Targeting the autophagy-lysosome system to block pancreatic cancer

靶向自噬-溶酶体系统来阻止胰腺癌

• 批准号: 10358483
• 负责人: Rushika Miriam Perera
• 金额: $ 36.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10358483
• 关键词: Address; Affinity; Antigen Presentation; Autophagocytosis; Autophagosome; BRCA1 gene; Binding; Biochemistry; Biogenesis; Cell Line; Cell membrane; Cell surface; Cells; Complex; Coupled; Endoplasmic Reticulum; Ensure; Epithelial; Evolution; Excision; Genetic; Goals; Golgi Apparatus; Grant; Growth; Human; Immune; Immune Evasion; Immunofluorescence Immunologic; In Vitro; Location; Lysosomes; Major Histocompatibility Complex; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Molecular; Molecular Genetics; Mus; Neoplasm Metastasis; Normal Cell; Organelles; Pancreas; Pancreatic Ductal Adenocarcinoma; Pancreatic Intraepithelial Neoplasia; Pathway interactions; Patients; Peptides; Post-Translational Protein Processing; Proteins; Proteomics; Publications; Recycling; Role; Route; Sampling; Specimen; Stains; Surface; System; T-Lymphocyte; Testing; Tissue Banks; Tumor Tissue; Ubiquitin; Work; base; cancer cell; fitness; genetic approach; immune checkpoint blockade; immunogenicity; in vivo; metabolic fitness; mouse model; novel; pancreatic ductal adenocarcinoma cell; pancreatic ductal adenocarcinoma model; peptide I; protein degradation; receptor; trafficking; tumor; ubiquitin ligase

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.

项目总结 癌细胞选择自噬--一种进化上保守的细胞循环途径--来维持 新陈代谢健康。先前的研究表明，胰腺导管腺癌(PDA)上调表达 自噬和溶酶体--自噬货物被降解的酸性细胞器--急剧增加 不同胞内底物的整体分解和回收。另一个不太为人所知的 自噬的功能是选择性地去除特定的蛋白质，以便赋予PDA细胞 增强了体能。 我们现在表明，自噬-溶酶体途径选择性地针对主要组织相容性。 复合I类(MHC-I)蛋白作为免疫逃避的一种降解机制。通过亲和力- 基于蛋白质组学的正常和PDA细胞系完整溶酶体(LysoIP)捕获 分析表明，MHC-I是PDA细胞中显著丰富的溶酶体底物。与此一致 发现，免疫荧光染色表明，与正常细胞不同，MHC-I定位于 质膜(PM)，在PDA细胞系和原发患者的PDA标本中，MHC-I被困在里面 自噬小体和溶酶体。重要的是，肿瘤特异性的自噬抑制足以1) 稳定和重新定位MHC-I到PM，2)增加抗原提呈，3)促进T细胞介导的肿瘤 在体外和体内杀死。基于这些发现，这项研究的目标是确定分子 MHC-I异常贩运的潜在机制，并确定可被操纵的靶向节点 在PDA细胞表面恢复MHC-I。我们修订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、一种新的小鼠模型和患者PDA样本 为了解决以下具体目标：1)确定MHC-I的翻译后修改如何合作 利用自噬机制来促进自噬捕获，2)识别其贩运的地点 MHC-I途径被转移到自噬体内，3)确定在PDA进化过程中何时发生 MHC-I失调的发生，我们是否可以利用这些信息来建立更有效的策略来增强 抗原提呈和免疫介导的肿瘤杀伤。 总之，我们发现的依赖自噬的MHC-I降解突出了一个重要的新的 PDA和其他潜在癌症的免疫逃逸范例。我们建议的研究结果将 确定MHC-I失调的关键分子机制并建立新的节点 目标是恢复PDA中的抗原呈递。