Mitochondrial Dysfunction and Chemoresistance in Malignant Gliomas

描述(申请人提供)：线粒体在细胞能量产生、细胞凋亡和自由基生成中起核心作用。线粒体功能障碍与许多癌症的发生有关，包括脑瘤。多形性胶质母细胞瘤(GBM)是最常见的颅内原发肿瘤，其致死率几乎一致，中位生存期为12-15个月。目前的治疗包括手术、放射治疗和化疗的组合。尽管采取了积极的治疗方法，90%的GBM患者仍会复发。这种不良结局的一个原因是出现了多药耐药(MDR)表型。我们之前详细描述了胶质瘤生长和进展的生物能量途径。最引人注目的观察之一是，依赖糖酵解代谢的胶质瘤细胞很容易通过参与线粒体途径来适应生物能量压力，以求生存和生长。这表明线粒体功能在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多药耐药表型形成中的作用尚不清楚。我们在这项探索性拨款中的目标是确认和扩大我们的初步发现，即线粒体功能缺陷支持MDR表型的发展，而MDR表型会导致恶性脑瘤的进展。从长远来看，我们相信这将为合理的治疗和诊断策略的发展提供信息，这些策略可以有效地应用于这种疾病。我们将检验我们的中心假设，即线粒体功能受损通过实现三个特定目标推动胶质瘤多药耐药表型的发展：(1)我们将使用人脑胶质瘤细胞和显示多药耐药表型增加的等基因A0(mtDNA缺失)衍生物来确定：i)通过研究药物对细胞生长和存活、细胞凋亡和细胞周期分布的影响，确定细胞对替莫唑胺(TZM)和卡莫司汀(BCNU)的相对敏感性；ii)MDR相关蛋白，尤其是在A0模型中高表达的主要保险库蛋白(MVP)的表达模式；以及iii)MVP在MDR中的作用。(2)我们将检查已建立的对替莫唑胺耐药的人脑胶质瘤细胞，以确定与化疗敏感的同基因细胞相比，耐药胶质瘤细胞的线粒体功能是否受损。我们将量化和比较：i)呼吸速率；ii)线粒体复合体的酶活性；iii)线粒体蛋白质组和iv)多药耐药相关蛋白。(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。这些研究将关键地检验线粒体功能在胶质瘤多药耐药形成中的作用，这些发现将为未来的研究奠定基础，以解决为恶性胶质瘤患者开发更有效、更有针对性的治疗方式和诊断策略。 与公共卫生相关：众所周知，恶性胶质瘤通常对化疗方式非常耐药，在积极的治疗方案后，患者的无进展或总存活率改善很小。有效治疗的一个重要障碍被认为存在于这些细胞表达的多药耐药表型中。我们的初步数据显示，胶质瘤细胞系线粒体功能受损导致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多药耐药调节中的作用。这些信息将为后续研究提供基础，旨在开发合理的方法来改善脑肿瘤患者的化疗方案，因此具有非常重要的意义。

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.