Calcium dynamics and signaling in glioma

项目总结/摘要 脑胶质瘤是最常见、最具侵袭性的原发性脑恶性肿瘤， 接近。最近的研究表明，神经元活动促进肿瘤-脑界面的肿瘤增殖 通过激发肿瘤细胞表达的钙（Ca 2+）渗透性谷氨酸受体， 细胞内钙波。这些令人兴奋的观察结果归因于神经胶质瘤生长中Ca 2+内流的关键作用，但它们 不能解释Ca 2+波是否也与肿瘤核心有关，在那里增殖发生在没有Ca 2+波的情况下。 神经元活动和突触输入。我们的初步数据揭示了一种新的机制， 在没有神经元-肿瘤突触的情况下，利用Ca 2+流向它们的增殖。事实上，我们发现神经胶质瘤 细胞表达几种类型的Ca 2+通道，并在缺乏Ca 2+通道的情况下产生肿瘤细胞自主的Ca 2+波。 神经元或神经胶质。这种波可以用培养的患者源性胶质母细胞瘤细胞的Ca 2+成像观察到 在体外没有其他细胞类型的情况下，以及在急性脑缺血中小鼠脑中患者来源的异种移植物的成像。 脑切片制备。这些Ca 2+波被Ca 2+通道的离子抑制剂Co 2+和Cd 2+阻断。寻找 对于Ca 2+波的可能效应物，我们进行了一个无偏的脱落CRISPR筛选，目标是“可药物化的”。 Ca 2 +/钙调素依赖性蛋白激酶2B（CAMK 2b）是一种新的必需组分 神经胶质瘤的生长。事实上，CAMK 2b的药理学抑制或短发夹RNA（shRNA）敲低损害了 在体外的患者来源的胶质母细胞瘤培养物的增殖，表明CAMK 2b的必要性。这些初步 观察使我们假设肿瘤细胞自主的Ca 2+波通过激活 CAMK 2b和调节肿瘤发生的蛋白质底物的磷酸化。为了阐明这一机制， 我们提出了以下具体目标：1）表征神经胶质瘤细胞中产生Ca 2+波的Ca 2+通道 并确定它们对体外和体内肿瘤生长的影响; 2）检验CAMK 2b代表 是Ca 2+波和磷酸化底物的关键效应物，促进肿瘤增殖。我们有 组建了一个多学科专家小组来验证这一新假设。拟议的工作将奠定 将神经胶质瘤细胞电反应性与致瘤信号传导机制联系起来的基础， 在这种耐药肿瘤中发现新的靶向弱点。

PROJECT SUMMARY/ABSTRACT Glioma, the most common and aggressive primary brain malignancy, is in dire need of novel therapeutic approaches. Recent findings suggest that neuronal activity promotes tumor proliferation at the tumor-brain interface via excitation of calcium (Ca2+)-permeable glutamate receptors expressed by tumor cells, leading to robust intracellular Ca2+ waves. These exciting observations ascribe a critical role for Ca2+ influx in glioma growth, but they do not explain whether Ca2+ waves are also relevant in the core of tumors, where proliferation occurs in the absence of neuronal activity and synaptic input. Our preliminary data reveal a novel mechanism that allows glioma cells to utilize Ca2+ flux toward their proliferation in the absence of neuron-tumor synapses. Indeed, we found that glioma cells express several types of Ca2+ channels and generate tumor cell-autonomous Ca2+ waves in the absence of neurons or glia. Such waves can be observed with Ca2+ imaging of patient-derived glioblastoma cells cultured without other cell types in vitro, as well as imaging of patient-derived xenografts in the mouse brain in the acute brain slice preparation. These Ca2+ waves are blocked by Co2+ and Cd2+, ionic inhibitors of Ca2+ channels. In search for possible effectors of Ca2+ waves, we performed an unbiased dropout CRISPR screen targeting the “druggable genome” and identified Ca2+/calmodulin-dependent protein kinase 2B (CAMK2b) as a novel essential component of glioma growth. Indeed, pharmacologic inhibition or short hairpin RNA (shRNA) knockdown of CAMK2b impair proliferation of patient-derived glioblastoma cultures in vitro, suggesting CAMK2b essentiality. These preliminary observations lead us to hypothesize that tumor cell-autonomous Ca2+ waves promote glioma growth via activation of CAMK2b and phosphorylation of protein substrates that regulate tumorigenesis. To elucidate this mechanism, we propose the following specific aims: 1) characterize the Ca2+ channels that generate Ca2+ waves in glioma cells and determine their impact on tumor growth in vitro and in vivo; and 2) test the hypothesis that CAMK2b represents a crucial effector of Ca2+ waves and phosphorylates substrates that promote tumor propagation. We have assembled a multidisciplinary team of experts to test this novel hypothesis. The proposed work will lay the foundation for linking glioma cell electroresponsiveness to tumorigenic signaling mechanisms and holds promise for discovery of novel targetable vulnerabilities in this therapy-resistant tumor.