W1744 The Extracellular Calcium-Sensing Receptor Regulates the Proliferation of Normal Colonic Epithelial Cells and Reverses the Malignant Phenotype of Colon Cancer Cells

Abstract

Background: EGFR family members are all thought to signal through the MAPK and PI3K/ Akt pathways. Upregulation of EGFR, HER2, HER3 and MAPK signaling is well documented in colorectal tumors. Similarly, PI3K activity is elevated in the vast majority of colorectal cancers and over-expression of Akt isoforms has also been detected in CRC. We previously reported that the sulfide and sulfone metabolites of the non-steroidal anti-inflammatory drug (NSAID) sulindac downregulate the expression of EGFR, inhibit EGFR signaling through the MAPK pathway, and induce apoptotic cell death in CRC cells. Goals: 1) To examine the effects of sulindac metabolites (SMs) on protein levels and phosphorylation status of the other EGFR family members, HER2 and HER3, and the downstream signaling effector, Akt and 2) To determine if downregulation of EGFR family members is required for the apoptotic effects of SMs. Results: SMs downregulated both total and phosphorylated forms of HER2 and HER3 in a manner similar to their effects on EGFR. This downregulation was seen by 12h after treatment and persisted through at least 48h. Unexpectedly bothmetabolites significantly increased Akt phosphorylation as early as 1h after treatment in all cell lines tested. SW620 CRC cells which lack EGFR and do not signal through HER2 remained sensitive to both the apoptotic effects and the pAkt stimulatory effects of SMs. siRNA treatment resulted in a 76% knockdown of HER3 expression in HT29 (EGFR+) cells and 84% in SW620 (EGFR-) cells. HER3 knockdown failed to induce significant levels of apoptosis in either cell line but it did not prevent the apoptotic effect or the pAkt induction by SMs. Pre-treatment of HT29 cells with LY294002 prevented Akt phosphorylation by SMs and potentiated the growth inhibitory effects of sulindac sulfide in this cell line. Conclusion: These results demonstrate that I) EGFR, HER2, and HER3 appear to be similarly downregulated by SMs; II) Downregulation of EGFR family members does not appear to be sufficient for the apoptotic effect of sulindacmetabolites; III) Sulindacmetabolites induce an unexpected stimulation of Akt phosphorylation through an EGFR family member-independent effect and IV) Prevention of NSAID-induced AKT phosphorylation might potentiate the growth inhibitory activity of these drugs.