Calcium dynamics and signaling in glioma

项目摘要/摘要 胶质瘤是最常见和最具侵袭性的脑部原发恶性肿瘤，迫切需要新的治疗方法。 接近了。最近的发现表明，神经元活动促进了肿瘤-脑交界处的肿瘤增殖 通过激发肿瘤细胞表达的钙(Ca~(2+))-渗透性谷氨酸受体，导致 细胞内钙波。这些令人兴奋的观察归因于钙离子内流在胶质瘤生长中的关键作用，但他们 不解释钙波是否也与肿瘤的核心有关，因为肿瘤的核心在没有钙波的情况下发生增殖。 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，使胶质瘤细胞能够 在没有神经元-肿瘤突触的情况下，利用钙离子流量促进它们的增殖。事实上，我们发现胶质瘤 细胞表达多种类型的钙离子通道，并在没有钙离子的情况下产生肿瘤细胞自主的钙离子波 神经元或神经胶质细胞。这种波可以用培养的患者来源的胶质母细胞瘤细胞的钙离子成像观察到 没有其他细胞类型的体外实验，以及患者来源的异种移植在急性脑损伤小鼠脑内的成像 脑片准备。这些钙波可被钙离子通道的离子抑制剂Co2+和Cd2+阻断。在搜索中 对于可能的钙波效应器，我们进行了一次无偏见的辍学CRISPR筛查，目标是 并鉴定了一种新的必需成分--钙/钙调蛋白依赖的蛋白激酶2B(CAMK2b 神经胶质瘤生长的迹象。事实上，CAMK2b的药理抑制或短发夹状RNA(ShRNA)敲除会损害 患者来源的胶质母细胞瘤培养物体外增殖，提示CAMK2b的重要性。这些初步的 观察结果使我们假设，肿瘤细胞自主的钙波通过激活促进胶质瘤生长 CAMK2b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)表征在胶质瘤细胞中产生钙波的钙通道 并确定它们对体外和体内肿瘤生长的影响；以及2)检验CAMK2b代表的假设 钙波和促进肿瘤生长的磷酸化底物的关键效应器。我们有 组建了一个由多学科专家组成的团队来验证这一新颖的假设。拟议的工作将为 将胶质瘤细胞的电反应与致瘤信号机制联系起来的基础和前景 在这种耐药肿瘤中发现新的靶向脆弱性。

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.