Calcium Channels in Glioblastoma

摘要 胶质母细胞瘤(GBM)是最常见、最致命的原发脑肿瘤。钙信号转导 调节过多的与癌症相关的分子和细胞过程，包括细胞增殖，细胞凋亡， 运动、血管生成、分化、基因转录以及神经传递和突触可塑性。 T-型钙通道(TTCC)调节钙离子从细胞外到胞浆的内流。我们的 初步数据显示，TTCC在GBM细胞、干细胞(GSC)和人类肿瘤中上调，并且 它们的阻断导致肿瘤细胞中促癌参数的抑制-内在和 依赖微环境的方式。基于这些数据，我们假设TTCC强烈监管 GBM分子事件和GBM-微环境相互作用驱动肿瘤生长 靶向TTCC联合其他治疗方法是一种很有前途的GBM治疗方法。为了检验这一假设， 我们建议研究肿瘤细胞的内在和微环境依赖的功能，机制 TTCC在GBM中的作用和治疗靶向性。在目标1中，我们将确定GBM细胞的内在作用 以及TTCC在微环境完好的新小鼠模型中的作用机制。我们将发展 新的RCAS/TVA和转基因免疫活性TTCC小鼠模型及其在TTCC中的作用 在完整的GBM微环境中。我们还将使用基因组和蛋白质组筛选以及分子和 功能途径以揭示TTCC在这些GBM肿瘤中的作用机制。在目标2中，我们将 揭示肿瘤微环境TTCC在介导促肿瘤神经元/GBM中的作用 互动。我们假设神经元TTCC和GBM TTCC协同调节促肿瘤作用 最近发现的基底膜细胞和神经元之间的相互作用。为了检验这一假设，我们将使用 共培养和GBM动物模型研究TTCC在调节神经元/GBM突触中的作用 形成，钙离子流入肿瘤细胞，以及肿瘤的生长和恶性。在目标3中，我们将开发和测试 肾小管上皮癌靶向治疗的新策略。我们有一个重新调整用途的FDA批准的TTCC 阻滞剂米贝拉地尔，在I期复发的GBM试验中被证明是安全的和可能有效的。我们 将测试米贝地尔对GBM移植瘤、同基因肿瘤和RCAS/TVA GBM小鼠生长的影响 采用标准的临床Stupp方案。我们还将在体外和体内进行合成致死CRISPR 筛选以发现可用药的靶点和与米贝拉地尔协同的药物。然后我们将测试组合 米贝拉地尔和合成致死药物在GBM动物模型中的作用。总之，这些发现将产生新的 了解TTCC在GBM及其微环境中的作用和作用机制，开发新的 研究TTCC的工具，揭示TTCC在介导促肿瘤神经元/GBM相互作用中的作用， 并开发和测试新的有效的GBM联合疗法，这些疗法可以转化为临床试验。

ABSTRACT Glioblastoma (GBM) is the most common and most deadly primary malignant brain tumor. Calcium signaling regulates a plethora of cancer-associated molecular and cellular processes including cell proliferation, apoptosis, motility, angiogenesis, differentiation, gene transcription as well as neurotransmission and synaptic plasticity. Calcium influx from the extracellular space to the cytosol is regulated by T-Type calcium channels (TTCC). Our preliminary data show that TTCC are upregulated in GBM cells, stem cells (GSC) and human tumors and that their blockage leads to inhibition of cancer-promoting parameters in tumor cell-intrinsic and microenvironment-dependent manners. Based on these data, we hypothesize that TTCC strongly regulate GBM molecular events and GBM-microenvironment interactions to drive tumor growth, and that targeting TTCC in combination with other modalities is a promising GBM therapy. To test this hypothesis, we propose to investigate the tumor cell-intrinsic and microenvironment-dependent functions, mechanisms of action, and therapeutic targeting of TTCC in GBM. In Aim 1, we will determine the GBM cell-intrinsic role and mechanisms of action of TTCC in new mouse models with intact microenvironment. We will develop new RCAS/Tva and transgenic immune competent TTCC mouse models and use them to study the role of TTCC in an intact GBM microenvironment. We will also use genomic and proteomic screenings and molecular and functional approaches to uncover the mechanisms of action of TTCC in these GBM tumors. In Aim 2, we will uncover the role of tumor microenvironment TTCC in mediating tumor-promoting neuron/GBM interactions. We hypothesize that neuronal TTCC and GBM TTCC cooperate to regulate the tumor-promoting interactions between GBM cells and neurons that were recently discovered. To test this hypothesis, we will use co-cultures and GBM animal models to investigate the role of TTCC in regulating neuron/GBM synaptic formation, calcium influx into tumor cells, and tumor growth and malignancy. In Aim 3, we will develop and test new strategies for the therapeutic targeting of TTCC in GBM. We have a repurposed FDA approved TTCC blocker, mibefradil, that was demonstrated to be safe and possibly effective in a phase I recurrent GBM trial. We will test the effects of mibefradil on the growth of GBM xenografts, syngeneic tumors and RCAS/Tva GBM mice using the standard clinical Stupp Regimen. We will also perform in vitro and in vivo synthetic lethal CRISPR screens to uncover druggable targets and drugs that synergize with mibefradil. We will then test combinations of mibefradil and the synthetic lethal drugs in GBM animal models. Altogether, the findings will generate new knowledge on the functions and mechanisms of action of TTCC in GBM and its microenvironment, develop new tools for the study of TTCC, uncover the role of TTCC in mediating tumor-promoting neuron/GBM interactions, and develop and test new efficacious GBM combination therapies that could be translated into clinical trials.