Regulation of ClC-3 in Human Malignant Glioma

描述（由申请人提供）： 恶性神经胶质瘤是最常见和致命的原发性脑癌，每10万人中约有5人患病。尽管数十年的研究和积极的治疗包括手术，放疗和化疗，这种癌症的中位生存期只有大约1年。为了在这种毁灭性神经系统疾病的临床管理方面取得进展，了解胶质瘤独特病理生理学的研究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-通道的表达赋予胶质瘤细胞增强的同时挤出K+和Cl-的能力，导致强制性水释放和动态细胞体积变化，这是细胞侵袭和增殖所必需的。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在胶质瘤细胞迁移和增殖中起主要作用，但ClC-3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可能是由Ca 2 +/钙调素依赖性蛋白激酶II，一个Ca 2+敏感的激酶。CaMKII对ClC-3的调节特别有趣，因为调节胶质瘤Ca 2+水平的配体和通道在胶质瘤迁移和增殖中也起关键作用。因此，本研究的目的是了解Ca 2+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。这将通过在特异性目标1中首先确定胶质瘤细胞中的ClC-3电流是否通过使用全细胞膜片钳电生理学、免疫细胞化学和其他生物化学测定法的CaMKII磷酸化而增强来实现。接下来，在具体目标2中，将进行成像、遗传敲低和电生理学实验以确定细胞内Ca 2+的增加是否通过神经胶质瘤细胞中的CaMKII磷酸化激活ClC-3电导。最后，特异性目的3旨在确定CaMKII介导的ClC-3活化是否在胶质瘤细胞的迁移和增殖中发挥作用。CaMKII可能是一种分子翻译器，通过ClC-3磷酸化将细胞内Ca 2+信号转化为氯离子电导的变化。因此，干扰C1 C-3活性或C1 C-3的CaMKII活化的新型治疗剂可能导致更好的临床结果。事实上，氯毒素，一种氯电流抑制剂，目前正在进入治疗恶性神经胶质瘤的III期试验。 公共卫生相关性： 尽管进行了积极的治疗，但患有多形性胶质母细胞瘤（一种IV级原发性脑癌）的患者的预后非常差。因此，了解胶质母细胞瘤生物学的独特特征将导致识别用于开发治疗剂的新靶点，从而导致更好的临床结果。

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.