。生物信息学提示mTOR通路及下游的自噬相关蛋白在两组间差异最显著。随后的CO-IP和pulldown发现自噬的关键蛋白ATG13可与PD1结合。结合研究发现自噬与肝癌术后复发的密切关系，我们提出“内源性的PD-1/PD-L1通过mTOR通路调节自噬促进肝癌复发转移”的假说。本课题，在明确PD-1与ATG13的直接作用后，我们通过体内外实验，分子生物学方法探索PD-1如何通过ATG13来抑制自噬，促进肝癌复发转移，PD-1抗体与mTOR抑制剂联用能否增加疗效等问题。最后在扩大的临床样本中对上述发现进行验证，并在自噬的基础上探索索拉菲尼与耐药的关系。

本课题在前期基础上，深入研究了内源性PD-1和下游自噬相关基因，ATG13蛋白磷酸化水平以及预后的相关性。同时我们还对PD-1/PD-L1上游的调控基因进行了相关探索。我们证实内源性的PD-1/PD-L1可通过调节mTOR 通路下游的ATG13 蛋白磷酸化对肝癌细胞自噬状态进行调节，从而促进肝癌生长、转移。mTOR 抑制剂联合PD-1抗体对于抑制小鼠原位癌的生长和转移具有累积效应，有助于提高免疫治疗的抗肿瘤效果。该发现为肝癌临床诊治的提供了新的思路新。另外，我们的研究还发现天然免疫的重要蛋白TLR9在肝癌细胞内激活后可调节了PARP1 自身 PARylation 和泛素化以及 STAT3 PARylation 和磷酸化之间的转换，共同上调PD-L1的表达并最终诱导肝癌细胞免疫逃逸。提示了天然免疫与肿瘤免疫之间的相关性。临床上，MET抑制剂未能在肝癌患者中产生令人满意的结果，从而限制了其应用。本课题发现MET抑制剂会导致PDL1 上调从而引起免疫抑制。采用MET抑制剂和抗 PD1 疗法的组合能有效抑制肿瘤生长，延长生存期。目前已经有临床试验应用该方案治疗肝癌。在对PD-L1稳定性的研究时，我们发现双硫仑及铜离子复合物作为PARP抑制，可诱导的GSK3β在Ser9的磷酸化，从而稳定PD-L1，促进免疫细胞杀死肿瘤。总而言之，该课题对于PD-1/PD-L1如何有效地影响肝癌免疫逃避的提供了相关理论。特别是对于上下游关键调控基因的发现，对于临床上如何改善PD-1抑制剂的治疗效果，如何联合用药等提供了新的思路与理论依据。