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有治疗耐药肿瘤的进展。对…的紧急抵抗 具有不同作用机制的不同治疗方法，称为多药耐药，是治疗 然而，其原因在很大程度上仍不清楚。癌症线粒体在治疗抵抗中的作用一直是 因为这些细胞器整合了压力和生存信号来决定细胞的命运。事实上，大多数 有效的癌症治疗诱导的应激信号足以激活线粒体凋亡信号，而 在肿瘤进展过程中选择抑制这一过程的改变。为了直接审问这一点，我们 优化了一种创新的检测方法，即从癌细胞中分离出功能性线粒体，并选择性地 暴露在TBID和/或Bim下，向线粒体传递的死亡刺激对大多数治疗性 压力源。这提供了它们相对应力敏感性的读数。我们使用这个工具来研究抗药性 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 在随后复发为耐多药疾病之前。我们创建了一个国家基础设施来派生 肿瘤细胞系和患者来源的异种移植在最初诊断时均来自同一患者 (治疗前)和治疗后复发时再进行一次。这些近等基因的肿瘤对提供了 独特的资源作为复发后肿瘤表现出深刻的多药耐药已被选为 在强化综合治疗的过程中。将我们的线粒体图谱技术应用于这些 肿瘤使人们发现，来自复发治疗后耐药肿瘤的线粒体 与治疗敏感的肿瘤相比，对TBID和BIM的反应严重钝化了细胞凋亡信号。 我们在这里工作的目的是确定这种治疗抵抗的线粒体决定因素。我们的 基于我们初步数据的中心假设是内质之间物理连接的丧失 网状结构和线粒体是多药耐药的主要驱动因素。内质网线粒体系绳(也称为 线粒体相关内质网膜形成IP3R/GRP75/VDAC富集区进行转移 钙到线粒体，它们的缺失减弱了细胞凋亡信号。为了测试这一点，我们将量化ER- 线粒体与化疗和激酶抑制剂耐药的肿瘤接触，使用 遗传和生化途径，并评估其对线粒体活性和耐药性的影响 (目标1)，并定义钙在这种表型中的作用(目标2)。虽然这种新的抵抗机制 在不同的应激源下游提供了一种生存偏见，它并不是其他抵抗机制所独有的。的 注意，内质网线粒体连接改变也与糖尿病和神经退行性变有关，因此其 放松管制与人类健康有着广泛的相关性。这些目标的结果将揭示其贡献 这种ER-线粒体对癌症治疗耐药性的表型，为开发工具提供了一种新的模式 测量这一点，并确定逆转耐药性的治疗机会。

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.