Targeting the autophagy-lysosome system to block pancreatic cancer

项目摘要 癌细胞共同选择自噬-一种进化上保守的细胞再循环途径-以维持 代谢健康先前的研究表明，胰腺导管腺癌（PDA）上调 自噬和溶酶体--自噬货物降解的酸性细胞器--急剧增加 大量的分解和多种细胞内底物的再循环。一个额外的，不太好理解的 自噬的功能是选择性地去除特定的蛋白质，以赋予PDA细胞 增强健身能力。 我们现在表明自噬-溶酶体途径选择性地靶向主要组织相容性 复合物I类（MHC-I）蛋白作为免疫逃避机制而降解。通过亲和力- 从正常和PDA细胞系中捕获完整溶酶体（LysoIP）， 通过分析，我们鉴定MHC-I为PDA细胞中显著富集的溶酶体底物。符合本 免疫荧光染色表明，与正常细胞不同，MHC-I定位于 在PDA细胞系和原发性患者PDA标本中，MHC-I被捕获在质膜（PM）内， 自噬体和溶酶体。重要的是，自噬的肿瘤特异性抑制足以1） 稳定并重新定位MHC-I至PM，2）增加抗原呈递和3）加强T细胞介导的肿瘤 体外和体内杀伤。基于这些发现，本研究的目标是确定 异常MHC-I运输的潜在机制，并确定可以操纵的靶向节点， 修复PDA细胞表面的MHC-I。我们修改后的应用程序将利用 生物化学、遗传学、细胞器纯化和蛋白质组学、新型小鼠模型和患者PDA样本 1）确定MHC-I的翻译后修饰如何协同作用， 与自噬机制，以促进捕获的自噬体，2）确定何处沿着其贩运 途径是MHC-I转向自噬体和3）确定何时在PDA进化过程中 MHC-I失调发生，我们能否利用这些信息建立更有效的策略，以提高 抗原递呈和免疫介导的肿瘤杀伤。 总之，我们发现MHC-I的自噬依赖性降解突出了一个重要的新发现， PDA和潜在的其他癌症中的免疫逃避的范例。我们的研究结果将 确定MHC-I失调的关键分子机制，并建立新的节点， 靶向恢复PDA中的抗原呈递。

PROJECT SUMMARY Cancer cells co-opt autophagy - an evolutionarily conserved cellular recycling pathway - to maintain metabolic fitness. Prior studies have shown that Pancreatic Ductal Adenocarcinoma (PDA) up-regulates autophagy and lysosomes – acidic organelles where autophagic cargo is degraded – to dramatically increase the bulk breakdown and recycling of diverse intracellular substrates. An additional, less well understood function of autophagy is the selective removal of specific proteins in order to endow PDA cells with enhanced fitness. We now show that the autophagy-lysosome pathway selectively targets major histocompatibility complex class I (MHC-I) protein for degradation as a mechanism of immune evasion. Through affinity- based capture of intact lysosomes (LysoIP) from normal and PDA cell lines coupled with proteomics-based analysis, we identified MHC-I as a significantly enriched lysosomal substrate in PDA cells. Consistent with this finding, immuno-fluorescence staining demonstrates that, unlike normal cells where MHC-I localizes to the plasma membrane (PM), in PDA cell lines and primary patient PDA specimens, MHC-I is trapped inside autophagosomes and lysosomes. Importantly, tumor-specific suppression of autophagy is sufficient to 1) stabilize and re-localize MHC-I to the PM, 2) increase antigen presentation and 3) boost T cell mediated tumor killing in vitro and in vivo. Building on these findings the goal of this study is to determine the molecular mechanisms underlying aberrant MHC-I trafficking, and to identify targetable nodes that can be manipulated to restore MHC-I on the cell surface of PDA cells. Our revised application will leverage the combined power of biochemistry, genetics, organelle purification and proteomics, a novel mouse model and patient PDA samples to address the following specific aims: 1) determine how post-translational modifications of MHC-I cooperate with the autophagy machinery to facilitate capture by autophagosomes, 2) identify where along its trafficking route is MHC-I diverted to autophagosomes and 3) determine when during the course of PDA evolution does MHC-I dysregulation occur and can we exploit this information to establish more effective strategies to enhance antigen presentation and immune mediated tumor killing. In summary, our discovery of autophagy dependent degradation of MHC-I highlights an important new paradigm for immune evasion in PDA and potentially other cancers. Findings from our proposed studies will determine key molecular mechanisms underlying MHC-I dysregulation and establish new nodes that can be targeted to restore antigen presentation in PDA.