Therapeutic Targeting of Autophagey-Dependent Cancer

自噬依赖性癌症的治疗靶向

• 批准号: 10472048
• 负责人: Christina G Towers
• 金额: $ 20.37万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-11 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10472048
• 关键词: Acute; Affect; Aftercare; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Apoptotic; Autophagocytosis; Binding; Biological Assay; Biology; CRISPR/Cas technology; Cancer cell line; Catabolic Process; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Cellular biology; Chemosensitization; Clinic; Clinical Treatment; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Dependence; Excision; Foundations; Genes; Growth; Homeostasis; Human; In Vitro; Knock-out; Knowledge; Link; Lysosomes; Malignant Neoplasms; Mediating; Mentors; Mentorship; Methods; Microscopy; Mitochondria; Mitochondrial Proteins; Mus; Parkin; Pharmaceutical Preparations; Phase; Predisposition; Process; Proteins; Publishing; Recycling; Regulation; Reporting; Research; Resistance; Resolution; Role; Signal Transduction; Stress; Techniques; Testing; Toxin; Training; Up-Regulation; Vesicle; Work; cancer cell; chemotherapeutic agent; chemotherapy; improved; inhibition of autophagy; inhibitor; insight; knock-down; neoplastic cell; novel; optogenetics; pre-clinical; preclinical study; pressure; pro-apoptotic protein; programs; proteostasis; resistance mechanism; response; targeted treatment; therapeutic target; tumor; tumorigenic

Project Summary/Abstract Autophagy is an essential catabolic process that cells rely on to breakdown cytoplasmic constituents. In-vitro and pre-clinical animal studies indicate a pro-tumorigenic role for autophagy, leading to the launch of over 60 clinical trials utilizing autophagy inhibition. Initial results from clinical trials have been promising, however, these studies already indicate both inherent and acquired resistance to autophagy inhibition in certain tumors. I hypothesize that understanding how tumors circumvent autophagy inhibition will improve the use of autophagy-targeted therapeutics. In my recently published studies, I created a dynamic, Live-Cell CRISPR/Cas9 assay (LC-CRISPR) to target 12 core autophagy genes in a panel of 8 cancer cell lines. I identified autophagy dependent cell lines, however, I also discovered that, with enough selective pressure, even highly autophagy dependent cells can survive loss of autophagy – specifically loss of the core autophagy proteins ATG7 and FIP200. I discovered that the newly-acquired, autophagy-independent, ATG7-/- and FIP200-/- cells acquired an increased dependence on NRF2 cytoprotective and antioxidant signaling to maintain protein homeostasis. My initial studies also suggest the ATG7-/- and FIP200-/- cells have increased additional cytoprotective processes such as mitochondrial fusion and mitochondrial derived vesicles (MDVs). Previous reports have linked autophagy and NRF2 signaling, however, this will be the first study to understand the role of NRF2 in dictating autophagy dependence. These studies will utilize novel techniques that I have developed including LC-CRISPR and rapid optogenetic inhibition of autophagy to understand the necessity and sufficiency, as well as the in-depth mechanism, rate, and duration of NRF2 antioxidant signaling in mediating autophagy dependence in cancer. I hypothesize that NRF2 expression, both basal and induced expression after autophagy inhibition will dictate autophagy dependence in cancer cells. The 2nd aim and 3rd aims will utilize the isogeneic autophagy dependent WT and ATG7-/- and FIP200-/- autophagy-independent cell lines as well as Atg5-/- mouse tumor cells that circumvented autophagy inhibition in a live animal to understand the links between autophagy dependence, apoptosis, and mitochondrial dynamics. In a previous study we identified the critical link between autophagy and apoptosis and showed that autophagy inhibition leads to increased FOXO3a binding to PUMA and increases sensitivity to other chemotherapeutic agents. The studies proposed here will test the hypothesis that cells that circumvent autophagy dependence also circumvent the FOXO3a-PUMA mediated link between autophagy and apoptosis due to increased mitochondrial fusion. The final aim will test the hypothesis that MDVs are critical for the removal of damaged portions of mitochondria independent of canonical mitophagy and this activity therefore maintains mitochondrial homeostasis. Together these studies will elucidate how cancer cells can circumvent autophagy dependence and provide insight into mechanisms of resistance to autophagy inhibition. !

项目摘要/摘要 自噬是细胞分解细胞质成分所依赖的一个基本的分解代谢过程。 体外和临床前的动物研究表明，自噬具有促肿瘤作用，导致了 超过60项利用自噬抑制的临床试验。临床试验的初步结果令人振奋， 然而，这些研究已经表明对自噬抑制的先天和后天抵抗。 某些肿瘤。我推测，了解肿瘤是如何避开自噬抑制的 改进自噬靶向疗法的使用。在我最近发表的研究中，我创造了一种动态， 针对8个癌细胞中12个核心自噬基因的活细胞CRISPR/Cas9分析(LC-CRISPR) 台词。我鉴定了依赖自噬的细胞系，然而，我也发现，有足够的选择性 压力，即使高度依赖自噬的细胞也可以在自噬丧失--特别是核心丧失--的情况下存活下来 自噬蛋白ATG7和FIP200。我发现新获得的、不依赖自噬的ATG7-/- FIP200-/-细胞对NRF2细胞保护和抗氧化信号的依赖增加 维持蛋白质动态平衡。我的初步研究还表明，ATG7-/-和FIP200-/-细胞数量增加 其他细胞保护过程，如线粒体融合和线粒体衍生小泡(MDV)。 以前的报告已经将自噬和NRF2信号联系在一起，然而，这将是第一次研究 了解NRF2在决定自噬依赖性中的作用。这些研究将利用新的技术 包括LC-CRISPR和自噬的快速光遗传抑制，以了解 NRF2抗氧化信号的必要性和充分性以及深入的机制、速率和持续时间 在调节癌症自噬依赖方面的作用。我假设NRF2的表达，无论是基础还是 自噬抑制后的诱导表达将决定癌细胞对自噬的依赖性。 第二个目标和第三个目标将利用同源自噬依赖的WT和ATG7-/-和FIP200-/- 自噬非依赖性细胞系以及ATG5/-小鼠肿瘤细胞绕过自噬抑制 了解自噬依赖、细胞凋亡和线粒体动力学之间的联系的活动物。 在之前的一项研究中，我们确定了自噬和细胞凋亡之间的关键联系，并表明自噬 抑制导致FOXO3a与PUMA结合增加，并增加对其他化疗药物的敏感性 探员们。这里提出的研究将检验这样一种假设，即规避自噬的细胞 依赖也绕过FOXO3a-PUMA介导的自噬和细胞凋亡之间的联系 以增加线粒体的融合。最终目标将检验MDV对 去除线粒体受损部分，不依赖于正常的有丝分裂和这一活性 因此维持线粒体的动态平衡。这些研究将共同阐明癌细胞是如何 避免自噬依赖，并提供对自噬抑制抵抗机制的洞察。 好了！