Inositol-trisphosphate 3-kinase and colorectal cancer cell adhesion

 描述（由申请方提供）：细胞溶质Ca 2+（[Ca 2 +]i）的增加与肿瘤发生的许多方面有关，包括细胞增殖、粘附和迁移。产生这些Ca 2+反应的途径和下游效应分子已被广泛研究。相反，终止Ca 2+信号的分子在致癌作用中的作用较少受到关注，可能代表治疗癌症的新治疗靶点或转移潜能的预后指标。我们的初步数据表明，肌醇1，4，5-三磷酸激酶C型（ItpkC）在调节肿瘤细胞粘附到微血管的作用。Itpk酶家族将1，4，5-三磷酸肌醇（InsP 3）转化为1，3，4，5-三磷酸肌醇（InsP 4），并且是受体偶联磷脂酶C（PLC）介导的Ca 2+增加的“关闭”信号机制的一部分。与源自原发性结肠肿瘤的细胞相比，Itpk的蛋白质水平在源自淋巴结转移的结肠癌细胞中显著降低。Itpk在转移性变异中的下调与对E-选择素刺激的敏感性增加和更大幅度的Ca 2+瞬变相关。此外，ItpkC的过表达抑制人结肠癌细胞与肝内皮细胞的粘附。两者合计，这些数据表明，Itpk的表达的减少可能会促进肿瘤细胞粘附，通过增强激动剂诱导的Ca 2+增加。在我们的模型中，肝内皮细胞上的E-选择素粘附受体与结肠癌细胞上的反受体的结合诱导双向激活信号，导致PLC的激活，InsP 3形成的刺激和癌细胞和肝细胞中Ca 2+的增加。我们假设ItpkC的下调为微血管粘附于循环结肠癌细胞提供了优势，因此为转移到肝脏提供了优势。这项提议的目的是检验新的假设，即ItpkC的表达水平 调节粘附诱导的Ca 2+增加，从而调节结直肠癌的转移潜力。我们将研究与无转移的肿瘤相比，ItpkC在有转移的原发性结肠癌中的表达是否失调。这些研究将使用标准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性和转移性结直肠癌中的表达和细胞分布。此外，我们将测试的猜想，下调ItpkC在结直肠癌细胞系将增强粘附依赖性的Ca 2+信号，并增加肿瘤细胞的结合到肝内皮。这些研究将使用体内癌症 细胞粘附测定和宽视野荧光成像技术来测量胞质Ca 2+反应，以及新的共聚焦显微镜技术来真实的实时监测结肠癌细胞和肝细胞[Ca 2 +]i在癌细胞通过肝窦时的反应 并粘附于内皮。

 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.