Impaired Mineral Ion Metabolism in a Mouse Model of Targeted Calcium-Sensing Receptor (CaSR) Deletion from Vascular Smooth Muscle Cells.

BACKGROUND: Impaired mineral ion metabolism is a hallmark of CKD-metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR. METHODS: To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells ( SM22α CaSR Δflox/Δflox ). RESULTS: Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D3), and parathyroid hormone levels. Renal tubular α-Klotho protein expression was increased in KO mice but vascular α-Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice. CONCLUSIONS: These results suggest that, in addition to CaSR's established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis.

International Union of Basic and Clinical Pharmacology. CVIII. Calcium-Sensing Receptor Nomenclature, Pharmacology, and Function.

The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor that responds to multiple endogenous agonists and allosteric modulators, including divalent and trivalent cations, L-amino acids, γ-glutamyl peptides, polyamines, polycationic peptides, and protons. The CaSR plays a critical role in extracellular calcium (Ca2+ o) homeostasis, as demonstrated by the many naturally occurring mutations in the CaSR or its signaling partners that cause Ca2+ o homeostasis disorders. However, CaSR tissue expression in mammals is broad and includes tissues unrelated to Ca2+ o homeostasis, in which it, for example, regulates the secretion of digestive hormones, airway constriction, cardiovascular effects, cellular differentiation, and proliferation. Thus, although the CaSR is targeted clinically by the positive allosteric modulators (PAMs) cinacalcet, evocalcet, and etelcalcetide in hyperparathyroidism, it is also a putative therapeutic target in diabetes, asthma, cardiovascular disease, and cancer. The CaSR is somewhat unique in possessing multiple ligand binding sites, including at least five putative sites for the "orthosteric" agonist Ca2+ o, an allosteric site for endogenous L-amino acids, two further allosteric sites for small molecules and the peptide PAM, etelcalcetide, and additional sites for other cations and anions. The CaSR is promiscuous in its G protein-coupling preferences, and signals via Gq/11, Gi/o, potentially G12/13, and even Gs in some cell types. Not surprisingly, the CaSR is subject to biased agonism, in which distinct ligands preferentially stimulate a subset of the CaSR's possible signaling responses, to the exclusion of others. The CaSR thus serves as a model receptor to study natural bias and allostery. SIGNIFICANCE STATEMENT: The calcium-sensing receptor (CaSR) is a complex G protein-coupled receptor that possesses multiple orthosteric and allosteric binding sites, is subject to biased signaling via several different G proteins, and has numerous (patho)physiological roles. Understanding the complexities of CaSR structure, function, and biology will aid future drug discovery efforts seeking to target this receptor for a diversity of diseases. This review summarizes what is known to date regarding key structural, pharmacological, and physiological features of the CaSR.

Novel Strategies in the Development of New Therapies, Drug Substances, and Drug Carriers Volume II.

The highly successful previous Volume 1 [...].

Novel Strategies in the Development of New Therapies, Drug Substances, and Drug Carriers Volume I.

At present, there is a strong need for new therapies that are effective and safe for widespread diseases [...].

AOM/DSS Induced Colitis-Associated Colorectal Cancer in 14-Month-Old Female Balb/C and C57/Bl6 Mice-A Pilot Study.

Colitis is a major risk factor for the development of colorectal cancer, leading to colitis-associated colorectal cancer (CAC). The most commonly used animal model to study CAC is the azoxymethane-dextran sulphate-sodium (AOM/DSS) model. The ideal experimental conditions of this model depend on several factors, including the used mouse strain. No data on feasibility and conditions for older mice, e.g., for aging studies, have yet been reported. Thus, we conducted a descriptive, observational pilot study where CAC was induced in 14-month-old female Balb/C and C57/Bl6 mice using 12.5 mg/kg AOM i.p. and three different concentrations of DSS (1, 2, and 3%) in drinking water (ad. lib.). The mice were monitored regularly during the three-month experimental phase. After euthanasia, the colons of the mice were evaluated macroscopically and microscopically. Both the mouse strains showed a DSS-concentration-dependent induction of CAC. Carcinomas were only observed at 3% DSS. The DSS dose was found to be significantly correlated with the histology score and % Ki67 positive cells only in C57/Bl6 mice but not in Balb/C mice, which showed a variable response to the CAC induction. No differences in colon length, weight, or mucin content were observed. Optimal conditions for CAC induction in these aged animals are thus considered to be 3% DSS, as carcinomas did not develop when 2% DSS was used. On the other hand, Balb/C mice reacted severely to 3% DSS, indicating that 2.5% DSS may be the "sweet spot" for future experiments comparing CAC in aged Balb/C and C57/Bl6 mice. This model will allow investigation of the effect of aging on CAC development and therapy.

Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells.

BACKGROUND: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. METHODS: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D3 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). RESULTS: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC50 = 2.98 ± 1.10 nmol/L, in 14433 cells: EC50 = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC50 = n/d, in 14433 cells: EC50 = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. CONCLUSIONS: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs' structure might lead to new treatment options against ovarian cancer.

Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells.

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on ß-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.

Sensing Extracellular Calcium - An Insight into the Structure and Function of the Calcium-Sensing Receptor (CaSR).

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a key role in calcium homeostasis, by sensing free calcium levels in blood and regulating parathyroid hormone secretion in response. The CaSR is highly expressed in parathyroid gland and kidney where its role is well characterised, but also in other tissues where its function remains to be determined. The CaSR can be activated by a variety of endogenous ligands, as well as by synthetic modulators such as Cinacalcet, used in the clinic to treat secondary hyperparathyroidism in patients with chronic kidney disease. The CaSR couples to multiple G proteins, in a tissue-specific manner, activating several signalling pathways and thus regulating diverse intracellular events. The multifaceted nature of this receptor makes it a valuable therapeutic target for calciotropic and non-calciotropic diseases. It is therefore essential to understand the complexity behind the pharmacology, trafficking, and signalling characteristics of this receptor. This review provides an overview of the latest knowledge about the CaSR and discusses future hot topics in this field.

Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium.

Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca2+]o) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca2+]o identified significant changes in expression of 1571 probe sets (ANOVA, p<10-5). The main biological processes affected by [Ca2+]o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca2+]o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca2+]o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor.

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 plasma membrane Ca2+ pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells.

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca2+ signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca2+ ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca2+ ]i clearance from cells after Ca2+ entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor.

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.

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.

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.

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

BACKGROUND: The 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 RESULTS: In 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 (HT29(CaSR)) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29(EMP)). The HT29(CaSR) cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/ß-catenin pathway as measured by a decrease in nuclear translocation of ß-catenin and transcriptional regulation of genes like GSK-3ß and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29(CaSR) 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. CONCLUSIONS: The 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.

Calcium sensing receptor signalling in physiology and cancer.

The calcium sensing receptor (CaSR) is a class C G-protein-coupled receptor that is crucial for the feedback regulation of extracellular free ionised calcium homeostasis. While extracellular calcium (Ca(2+)o) is considered the primary physiological ligand, the CaSR is activated physiologically by a plethora of molecules including polyamines and l-amino acids. Activation of the CaSR by different ligands has the ability to stabilise unique conformations of the receptor, which may lead to preferential coupling of different G proteins; a phenomenon termed 'ligand-biased signalling'. While mutations of the CaSR are currently not linked with any malignancies, altered CaSR expression and function are associated with cancer progression. Interestingly, the CaSR appears to act both as a tumour suppressor and an oncogene, depending on the pathophysiology involved. Reduced expression of the CaSR occurs in both parathyroid and colon cancers, leading to loss of the growth suppressing effect of high Ca(2+)o. On the other hand, activation of the CaSR might facilitate metastasis to bone in breast and prostate cancer. A deeper understanding of the mechanisms driving CaSR signalling in different tissues, aided by a systems biology approach, will be instrumental in developing novel drugs that target the CaSR or its ligands in cancer. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

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.

Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer.

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3 ). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo. Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR. In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

The Effect of Vitamin D and Its Analogs in Ovarian Cancer.

Ovarian cancer is one of the deadliest cancers in women, due to its heterogeneity and usually late diagnosis. The current first-line therapies of debulking surgery and intensive chemotherapy cause debilitating side effects. Therefore, there is an unmet medical need to find new and effective therapies with fewer side effects, or adjuvant therapies, which could reduce the necessary doses of chemotherapeutics. Vitamin D is one of the main regulators of serum calcium and phosphorus homeostasis, but it has also anticancer effects. It induces differentiation and apoptosis, reduces proliferation and metastatic potential of cancer cells. However, doses that would be effective against cancer cause hypercalcemia. For this reason, synthetic and less calcemic analogs have been developed and tested in terms of their anticancer effect. The anticancer role of vitamin D is best understood in colorectal, breast, and prostate cancer and much less research has been done in ovarian cancer. In this review, we thus summarize the studies on the role of vitamin D and its analogs in vitro and in vivo in ovarian cancer models.