Regulation of ClC-3 in Human Malignant Glioma

描述(申请人提供)：恶性胶质瘤是最常见和最致命的原发脑癌类型，大约每10万人中就有5人受到影响。尽管经过了几十年的研究和积极的治疗，包括手术、放射治疗和化疗，这种癌症的中位生存期只有大约一年。为了在这种毁灭性的神经疾病的临床治疗方面取得进展，了解胶质瘤独特的病理生理机制的研究可能会揭示治疗的新靶点。最近的研究表明，离子通道与胶质瘤细胞的侵袭性迁移和增殖能力有关。更具体地说，某些K和Cl-通道的表达增强了胶质瘤细胞同时排出K和Cl-的能力，导致了对细胞侵袭和增殖至关重要的强制性水分释放和动态细胞体积变化。神经胶质瘤细胞表达的离子通道之一是ClC-3，这是一种电压门控氯离子通道。ClC-3在胶质瘤细胞的迁移和增殖中起主要作用，但其在胶质瘤细胞中被激活的机制尚不清楚。一些证据表明ClC-3可能受钙/钙调蛋白依赖的蛋白激酶II的调节，这是一种对钙敏感的激酶。CaMKII对ClC-3的调节特别有趣，因为调节胶质瘤钙水平的配体和通道在胶质瘤的迁移和增殖中也发挥着关键作用。因此，本研究的目的是了解CaMKII的钙激活导致ClC-3磷酸化是否会促进胶质瘤细胞的迁移和增殖。这将通过使用全细胞膜片钳电生理学、免疫细胞化学和其他生化分析，首先在特定目标1中确定CaMKII磷酸化是否增强胶质瘤细胞中的ClC-3电流来实现这一点。接下来，在特定的目标2中，将进行成像、基因敲除和电生理实验，以确定细胞内钙离子的增加是否通过CaMKII磷酸化激活CLC-3电导。最后，特异靶3被设计来确定CaMKII介导的ClC-3的激活是否确实在胶质瘤细胞的迁移和增殖中发挥作用。CaMKII可能是一个分子翻译器，通过ClC-3的磷酸化将细胞内的钙信号转化为氯离子电导的变化。因此，干扰ClC-3活性或CaMKII激活ClC-3的新疗法可能会导致更好的临床疗效。事实上，氯毒素，一种氯电流的抑制剂，目前正在进入治疗恶性胶质瘤的第三阶段试验。 公共卫生相关性：尽管积极治疗，多形性胶质母细胞瘤患者的预后非常差，这是一种IV级原发脑癌。因此，了解胶质母细胞瘤生物学的独特特征将有助于确定新的靶点，以开发治疗药物，从而获得更好的临床结果。

DESCRIPTION (provided by applicant): Malignant glioma, the most common and lethal type of primary brain cancer, affects about 5 in 100,000 people. Despite decades of research and aggressive treatments consisting of surgery, radiotherapy, and chemotherapy, the median survival for this cancer is only about 1 year. To make headway in the clinical management of this devastating neurological disease, research to understand the unique pathophysiology of gliomas may reveal new targets for therapy. Recent research has implicated ion channels in the ability of glioma cells to aggressively migrate and proliferate. More specifically, expression of certain K+ and Cl- channels endows glioma cells with an enhanced ability to concomitantly extrude K+ and Cl-, leading to obligated water release and dynamic cell volume change that are essential for cell invasion and proliferation. One of the ion channels expressed by glioma cells critical to this process is ClC-3, a voltage-gated chloride channel. ClC-3 plays a major role in glioma cell migration and proliferation, but the mechanism by which ClC-3 is activated in glioma cells is not known. Several lines of evidence suggest that ClC-3 may be regulated by Ca2+/calmodulin-dependent protein kinase II, a Ca2+-sensitive kinase. Regulation of ClC-3 by CaMKII is particularly interesting, given that ligands and channels regulating glioma Ca2+ levels also play critical roles in glioma migration and proliferation. Therefore the goal of the current study is to understand if Ca2+ activation of CaMKII leading to ClC-3 phosphorylation will lead to enhanced glioma cell migration and proliferation. This will be accomplished by first determining in Specific Aim 1 if ClC-3 currents in glioma cells are enhanced by CaMKII phosphorylation by using whole-cell patch clamp electrophysiology, immunocytochemistry, and other biochemical assays. Next, in Specific Aim 2, imaging, genetic knockdown, and electrophysiological experiments will be performed to determine if increases in intracellular Ca2+ activate ClC-3 conductance via CaMKII phosphorylation in glioma cells. Finally, Specific Aim 3 is designed to determine if CaMKII-mediated activation of ClC-3 actually plays a role in the migration and proliferation of glioma cells. CaMKII may be a molecular translator, converting intracellular Ca2+ signals into changes in chloride conductance via ClC-3 phosphorylation. Therefore novel therapeutics interfering with ClC-3 activity or CaMKII activation of ClC-3 may lead to better clinical outcomes. Indeed, Chlorotoxin, an inhibitor of chloride currents, is currently entering Phase III trials for the treatment of malignant gliomas. PUBLIC HEALTH RELEVANCE: Despite aggressive treatment, the prognosis for patients suffering from glioblastoma multiforme, a Grade IV primary brain cancer, is very poor. Therefore understanding the unique features of glioblastoma biology will lead to the identification of novel targets for the development of therapeutic agents leading to better clinical outcomes.