inositol-1-4-5-trisphosphate and Colorectal-Neoplasms

The GTPase Ras is a molecular switch engaged downstream of G-protein-coupled receptors and receptor tyrosine kinases that controls multiple cell-fate-determining signalling pathways. Ras signalling is frequently deregulated in cancer, underlying associated changes in cell phenotype. Although Ca(2+) signalling pathways control some overlapping functions with Ras, and altered Ca(2+) signalling pathways are emerging as important players in oncogenic transformation, how Ca(2+) signalling is remodelled during transformation and whether it has a causal role remains unclear. We have investigated Ca(2+) signalling in two human colorectal cancer cell lines and their isogenic derivatives in which the allele encoding oncogenic K-Ras (G13D) was deleted by homologous recombination. We show that agonist-induced Ca(2+) release from the endoplasmic reticulum (ER) intracellular Ca(2+) stores is enhanced by loss of K-Ras(G13D) through an increase in the Ca(2+) content of the ER store and a modification of the abundance of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) subtypes. Consistently, uptake of Ca(2+) into mitochondria and sensitivity to apoptosis was enhanced as a result of K-Ras(G13D) loss. These results suggest that suppression of Ca(2+) signalling is a common response to naturally occurring levels of K-Ras(G13D), and that this contributes to a survival advantage during oncogenic transformation.

Topics: Apoptosis; Calcium; Calcium Signaling; Cell Line, Tumor; Colorectal Neoplasms; Endoplasmic Reticulum; Genes, ras; HCT116 Cells; Humans; Inositol 1,4,5-Trisphosphate; Protein Isoforms; ras Proteins

We have investigated the molecular basis of intracellular Ca(2+) handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca(2+) stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca(2+) levels, agonist-induced Ca(2+) increases, store-operated Ca(2+) entry (SOCE), and store-operated currents (ISOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca(2+) stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca(2+) release-activated Ca(2+) currents (ICRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced ICRAC plus a nonselective ISOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca(2+) store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca(2+) store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca(2+) remodeling and tumor features in colon cancer.

Topics: Apoptosis; Calcium; Carcinogenesis; Cell Adhesion Molecules; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colon; Colorectal Neoplasms; Electrophysiological Phenomena; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Inositol 1,4,5-Trisphosphate; Intestinal Mucosa; Membrane Proteins; Neoplasm Proteins; Stromal Interaction Molecule 1; Stromal Interaction Molecule 2; TRPC Cation Channels

The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate is mediated by activated phosphatidylinositol-specific phospholipase C (PLC) enzymes. We report the enhancement of the phosphoinositide metabolism pathway in KMS-4 and KMS-8 cells, both of which are human colorectal carcinoma cell lines derived from familial adenomatous polyposis patients. In these cells, the cellular contents of diacylglycerol and inositol 1,4,5-trisphosphate were constitutively increased and the PLC activity in vitro was significantly high, as compared with those in normal colon cells or in other sporadic colorectal carcinoma cells. Northern and Western analyses showed the high expression levels of both PLC-gamma 1 and PLC-delta 1 in KMS-4 and KMS-8 cells. Moreover, we detected the enhancement of protein-tyrosine kinase activity and tyrosine phosphorylation of PLC-gamma 1 in these KMS cells. These results suggest the involvement of activated phosphoinositide signaling pathways in the colorectal tumorigenesis of familial adenomatous polyposis.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli; Cell Line; Chromatography, Affinity; Colorectal Neoplasms; Diglycerides; Electrophoresis, Polyacrylamide Gel; Gene Expression; Humans; Inositol 1,4,5-Trisphosphate; Isoenzymes; Phosphatidylinositols; Phosphoproteins; Phosphorylation; Phosphotyrosine; RNA, Messenger; Second Messenger Systems; Tumor Cells, Cultured; Type C Phospholipases; Tyrosine

Addition of purified plasmin or plasminogen (0.1 microM) to serum-free culture media elevated cellular D-myo-inositol 1,4,5-trisphosphate (InsP3) levels in human colorectal carcinoma cells within 1 h to double those of control cells. This was accompanied by decreases in cellular phosphatidylinositol bisphosphate by 40% in cells exposed to fibrinolytic ligands for up to 1 h. The effect was not due to opening of Ca2+ channels of the type blocked by 5 microM nifedipine, and 100 microM EGTA, a Ca2+ chelator, did not suppress plasmin's ability to elevate InsP3. Binding assays at 4 degrees C with 125I-labelled plasmin indicated maximum binding within 1 h suggesting that the effects of plasmin may be associated with its cell-binding function. These cells could convert exogenous plasminogen to plasmin with endogenous activation and this was accompanied by a decrease in radioactive phosphatidylinositol well below control levels (13% of control). Our results contribute to evidence for the association of plasmin-binding sites with a signalling system. A cell signalling system indirectly or directly associated with plasmin binding, would permit carcinoma cells to coordinate extracellular fibrinolysis with cell migration and motility through second messengers.

Topics: Cell Communication; Colorectal Neoplasms; Egtazic Acid; Fibrinolysin; Fibrinolysis; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Neoplasm Invasiveness; Nifedipine; Phosphatidylinositols; Plasminogen; Signal Transduction; Tumor Cells, Cultured