Inositol-trisphosphate 3-kinase and colorectal cancer cell adhesion

 描述(由申请人提供)：细胞内钙离子([Ca~(2+)]i)增加与肿瘤发生的许多方面有关，包括细胞增殖、黏附和迁移。产生这些钙离子反应的途径和下游效应分子已经被广泛研究。相反，终止钙信号的分子在癌症发生中的作用受到的关注较少，可能代表着治疗癌症的新的治疗靶点或转移潜力的预后指标。我们的初步数据表明，肌醇1，4，5-三磷酸C型激酶(ItpkC)在调节肿瘤细胞与微血管系统的黏附中发挥了作用。Itpk酶家族将1，4，5-三磷酸(InsP3)转化为1，3，4，5-三磷酸(InsP4)，是受体偶联磷脂酶C(PLC)介导的钙升高的“关”信号机制的一部分。与原发源于结肠癌的细胞相比，来自淋巴结转移的结肠癌细胞中Itpk的蛋白水平显著降低。转移变异体中Itpk的下调与对E-选择素刺激的敏感性增加和更大的钙瞬变幅度有关。此外，ItpkC的过表达抑制了人结肠癌细胞与肝内皮细胞的黏附。综上所述，这些数据表明，Itpk表达的减少可能通过增强激动剂诱导的钙离子增加来促进肿瘤细胞的黏附。在我们的模型中，肝内皮细胞上的E-选择素黏附受体与结肠癌细胞上的对抗性受体结合，诱导了双向激活信号，从而激活了PLC，刺激了InsP3的形成，并增加了癌细胞和肝细胞中的钙离子。我们推测，ItpkC的下调有利于微血管与循环中的结肠癌细胞的黏附，从而有利于转移到肝脏。这项提议的目标是检验新的假设，即ItpkC的表达水平 调节黏附诱导的钙离子增加，从而调节结直肠癌的转移潜能。我们将研究ItpkC在有转移的原发结肠癌和无转移的肿瘤中的表达是否失调。这些研究将使用标准的分子生物学和免疫组织化学技术来确定ItpkC在正常人结肠上皮和患者匹配的原发和转移性结直肠癌中的表达和细胞分布。此外，我们还将验证这样一种猜想，即结直肠癌细胞系中ItpkC的下调将增强黏附依赖的钙信号，并增加肿瘤细胞与肝内皮细胞的结合。这些研究将使用体内癌症 用于测量胞浆钙反应的细胞黏附分析和广域荧光成像技术，以及实时监测结肠癌细胞和肝细胞[Ca~(2+)]i反应的新型共聚焦显微镜技术 并附着于内皮细胞。

 DESCRIPTION (provided by applicant): Increases in cytosolic Ca2+ ([Ca2+]i) have been implicated in many aspects of tumorigenesis including cell proliferation, adhesion and migration. The pathways generating these Ca2+ responses and the downstream effector molecules have been extensively studied. In contrast, the role of molecules terminating Ca2+ signals have received less attention in carcinogenesis and may represent new therapeutic targets to treat cancer or prognostic indicators for metastatic potential. Our preliminary data indicate a role for inositol 1,4,5-trisphosphate kinase type C (ItpkC) in regulating tumor cell adhesion to the microvasculature. The Itpk enzyme family converts inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-trisphosphate (InsP4) and is part of the "off" signaling mechanism for receptor-coupled phospholipase C (PLC) mediated Ca2+ increases. The proteins levels of Itpk are significantly decreased in colon cancer cells derived from a lymph node metastases compared to a cells originating from the primary colon tumor. The down regulation of Itpk in the metastatic variant was associated with increased sensitivity to E-selectin stimulation and larger amplitude Ca2+ transients. Moreover, the overexpression of ItpkC inhibits the adhesion of human colon cancer cells to liver endothelial cells. Taken together, these data suggest that decreases in the expression of Itpk may promote tumor cell adhesion by potentiating agonist-induced Ca2+ increases. In our model, the binding of E-selectin adhesion receptors on the liver endothelium to counter-receptors on colon cancer cells induces a bidirectional activation signal resulting in the activation of PLC, stimulation of InsP3 formation and increases in Ca2+ in both cancer and liver cells. We hypothesize that the down-regulation of ItpkC provides an advantage for microvascular adhesion to circulating colon cancer cells and therefore for metastasizing into the liver. The goal of this proposal is to test the novel hypothesis that the level of ItpkC expression regulates adhesion-induced Ca2+ increases and, thereby the metastatic potential of colorectal cancers. We will investigate if the expression ItpkC is dysregulated in primary colon cancers with metastases compared to tumors without metastases. These studies will use standard molecular biology and immunohistochemical techniques to determine the expression and cellular distribution of ItpkC in normal human colonic epithelium and patient matched primary and metastatic colorectal cancers. Moreover, we will test the conjecture that down regulation of ItpkC in colorectal cancer cell lines will potentiate adhesion-dependent Ca2+ signals and increase the binding of tumor cells to the liver endothelium. These studies will use in vivo cancer cell adhesion assays and wide-field fluorescent imaging techniques to measure cytosolic Ca2+ responses, as well as novel confocal microscopy techniques to monitor, in real time, colon cancer cell and liver cell [Ca2+]i responses as the cancer cell travels through the liver sinusoids and adheres to the endothelium.