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.

PUBLIC HEALTH RELEVANCE Colorectal cancers frequently metastasize to the liver; however, the mechanisms required for implantation and colonization of tumor cells in the liver have not been fully delineated. In this application, we will investigate the role of inositol 1,4,5-trisphosphate kinase isoform C in adhesion of colorectal cancer cells to liver endothelium.