Regulation of ClC-3 in Human Malignant Glioma

描述（由申请人提供）：恶性胶质瘤是最常见和最致命的原发性脑癌类型，大约10万人中有5人患病。尽管经过数十年的研究和包括手术、放疗和化疗在内的积极治疗，这种癌症的中位生存期仅为1年左右。为了在这种毁灭性的神经系统疾病的临床治疗中取得进展，研究胶质瘤独特的病理生理学可能会揭示新的治疗靶点。最近的研究表明，离子通道与胶质瘤细胞积极迁移和增殖的能力有关。更具体地说，某些K+和Cl-通道的表达使胶质瘤细胞具有增强的同时挤出K+和Cl-的能力，从而导致强制性的水释放和细胞体积的动态变化，这是细胞侵袭和增殖所必需的。胶质瘤细胞表达的离子通道之一是ClC-3，这是一个电压门控的氯离子通道。ClC-3在胶质瘤细胞迁移和增殖中起重要作用，但其在胶质瘤细胞中的激活机制尚不清楚。一些证据表明，ClC-3可能受Ca2+/钙调素依赖性蛋白激酶II（一种Ca2+敏感激酶）的调节。CaMKII对ClC-3的调节特别有趣，因为调节胶质瘤Ca2+水平的配体和通道在胶质瘤迁移和增殖中也起着关键作用。因此，本研究的目的是了解Ca2+激活CaMKII导致ClC-3磷酸化是否会导致胶质瘤细胞迁移和增殖增强。这将首先通过使用全细胞膜片钳电生理、免疫细胞化学和其他生化分析，在Specific Aim 1中确定CaMKII磷酸化是否会增强胶质瘤细胞中的ClC-3电流，从而实现。接下来，在Specific Aim 2中，将进行成像、基因敲除和电生理实验，以确定细胞内Ca2+的增加是否通过CaMKII磷酸化激活胶质瘤细胞中的ClC-3电导。最后，Specific Aim 3旨在确定camkii介导的ClC-3激活是否在胶质瘤细胞的迁移和增殖中起作用。CaMKII可能是一个分子翻译者，通过ClC-3磷酸化将细胞内Ca2+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的新疗法可能会带来更好的临床结果。事实上，氯毒素，一种氯电流抑制剂，目前正在进入治疗恶性胶质瘤的III期试验。

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.