Abstract 5272: The tumor suppressor, calcium sensing receptor, regulates proliferation and promotes differentiation in colon cancer

Abstract

Abstract There is increasing evidence highlighting an inverse correlation between high dietary calcium intake and reduced risk of colorectal cancer. The classical role of the calcium sensing receptor (CaSR), a G protein coupled receptor, is regulation of calcium homeostasis in the parathyroid and kidney. In other tissue types, CaSR regulates different cellular processes like proliferation, differentiation, and apoptosis. We have demonstrated that CaSR expression is downregulated in colorectal cancer (CRC) leading us to hypothesize that loss of CaSR might provide a growth advantage to transformed cells, conferring them resistance to calcium-mediated growth inhibition. Therefore the aim of this study was to understand the role of CaSR in CRC. In vitro, HT29 (moderately differentiated) and Caco-2/15 (well differentiated) colon cancer cells were stably transfected with constructs to overexpress either the wild type CaSR (CaSRWT) or a dominant negative CaSR mutant (CaSRDN). The cells transfected with the CaSRWT construct grew significantly slower than the cells overexpressing the CaSRDN (Caco2-15: 40% slower; HT29: 50% slower; p<0.001) as assessed by cell counting. This effect was much more pronounced when cells were treated with the calcimimetic NPS R568, an allosteric modulator of the CaSR. Treating the cells with the calcilytic NPS 2143, a negative allosteric modulator of the receptor, we could reverse the antiproliferative effect. Furthermore, CaSRWT cells were more differentiated than CaSRDN cells as shown by the high Alkaline Phosphatase activity in culture (3-fold higher in Caco2-15; p<0.001). We also evaluated the tumor formation ability of these cells in hanging drop cultures. Indeed, CaSRDN formed large spheroids, whereas CaSRWT cells formed irregular shaped aggregates demonstrating a possible role of the CaSR in inhibiting tumor formation (in HT29 cells, CaSRDN spheroids had 10-fold increased volume compared with control cells; p<0.001). To translate these findings in vivo, we investigated the effects of global knock down of CaSR. Global ablation of CaSR in mice led to lethality within 1-2 weeks; therefore we analyzed the colon of ‘rescued’ CaSR-/PTH- double knockout mice, and compared them to colons of CaSR+/PTH- mice. CaSR-/PTH- mice had increased expression of proliferation related markers (for instance, 1.5-fold cell cycle division 6 (Cdc6) expression, p<0.01). Histological examination has revealed the presence of aberrant crypt foci in the colon of the double knockout mice. This suggests that loss of CaSR expression gives colon cancer cells a growth advantage during tumorigenesis. These data further our understanding of the molecular mechanism by which the CaSR protects colon epithelial cells from malignant transformation. These mechanisms, in part, account for the anti-neoplastic effects of calcium in the colon. Citation Format: Abhishek Aggarwal, Samawansha Tennakoon, Maximilian Wohlgenannt, Cedric Boudot, Romuald Mentaverri, Sabina Baumgartner-Parzer, Enikö Kállay. The tumor suppressor, calcium sensing receptor, regulates proliferation and promotes differentiation in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5272. doi:10.1158/1538-7445.AM2014-5272