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.

项目概要 癌细胞选择自噬（一种进化上保守的细胞回收途径）来维持 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（自噬货物被降解的酸性细胞器）急剧增加 不同细胞内底物的大量分解和回收。另外一个不太了解的 自噬的功能是选择性去除特定蛋白质，从而赋予 PDA 细胞 增强体质。 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 复杂的 I 类 (MHC-I) 蛋白降解作为免疫逃避的机制。通过亲和力—— 基于从正常细胞系和 PDA 细胞系中捕获完整溶酶体 (LysoIP) 并结合基于蛋白质组学的 通过分析，我们确定 MHC-I 是 PDA 细胞中显着富集的溶酶体底物。与此一致 研究发现，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 细胞系和原发性患者 PDA 标本中，MHC-I 被困在内部 自噬体和溶酶体。重要的是，肿瘤特异性的自噬抑制足以：1) 稳定 MHC-I 并将其重新定位到 PM，2) 增加抗原呈递，3) 增强 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分子 异常 MHC-I 贩运的潜在机制，并识别可被操纵的目标节点 恢复PDA细胞表面的MHC-I。我们修改后的应用程序将利用以下综合力量 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 实现以下具体目标：1) 确定 MHC-I 的翻译后修饰如何合作 利用自噬机制促进自噬体捕获，2) 确定其运输过程 MHC-I 转移至自噬体的路线，并且 3) 确定 PDA 进化过程中何时发生 MHC-I 失调发生，我们能否利用这些信息来制定更有效的策略来增强 抗原呈递和免疫介导的肿瘤杀伤。 总之，我们对 MHC-I 的自噬依赖性降解的发现凸显了一个重要的新发现 PDA 和其他潜在癌症的免疫逃避范例。我们拟议的研究结果将 确定 MHC-I 失调的关键分子机制并建立新的节点 旨在恢复 PDA 中的抗原呈递。