Mitochondrial Dysfunction and Chemoresistance in Malignant Gliomas

描述（由申请人提供）：线粒体在细胞能量产生、细胞凋亡和自由基产生中起核心作用。线粒体功能障碍与许多癌症的发展有关，包括脑肿瘤。多形性胶质母细胞瘤（GBM）是最常见的原发性颅内肿瘤，其几乎一致的致死率为12-15个月的中位生存期。目前的管理包括手术，放疗和化疗的组合。尽管有积极的治疗方法，但90%的GBM患者会复发。这种不良结果的一个原因是多药耐药（MDR）表型的发展。我们先前详细描述了神经胶质瘤生长和进展的生物能量途径。最引人注目的观察结果之一是，依赖于糖酵解代谢的胶质瘤细胞通过参与其线粒体途径容易地适应生物能应激，以便存活和生长。这表明线粒体功能在神经胶质瘤的生物学中起着关键作用。线粒体功能障碍在脑肿瘤MDR表型发展中的作用尚不清楚。我们在这项探索性资助中的目标是证实和扩展我们的初步发现，即线粒体功能缺陷支持导致恶性脑肿瘤进展的MDR表型的发展。从长远来看，我们相信这将为制定合理的治疗和诊断策略提供信息，这些策略可以有效地应用于这种疾病。我们将通过以下三个具体目的来验证我们的中心假设，即线粒体功能受损驱动胶质瘤中MDR表型的发展：（1）我们将使用人胶质瘤细胞和同基因A0显示增加的MDR表型的（mtDNA耗尽的）衍生物，以确定：i）通过研究药物对细胞生长和存活的作用，对替莫唑胺（TZM）和卡莫司汀（BCNU）的相对细胞敏感性，凋亡和细胞周期分布; ii）MDR相关蛋白的表达模式，特别是在A0模型中高度表达的主要穹窿蛋白（MVP），和; iii）MVP对MDR的贡献。(2)我们将检查已建立的替莫唑胺耐药的人脑胶质瘤细胞，以确定与化学敏感的同基因细胞相比，化学耐药的胶质瘤细胞中线粒体功能是否受损。我们将量化和比较：i）呼吸率; ii）线粒体复合物的酶活性; iii）线粒体蛋白质组和iv）MDR相关蛋白。(3)我们将确定线粒体功能障碍是否通过HIF-11转录活性导致MDR相关蛋白表达增加。这些研究将严格检查线粒体功能在神经胶质瘤多药耐药发展中的作用，这些发现将为未来的研究奠定基础，以解决恶性神经胶质瘤患者更有效，更有针对性的治疗方式和诊断策略的发展。 公共卫生相关性：众所周知，恶性胶质瘤通常对化疗方式非常耐药，在积极方案后患者无进展生存率或总生存率的改善极小。有效治疗的一个重要障碍被认为是这些细胞表达的多药耐药表型。我们的初步数据显示，胶质瘤细胞系中线粒体功能的损伤导致MDR样表型。这项研究的相关性在于我们计划更详细地了解线粒体在调节多药耐药性中的作用。这些信息将为后续研究提供基础，这些研究旨在开发合理的方法来改善脑肿瘤患者的化疗方案，因此非常重要。

DESCRIPTION (provided by applicant): Mitochondria play a central role in cellular energy production, apoptosis and free radical generation. Mitochondrial malfunctions have been associated with development of many cancers, including brain tumors. Glioblastoma multiforme (GBM) is the most common primary intracranial neoplasm and its almost uniform lethality is exemplified by a median survival of 12-15 months. Current management consists of a combination of surgery, radiotherapy and chemotherapy. Despite aggressive treatment approaches, recurrence occurs in 90% of GBM patients. One cause of this poor outcome is development of a multidrug-resistance (MDR) phenotype. We previously described in detail the bioenergetic pathways central to glioma growth and progression. One of the most striking observations is that glioma cells which rely on glycolytic metabolism readily adapt to bioenergetic stress by engaging their mitochondrial pathway in order to survive and grow. This suggests that mitochondrial function plays a critical role in the biology of gliomas. The role that mitochondrial dysfunction has in development of the MDR phenotype in brain tumors is unknown. Our goal in this exploratory grant is to confirm and extend our preliminary findings that defective mitochondrial function supports development of the MDR phenotype that leads to progression of malignant brain tumors. Long-term, we believe this will inform development of rational therapeutic and diagnostic strategies that can be applied effectively to this disease. We will test our central hypothesis that impairment of mitochondrial function drives development of the MDR phenotype in glioma by achieving three Specific Aims: (1) We will use human glioma cells and isogenic A0 (mtDNA-depleted) derivatives that display increased MDR phenotype to determine: i) relative cellular sensitivities to temozolomide (TZM) and carmustine (BCNU), by investigating drug effects on cell growth and survival, apoptosis and cell cycle distribution; ii) expression pattern of MDR-associated proteins in particular Major Vault Protein (MVP), highly expressed in the A0 model, and; iii) contribution of MVP to MDR. (2) We will examine established, temozolomide-resistant human glioma cells to determine whether mitochondrial function is impaired in chemoresistant glioma cells compared to chemosensitive isogenic cells. We will quantify and compare: i) respiration rates; ii) enzymatic activities of mitochondrial complexes; iii) mitochondrial proteome and iv) MDR associated proteins. (3) We will determine whether mitochondrial dysfunction results in increased expression of MDR-associated proteins via HIF-11 transcriptional activity. These studies will critically examine the contribution of mitochondrial function in development of multidrug- resistance in gliomas and the findings will produce a foundation for future studies to address development of more effective, targeted therapeutic modalities and diagnostic strategies for malignant glioma patients. PUBLIC HEALTH RELEVANCE: It is well-established that malignant gliomas are generally very resistant to chemotherapeutic modalities with minimal improvement in patient progression-free or overall survivals following aggressive regimens. An essential impediment to effective therapy is believed to reside in the Multi-Drug Resistant Phenotype expressed by these cells. Our preliminary data has revealed that impairment of mitochondrial function in glioma cell lines results in a MDR-like phenotype. The relevance of this research lies in our plan to develop a more detailed understanding of the role that mitochondria have in regulation of multidrug-resistance. This information will provide the foundation for subsequent studies designed to develop rational means to improve chemotherapy regimens for brain tumor patients and as such is highly significant.