Deciphering Autophagy-Dependent Secretion In Cancer Via Proximity-Based Biotinylation

通过基于邻近的生物素化破译癌症中自噬依赖性分泌

• 批准号: 9178012
• 负责人: Jayanta Debnath
• 金额: $ 17.24万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9178012
• 关键词: Ablation; Address; Advanced Malignant Neoplasm; Angiogenic Factor; Antimalarials; Antineoplastic Agents; Autophagocytosis; Biogenesis; Biological Markers; Biotinylation; Bypass; Cancer Patient; Cell Survival; Cell secretion; Cells; Clinical; Clinical Oncology; Defect; Endoplasmic Reticulum; Exhibits; Extracellular Space; Family; GORASP2 gene; GRASP gene; Genetic; Goals; Golgi Apparatus; Growth; Homeostasis; Human; Hydroxychloroquine; KRAS2 gene; Knowledge; Label; Laboratories; Link; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Metabolic; Molecular; Monitor; Pathogenesis; Pathway interactions; Patients; Process; Proteins; Proteomics; Publishing; Recording of previous events; Reporter; Role; Serum; Stress; Therapeutic Intervention; Toxic effect; Tumor Cell Invasion; Work; base; biomarker discovery; cancer biomarkers; cancer cell; cancer therapy; clinical biomarkers; cytokine; exosome; fitness; genetic approach; in vivo; inhibition of autophagy; inhibitor/antagonist; innovation; insight; interest; loss of function; mutant; neoplastic cell; novel; release factor; response; therapy resistant; trafficking; treatment response; tumor; tumor progression; tumorigenic

PROJECT SUMMARY Currently, there is immense interest in modulating autophagy against cancer. Anti-malarials, such as hydroxychloroquine (HCQ), have been aggressively repurposed as autophagy inhibitors in numerous clinical oncology trials. Despite this enthusiasm, we still do not fully understand how autophagy impacts cancer progression and treatment. To date, the primary rationale for targeting autophagy against cancer is because this catabolic pathway promotes tumor cell survival and metabolic adaptation. However, recent findings challenge this prevailing idea in the field. Although traditionally viewed as an “auto-digestive” pathway, emerging genetic evidence now implicates autophagy as an important regulator of cellular secretion. For example, we recently discovered new roles for autophagy regulators (ATGs) in promoting the coordinate secretion of cytokines required for tumor cell invasion. Nevertheless, our understanding of autophagy- dependent secretion in cancer remains rudimentary because of major conceptual and technical barriers that hamper deciphering how autophagy enables secretion. Most importantly, studies to date have exclusively relied on phenotypic analysis following genetic ablation of specific pathway components, such as ATGs; such loss-of-function approaches are limited because they fail to discern whether secretory defects represent a direct versus indirect consequence of impaired autophagy. To overcome these major obstacles in the field, we have created novel reporters utilizing proximity-based biotinylation (BioID) to specifically label proteins that rely upon the autophagy machinery for their release into the extracellular space. We will now leverage these innovative reporters to decipher the autophagy-dependent secretome and rigorously define the mechanisms by which autophagy promotes secretion. In Aim 1, we will elaborate the repertoire of autophagy-dependent secreted products using quantitative proteomics and assess their utility as biomarkers for monitoring tumor progression and treatment in vivo. For these studies, we will focus on KRAS mutant lung cancer cells exhibiting high levels of basal autophagy and secretion. In Aim 2, we will define the mechanisms by which autophagy controls tumor cell secretion. We will utilize proximity-based biotinylation strategies, combined with genetic approaches, to dissect the intracellular trafficking of autophagy-dependent secreted proteins as they exit the cell, with the long-term goal of identifying targets in these pathways for therapeutic intervention in cancer. Overall, these studies provide timely mechanistic insight into an entirely new role for autophagy in cancer progression and therapeutic response.

项目摘要 目前，人们对调节自噬对抗癌症有着极大的兴趣。抗疟疾药物，如 羟氯喹（HCQ）已在许多临床中被积极重新用作自噬抑制剂 肿瘤试验尽管有这种热情，我们仍然没有完全了解自噬如何影响癌症 进展和治疗。迄今为止，靶向自噬对抗癌症的主要理由是， 这种分解代谢途径促进肿瘤细胞存活和代谢适应。然而，最近的调查结果 挑战这个领域的主流观点。虽然传统上被视为“自动消化”途径， 新出现的遗传学证据表明自噬是细胞分泌的重要调节因子。为 例如，我们最近发现了自噬调节因子（ATGs）在促进协调 分泌肿瘤细胞侵袭所需的细胞因子。尽管如此，我们对自噬的理解- 癌症中的依赖性分泌仍然是基本的，因为主要的概念和技术障碍， 阻碍了解释自噬如何使分泌。最重要的是，迄今为止的研究只 依赖于特定途径组分（如ATG）的遗传消除后的表型分析; 功能丧失的方法是有限的，因为它们不能辨别分泌缺陷是否代表一种 自噬受损的直接与间接后果。为了克服实地的这些主要障碍，我们 利用基于邻近的生物素化（BioID）来特异性标记依赖于 在自噬机制的作用下，它们被释放到细胞外空间。我们现在将利用这些 创新的报告者破译自噬依赖的分泌组，并严格定义的机制， 自噬促进分泌。在目标1中，我们将详细阐述自噬依赖的 使用定量蛋白质组学的分泌产物，并评估它们作为监测肿瘤的生物标志物的效用 进展和体内治疗。对于这些研究，我们将重点关注KRAS突变型肺癌细胞 表现出高水平的基础自噬和分泌。在目标2中，我们将定义 自噬控制肿瘤细胞分泌。我们将利用基于邻近性的生物素化策略，结合 遗传方法，解剖细胞内运输的自噬依赖性分泌蛋白，因为它们 退出细胞，长期目标是确定这些途径中的靶点，用于治疗干预， 癌总的来说，这些研究为自噬在人类免疫系统中的全新作用提供了及时的机制性见解。 癌症进展和治疗反应。