Samawansha Tennakoon

The calcium-sensing receptor and the hallmarks of cancer.

The calcium-sensing receptor (CaSR) plays a pivotal role in systemic calcium metabolism by regulating parathyroid hormone secretion and urinary calcium excretion. The CaSR is ubiquitously expressed, implying a wide range of functions regulated by this receptor. Abnormal CaSR function affects the development of both calciotropic disorders such as hyperparathyroidism, and non-calciotropic disorders such as cardiovascular disease and cancer, which are the leading causes of mortality worldwide. The CaSR is able to bind a plethora of ligands; it interacts with multiple G protein subtypes, and regulates highly divergent downstream signalling pathways, depending on the cellular context. The CaSR is a key regulator for such diverse processes as hormone secretion, gene expression, inflammation, proliferation, differentiation, and apoptosis. Due to this pleiotropy, the CaSR is able to regulate cell fate and is implicated in the development of many types of benign or malignant tumours of the breast, prostate, parathyroid, and colon. In cancer, the CaSR appears to have paradoxical roles, and depending on the tissue involved, it is able to prevent or promote tumour growth. In tissues like the parathyroid or colon, the CaSR inhibits proliferation and induces terminal differentiation of the cells. Therefore, loss of the receptor, as seen in colorectal or parathyroid tumours, confers malignant potential, suggestive of a tumour suppressor role. In contrast, in prostate and breast tumours the expression of the CaSR is increased and it seems that it favours metastasis to the bone, acting as an oncogene. Deciphering the molecular mechanism driving the CaSR in the different tissues could lead to development of new allosteric drug compounds that selectively target the CaSR and have therapeutic potential for cancer. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

The calcium-sensing receptor: A promising target for prevention of colorectal cancer.

The inverse correlation between dietary calcium intake and the risk of colorectal cancer (CRC) is well known, but poorly understood. Expression of the calcium-sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is downregulated in CRC leading us to hypothesize that the CaSR has tumor suppressive roles in the colon. The aim of this study was to understand whether restoration of CaSR expression could reduce the malignant phenotype in CRC. In human colorectal tumors, expression of the CaSR negatively correlated with proliferation markers whereas loss of CaSR correlated with poor tumor differentiation and reduced apoptotic potential. In vivo, dearth of CaSR significantly increased expression of proliferation markers and decreased levels of differentiation and apoptotic markers in the colons of CaSR/PTH double knock-out mice confirming the tumor suppressive functions of CaSR. In vitro CRC cells stably overexpressing wild-type CaSR showed significant reduction in proliferation, as well as increased differentiation and apoptotic potential. The positive allosteric modulator of CaSR, NPS R-568 further enhanced these effects, whereas treatment with the negative allosteric modulator, NPS 2143 inhibited these functions. Interestingly, the dominant-negative mutant (R185Q) was able to abrogate these effects. Our results demonstrate a critical tumor suppressive role of CaSR in the colon. Restoration of CaSR expression and function is linked to regulation of the balance between proliferation, differentiation, and apoptosis and provides a rationale for novel strategies in CRC therapy.

Calcium-sensing receptor silencing in colorectal cancer is associated with promoter hypermethylation and loss of acetylation on histone 3

The calcium‐sensing receptor (CaSR) is suggested to mediate the antiproliferative effects of calcium in colon. However, in colorectal cancer (CRC) the expression of the CaSR is silenced and the underlying mechanisms leading to its loss are poorly understood. We investigated whether loss of the CaSR expression in colorectal tumors is caused by DNA hypermethylation and imbalance of transcriptionally permissive/repressive histone alterations. We observed significantly lower CaSR mRNA expression (n = 65, p < 0.001) in colorectal tumors compared with the adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein also. The CaSR promoter was methylated to a greater extent in tumors compared with adjacent mucosa as determined by bisulfite sequencing (n = 20, p < 0.01) and by pyrosequencing (n = 45, p < 0.001), and methylation correlated inversely with mRNA expression (n = 20, ρ = −0.310, p < 0.05 and n = 45, ρ = −0.588, p < 0.001). Treatments with 5‐aza‐2′‐deoxycytidine (DAC), a DNA methyltransferase inhibitor and/or with two different histone deacetylase inhibitors, trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) restored the expression of CaSR in colon cancer cells. Restored CaSR expression in Coga1A and HT29 cells was functional. Inhibition of lysine‐specific demethylase 1 (LSD1) to prevent demethylation of mono‐ and dimethylated H3K4, increased CaSR expression only marginally. Our data show that hypermethylation of the CaSR promoter and H3K9 deacetylation, but not H3K4me2 demethylation are important factors that cause silencing of the CaSR in colorectal cancer.

miR‐135b‐ and miR‐146b‐dependent silencing of calcium‐sensing receptor expression in colorectal tumors

Studies have shown that the calcium‐sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco‐2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain‐ and loss‐of‐function studies with the top candidates: miR‐9, miR‐27a, miR‐135b, and miR‐146b. Modulation of miR‐135b or miR‐146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR‐135b and miR‐146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR‐135b and miR‐146b, and this correlated inversely with CaSR expression (miR‐135b: r = −0.684, p < 0.001 and miR‐146b: r = −0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR‐135b and miR‐146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.

The calcium-sensing receptor suppresses epithelial-to-mesenchymal transition and stem cell- like phenotype in the colon

BackgroundThe calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown.Methods and resultsIn a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29CaSR) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29EMP). The HT29CaSR cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/β-catenin pathway as measured by a decrease in nuclear translocation of β-catenin and transcriptional regulation of genes like GSK-3β and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29CaSR cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype.ConclusionsThe results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.

Cytochrome P450 Vitamin D Hydroxylases in Inflammation and Cancer

Vitamin D insufficiency correlates with increased incidence of inflammatory disorders and cancer of the colon, breast, liver, and prostate. Preclinical studies demonstrated that the hormonally active form of vitamin D, 1,25(OH)2D3, has antiproliferative, proapoptotic, anti-inflammatory, and immunomodulatory effects. Tissue levels of 1,25(OH)2D3 are determined by expression and activity of specific vitamin D hydroxylases expressed at renal and extrarenal sites. In order to understand how perturbations in the vitamin D system affect human health, we need to understand the steps involved in the synthesis and catabolism of the active metabolite. This review provides an overview about recent findings on the altered vitamin D metabolism in inflammatory conditions and carcinogenesis. We will summarize existing data on the pathophysiological regulation of vitamin D hydroxylases and outline the role of adequate levels of 1,25(OH)2D3 on tissue homeostasis.

Impact of CYP24A1 overexpression on growth of colorectal tumour xenografts in mice fed with vitamin D and soy

Our previous studies showed that the 1,25‐dihydroxyvitamin D (1,25‐D3) catabolizing enzyme, 1,25‐dihydoxyvitamin D 24 hydroxylase (CYP24A1) was overexpressed in colorectal tumours and its level correlated with increased proliferation. We hypothesised that cells overexpressing CYP24A1 have growth advantage and a diet rich in vitamin D and soy would restore sensitivity to the anti‐tumourigenic effects of vitamin D. Soy contains genistein, a natural CYP24A1 inhibitor. To determine causality between CYP24A1 and tumour growth, we established xenografts in male SCID mice with HT29 cells stably overexpressing either GFP‐tagged CYP24A1 or GFP. Mice were fed with either high (2500 IU D3/kg) or low vitamin D (100 IU D3/kg) diet in the presence or absence of soy (20% diet). In vitro, cells overexpressing CYP24A1 grew faster than controls. 1,25‐D3, the active vitamin D metabolite, reduced cell number only in the presence of the CYP24A1 inhibitor VID400. Regardless of the amount of vitamin D in the diet, xenografts overexpressing CYP24A1 grew faster, were heavier and more aggressive. Soy reduced tumour volume only in the control xenografts, while the tumours overexpressing CYP24A1 were larger in the presence of dietary soy. In conclusion, we demonstrate that CYP24A1 overexpression results in increased aggressiveness and proliferative potential of colorectal tumours. Irrespective of the dietary vitamin D3, dietary soy is able to increase tumour volume when tumours overexpress CYP24A1, suggesting that combination of vitamin D3 and soy could have an anti‐tumourigenic effect only if CYP24A1 levels are normal.

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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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, Eniko Kallay. 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

Abstract 4003: Influence of extracellular calcium on MAPK signalling cascade in colon cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: The calcium sensing receptor (CaSR) is a G-protein coupled receptor, which is capable of sensing even minute changes in extracellular calcium levels. The main role of the CaSR is regulation of calcium homeostasis. Besides the organs responsible for calcium homeostasis the receptor is expressed in a number of tissues including colonic epithelium and can be activated by several ligands, stimulating various downstream signalling pathways. Thus the CaSR is involved in numerous biological processes such as cell proliferation, differentiation and lipid membrane metabolism. Previous studies demonstrated that calcium regulates cell proliferation in colonocytes but the pathways or factors involved in this process still need to be elucidated. Aim: The aim of this study is to investigate the impact of CaSR activation on the Mitogen-Activated Protein Kinase (MAPK) signalling cascade in colon cancer cell lines. We hypothesize that in colonocytes, activated CaSR down regulates proliferative pathways. Methods: We studied the effect of CaSR ligands on downstream pathways through gene expression assays and protein analysis. Results and Conclusion: We could show that the differentiation status of the cells (TC-7 a colon cancer cell line) determines whether a pathway is activated or rather inhibited by calcium treatment. Gene expression studies performed by luciferase assay and protein analysis by western blots reflect that extracellular calcium activated MAPK cascade in subconfluent TC-7 cells. On the other hand, short time (10 to 30 minutes) treatment of confluent/ differentiated cells with extracellular calcium showed no activation of ERK. Treatment for longer time (e.g 8h) inhibited ERK phosphorylation. The treatment of the cells with 100nM R-568, a calcimimetic that act only through the CaSR slightly inhibited ERK phosphorylation compared with the vehicle control, suggesting an anti-proliferative role of the CaSR. Citation Format: Samawansha Tennakoon, Johannes Schmid, Brigitte Marian, Eniko Kallay. Influence of extracellular calcium on MAPK signalling cascade in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4003. doi:10.1158/1538-7445.AM2013-4003

Abstract 685: Epigenetic modulators and calcium-sensing receptor expression in colorectal cancer cells.

Introduction: Epidemiological studies suggest a role for calcium in prevention of colorectal cancer. The antiproliferative action of calcium in colon might be mediated by the calcium-sensing receptor (CaSR). The CaSR expression is lost in human colorectal cancer. We hypothesized that DNA methylation and histone modifications may be the cause of CaSR silencing in tumors. Material and methods: We analyzed CaSR mRNA and protein expression in colorectal tumors and cell lines by real time qRT-PCR and immunofluorescence. We determined the methylation pattern in the second promoter of the CaSR that contains a large CpG island by bisulfite sequencing. To induce the expression of the CaSR we treated colon tumor cell lines with 5-aza-2-deoxycytidine (5-aza-dC) and RG108, two DNA methyltransferase inhibitors, three different histone deacetylase inhibitors (HDACIs) Trichostatin A, suberoylanilide hydroxamic acid, sodium butyrate in the presence or absence of a lysine specific demethylase 1 (LSD1) inhibitor to prevent demethylation of mono- and dimethylated histone H3 lysine 4. Results: In our patient cohort we observed significantly less CaSR mRNA expression (n=57, P Conclusion: In summary, DNA methylation and histone deacetylation seem to be important factors in silencing the expression of CaSR in colorectal cancer, although additional mechanism or factors might be also involved. Citation Format: Irfete Sh. Fetahu, Julia Hobaus, Abhishek Aggarwal, Samawansha Tennakoon, Ildiko Mesteri, Sabina Baumgartner-Parzer, Enikoe Kallay. Epigenetic modulators and calcium-sensing receptor expression in colorectal cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 685. doi:10.1158/1538-7445.AM2013-685