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

项目总结 目前，人们对调节自噬对抗癌症产生了极大的兴趣。抗疟疾药物，如 羟基氯喹(Hcq)在许多临床上被积极地用作自噬抑制剂。 肿瘤学试验。尽管有这种热情，我们仍然不能完全理解自噬如何影响癌症。 进展和治疗。到目前为止，以自噬为目标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，最近的发现 挑战这一领域的主流观点。尽管传统上被认为是一种“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)在促进协调中的新角色 分泌肿瘤细胞侵袭所需的细胞因子。然而，我们对自噬的理解是- 由于主要的概念和技术障碍，癌症中的依赖分泌仍然处于初级状态 阻碍破译自噬是如何促进分泌的。最重要的是，到目前为止的研究只有 依赖于遗传消融特定途径组件后的表型分析，如ATGs； 功能丧失的方法是有限的，因为它们无法辨别分泌缺陷是否代表 自噬受损的直接和间接后果。为了克服实地的这些主要障碍，我们 创造了新的记者，利用基于邻近的生物素化(BioID)专门标记依赖于 通过自噬机制将其释放到细胞外空间。我们现在将利用这些 创新的记者破译依赖自噬的分泌组，并严格定义机制 自噬促进分泌。在目标1中，我们将详细阐述依赖自噬的全套技术。 利用定量蛋白质组学分析分泌产物并评价其作为监测肿瘤生物标志物的价值 体内进展和治疗。在这些研究中，我们将重点研究KRAS突变的肺癌细胞 表现出高水平的基础自噬和分泌物。在目标2中，我们将定义 自噬控制着肿瘤细胞的分泌。我们将利用基于邻近的生物素化策略，结合 遗传学方法，剖析依赖自噬的分泌蛋白在细胞内的运输 退出细胞，长期目标是识别这些通路中的靶点，以便进行治疗干预 癌症。总体而言，这些研究为自噬提供了及时的机械性洞察，使其在 癌症进展和治疗反应。

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.