Secretory Mitophagy in Cancer Metabolic Reprogramming

肿瘤代谢重编程是肿瘤进展、生存和治疗耐药的标志。一个 靶向类别的癌症代谢适应利用有丝分裂吞噬，这是一种已知的特殊自噬途径 与癌症表型有关。有丝分裂通过靶向选择性地消除功能障碍的线粒体 它们通过自噬小体穿梭到溶酶体进行降解。癌细胞有丝分裂是由 缺氧、放疗、分子治疗引起的氧化应激和线粒体DNA损伤增加， 和免疫疗法。高的有丝分裂需求会压倒溶酶体的能力，导致 损伤的线粒体堆积，对细胞有害，并可抑制健康的生物发生 线粒体。我们推测，新发现的分泌性吞噬输出过程破坏了 线粒体分裂释放的片段，以减少溶酶体系统的过载压力，从而 在治疗性线粒体应激的情况下维持癌细胞存活。我们发现了一种形式的 在生长中的实体瘤体内发生的分泌性有丝分裂吞噬。我们对全套曲目的分子分析 细胞外小泡(EV)进入体内残留的肿瘤间质液(IF)提供了丰富的信息 关于肿瘤细胞和宿主细胞内线粒体的功能状态。在肿瘤间质液中， 在经历氧化应激的癌细胞的培养液中，我们鉴定出了EV包装的全套 指线粒体分子组成线粒体细胞器的外围分裂被掐断的片段。 最近发现，外周有丝分裂受线粒体裂变1蛋白(FIS1)调控， 是吞噬有丝分裂的关键基本调节因子，与中区线粒体分裂不同，与 线粒体的生物发生。我们还发现有丝分裂诱导剂PTEN诱导了蛋白激酶1(PINK1)的裂解状态 (全长与裂解)，显著反映在IF EVS内输出的一组线粒体蛋白质中， 可能构成一种新的定量测量工具来监测肿瘤细胞内的实时状态 有丝分裂。我们的发现提出了重要的机械性问题，我们将在目标中探索，涉及 未知的细胞内分泌性有丝分裂步骤以及出口的EVS的内容如何反映内部 有丝分裂状态。在目标1下，我们将检验这样的假设，即外周分裂分泌性有丝分裂是诱导的 通过高要求的有丝分裂吞噬来压倒溶酶体，并探索分泌性有丝分裂是否明显 来自小鼠4T1和人MDA-MB-231三阴性乳腺癌细胞的分泌性自噬 台词。在目标2下，我们将检验全长与裂解的PINK1的比率升高的假设 (线粒体损伤传感器)在输出的同基因小鼠乳腺肿瘤间质液EVS中，如下 使用丝裂原诱导剂Mito-CP和雷帕霉素治疗。其结果是对 分泌性有丝分裂吞噬的重要性，可以构成一个重要的治疗靶点，以及一个新的临床应用 监测肿瘤微环境中有丝分裂通量体内状态的相关手段。

Tumor metabolic reprogramming is a hallmark of cancer progression, survival, and therapeutic resistance. A targetable class of cancer metabolic adaptation exploits mitophagy, a specialized autophagy pathway known to be linked to the cancer phenotype. Mitophagy selectively eliminates dysfunctional mitochondria by targeting them, via autophagosome shuttling, to the lysosome for degradation. Cancer cell mitophagy is triggered by elevated oxidative stress and mitochondria DNA damage caused by hypoxia, radiotherapy, molecular therapy, and immunotherapy. A high mitophagy demand can overwhelm the lysosome capacity resulting in the accumulation of damaged mitochondria that is harmful to the cell, and can suppress biogenesis of healthy mitochondria. We hypothesize that the newly discovered process of secretory mitophagy exports damaged mitochondrial fission-released segments to reduce the overload pressure on the lysosomal system, and thereby sustains cancer cell survival in the face of therapeutic mitochondrial stress. We have discovered a form of secretory mitophagy occurring in vivo in a growing solid tumor. Our molecular analysis of the full repertoire of extracellular vesicles (EV) shed into the resident tumor interstitial fluid (IF) in vivo yielded a rich set of information about the functional state of mitochondria within the tumor cells, and the host cells. Within tumor interstitial fluid, and within the culture media of cancer cells undergoing oxidative stress, we identified an EV-packaged full set of mitochondria molecules comprising the peripheral fission pinched-off segment of the mitochondrial organelle. It has recently been found that peripheral mitophagy fission, regulated by mitochondrial fission 1 protein (FIS1), is a key essential regulator of mitophagy, and is distinct from mid-zone mitochondria fission associated with mitochondria biogenesis. We also found that mitophagy inducer PTEN induced kinase 1 (PINK1) cleavage status (full length versus cleaved), is prominently reflected in the set of mitochondrial proteins exported within IF EVs, and may constitute a new quantitative measurement tool to monitor the real-time state of tumor intracellular mitophagy. Our findings raise important mechanistic questions, that we will explore in the Aims, concerning the unknown intracellular steps of secretory mitophagy and how the content of the exported EVs reflects the internal mitophagy state. Under Aim 1 we will test the hypothesis that peripheral fission secretory mitophagy is induced by high mitophagy demands that overwhelm lysosomal engulfment, and explore if secretory mitophagy is distinct from secretory autophagy within the murine 4T1 and human MDA-MB-231 triple negative breast cancer cell lines. Under Aim 2 we will test the hypothesis that the ratio of full length to cleaved PINK1 is elevated (mitochondria damage sensor) within the exported murine syngeneic breast tumor interstitial fluid EVs, following treatment with mitophagy inducers Mito-CP and Rapamycin. The outcome is new understanding of the importance of secretory mitophagy that can constitute an important therapeutic target, and a new clinically relevant means of monitoring the in vivo state of mitophagic flux within the tumor microenvironment.