Altered mitochondria-ER signaling as a cause of chemotherapy resistance

项目摘要 大多数死于癌症的患者都患有耐药性肿瘤。紧急抵抗 具有不同活性机制的多种治疗，称为多药耐药，是治疗的最大障碍。 治愈，但其原因仍然在很大程度上未知。癌症线粒体在治疗抗性中的作用已经被证实。 因为这些细胞器整合了压力和生存信号以决定细胞命运。实际上几乎所有的 有效的癌症疗法诱导足以激活线粒体凋亡信号的应激信号，而 在肿瘤进展期间选择抑制该过程的改变。直接审问这个问题， 优化了一种创新的检测方法，在该方法中，功能性线粒体从癌细胞中分离出来， 暴露于tBid和/或Bim，响应于大多数治疗性药物而递送至线粒体的死亡刺激物， 压力源这提供了它们的相对应力灵敏度的读数。我们用这个工具来研究 神经母细胞瘤，一种高度致命的癌症，通常在放化疗后完全消退 随后复发为多药耐药疾病。我们建立了一个国家基础设施， 肿瘤细胞系和患者来源的异种移植物均来自初次诊断时的相同患者 （治疗前）和治疗后复发时再次进行。这些近等基因肿瘤对提供了一个 独特的资源，因为复发后肿瘤表现出深刻的多药耐药性， 在密集的多模式治疗过程中。将我们的线粒体分析技术应用于这些 肿瘤使人们发现，来自复发后耐药肿瘤的线粒体， 与治疗敏感性肿瘤相比，对tBid和Bim应答的凋亡信号传导严重减弱。 我们在这里工作的目的是确定这种治疗抗性的线粒体决定因素。我们 基于我们初步数据的中心假设是， 网状细胞和线粒体是多药耐药的主要驱动力。ER线粒体系链（也称为 与ER相关的膜或MAM）形成IP 3R/GRP 75/VDAC富集的结构域，以转移 钙的缺乏减弱了细胞凋亡信号传导。为了验证这一点，我们将量化ER- 线粒体接触肿瘤与化疗和激酶抑制剂耐药，操纵拴系使用 遗传和生化方法，并评估其对线粒体活性和耐药性的影响 (Aim 1），并定义钙在这种表型中的作用（目的2）。虽然这种新的抵抗机制 提供了一个生存偏见下游的各种压力，它不是排他性的其他阻力机制。的 值得注意的是，改变的内质网-线粒体束缚也与糖尿病和神经退行性变有关， 放松管制对人类健康具有广泛的相关性。这些目标的结果将揭示贡献 这种ER-线粒体表型对癌症治疗耐药性的影响，这是一种开发工具的新模型， 衡量这一点，并确定治疗的机会，以恢复耐药性。

Project Summary Most patients who die from cancer have progression of therapy resistant tumors. Emergent resistance to diverse treatments with distinct mechanisms of activity, termed multidrug resistance, is the greatest barrier to cure yet its causes remain largely unknown. A role for cancer mitochondria in therapy resistance has been sought since these organelles integrate stress and survival signals to determine cell fate. Indeed, most effective cancer therapies induce stress signals sufficient to activate mitochondrial apoptotic signaling, while alterations that repress this process are selected for during tumor progression. To interrogate this directly we optimized an innovative assay in which functional mitochondria are isolated from cancer cells and selectively exposed to tBid and/or Bim, the death stimuli delivered to mitochondria in response to most therapeutic stressors. This provides a read-out of their relative stress sensitivity. We use this tool to study resistance in neuroblastoma, a highly lethal cancer that often completely regresses in response to chemoradiotherapy before subsequently relapsing as multidrug resistant disease. We created a national infrastructure to derive tumor cell lines and patient-derived xenografts from the same patients both at the time of initial diagnosis (before therapy) and again at the time of relapse after treatment. These near-isogenic tumor pairs provide a unique resource as the post-relapse tumors manifest profound multidrug resistance that has been selected for during the course of intensive multimodality treatment. Applying our mitochondrial profiling technique to these tumors enabled the discovery that mitochondria derived from post-relapse therapy resistant tumors have severely blunted apoptotic signaling in response to tBid and Bim in comparison with therapy sensitive tumors. The objective of our work here is to identify the mitochondrial determinants of this therapy resistance. Our central hypothesis based on our preliminary data is that a loss of physical tethering between endoplasmic reticulum and mitochondria is the principal driver of multidrug resistance. ER mitochondria tethers (also termed mitochondria-associated ER membranes, or MAMs) form IP3R/GRP75/VDAC-enriched domains to transfer calcium to mitochondria, and their absence attenuates apoptotic signaling. To test this we will quantify ER- mitochondria contacts in tumors with chemotherapy and kinase inhibitor resistance, manipulate tethering using genetic and biochemical approaches and assess its impact on mitochondrial activities and drug resistance (Aim 1), and define the role calcium plays in this phenotype (Aim 2). While this novel resistance mechanism provides a survival bias downstream of diverse stressors it is not exclusive to other resistance mechanisms. Of note, altered ER-mitochondria tethering has been implicated in diabetes and neurodegeneration as well so its deregulation has broad relevance to human health. The outcomes of these Aims will reveal the contributions of this ER-mitochondria phenotype to cancer therapy resistance, a novel model for the development of tools to measure this, and the identification of therapeutic opportunities to revert resistance.