Calcium-sensing Receptor Variants Research Articles

The A986S calcium sensing receptor variant supports the adaptation of breast cancer cells to high calcium — a novel paradigm in breast cancer progression

Breast cancer and other solid tumors that metastasize to bone are frequently accompanied by a progressive increase in circulating calcium as the disease becomes more malignant. The increase in circulating calcium not only disrupts systemic calcium homeostasis but also alters the ability of cells to sense slight changes in extracellular Ca2+ via the ubiquitous calcium sensing receptor (CaSR). Of the CASR polymorphisms at rs1801725 (p.A986S), rs1801726 (p.Q1011E), and rs1042636 (p.R990G) in exon 7 of the receptor, previous studies have shown that only the A986S CaSR variant is associated with calcium in breast, prostate and skin cancers. In this study, we assessed if exon 7 inactivating A986S (AS-CaSR) and Q1011E (QE-CaSR) variants of the receptor influence the survival of breast tumor cells at higher Ca2+. We generated isogenic HEK293T and BT-549 cells expressing these CaSR variants and carried out bulk RNA sequencing of high Ca2+ adapted control and breast cancer cells. CaSR expression was detected by western blotting using anti-flag M2 (Sigma) antibody. By using phospho-ERK1/2 as the readout, the activation of AS-CaSR required higher concentrations of Ca2+ (EC50 ~5.28 mM) compared to the QE-CaSR (EC50 of ~3.81 mM) and the wild type (WT-CaSR; EC50 ~3.69 mM) receptor. When the CaSR variants transfected cells initially cultured at 7.5 mM for 6 days, and then sub-cultured in medium containing 7.5 to 20 mM Ca2+, the viability of empty vector control and WT-CaSR transfected cells strongly decreased at Ca2+ levels beyond 7.5 mM and these cells were mostly necrotic at ≥15 mM Ca2+. The QE-CaSR variant transfected cells on the other hand exhibited limited cell death beyond 10 mM Ca2+, while the surviving colonies of the AS-CaSR variant were viable at up to 15 mM Ca2+ with invasive tendencies. Analysis of differentially expressed genes in high Ca2+ adapted mesenchymal-like BT-549 and epithelial MDA-MB-468 breast cancer cells compared to MCF10A control cells revealed a small set of differentially expressed genes that were significantly associated with relapse free survival of basal-like breast cancer patients. Among these genes are ligands and negative regulators of the WNT/FZD pathway. Our data suggest that the A986S-CaSR variant with reduced sensitivity to Ca2+ supported adaptation of TNBC cells at high Ca2+, and that high Ca2+ adaptation of breast cancer cells may be mediated via a panel of high Ca2+ inducible genes with implications in bone metastasis. This study is supported by NIH SC1GM139814, T32AI007281, T32GM144927, T32HL007737; and the American Cancer Society. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Functional evaluation of a novel nonsense variant of the calcium-sensing receptor gene leading to hypocalcemia.

The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.

Association of TRPV5, CASR, and CALCR genetic variants with kidney stone disease susceptibility in Egyptians through main effects and gene–gene interactions

Kidney stone disease (KSD) represents an urgent medical problem because of increasing its prevalence. Several functional polymorphisms in genes involved in the renal handling of calcium were associated with KSD pathogenesis. Among those, the rs4236480 of transient receptor potential vanilloid member 5 (TRPV5) gene, the rs1801725 of calcium-sensing receptor (CASR) gene, and the rs1801197 of calcitonin receptor (CALCR) gene appear to be of great importance. Due to the scarce data on the Egyptians, this study aimed to evaluate the association of these candidate genetic variants with the risk of developing KSD in an Egyptian population. To do so, the biochemical parameters were measured along with the genotyping of the three polymorphisms using allelic discrimination assay in 134 KSD patients and 86 age and sex-matched healthy subjects. The results showed that the genotypic distributions and allelic frequencies of the studied variants were significantly different between cases and controls. The three polymorphisms increased the risk of KSD significantly under all the tested genetic models (OR ranges from 2.152 to 5.994), except for the recessive model of the CALCR rs1801197 polymorphism after Bonferroni correction. The gene–gene interaction analyzed by multifactor dimensionality reduction selected the three-locus combination as the best model associated with the susceptibility to KSD with OR 9.706. Further, synergistic interactions were identified between TRPV5 rs4236480 and CALCR rs1801197 variants and CASR rs1801725 and CALCR rs1801197 variants. In conclusion, the TRPV5 rs4236480, CASR rs1801725, and CALCR rs1801197 polymorphisms showed a significant association with the risk of KSD in the Egyptian population. Furthermore, their complex interactions might have an impact on the genetic susceptibility to develop KSD.

The Importance of Functionally Characterizing Calcium-Sensing Receptor Variants in Individuals With Hypercalcemia.

The Importance of Functionally Characterizing Calcium-Sensing Receptor Variants in Individuals With Hypercalcemia.

The three common polymorphisms p.A986S, p.R990G and p.Q1011E in the calcium sensing receptor (CASR) are not associated with chronic pancreatitis

BackgroundThe calcium sensing receptor (CASR) is a G protein-coupled receptor that is responsible for assessing extracellular Ca2+ levels and thus plays a crucial role in calcium homeostasis. Hypercalcemia is a metabolic risk factor for pancreatitis and rare CASR variants have been described in patients with chronic pancreatitis. At the carboxy-terminal tail of CASR, there is a cluster of three common polymorphisms, p.A986S (rs1801725), p.R990G (rs1042636) and p.Q1011E (rs1801726), which have been associated with chronic pancreatitis in various studies, but with conflicting results. MethodsWe examined 542 German and 339 French patients with chronic pancreatitis as well as 1025 German controls for the 3 common CASR polymorphism by melting curve analysis. For comparison, we used genotype data from 583 French controls from a previous study. In addition, we functionally analyzed the three variants by NFAT and SRE luciferase reporter systems as well as Western blotting and verified cell surface expression by ELISA. ResultsIn both cohorts, neither the genotype nor the allele frequencies differed significantly between patients and controls. In both luciferase assays, p.R990G showed a significant leftward shift, indicating an increased responsiveness of the receptor. p.A986S showed a leftward shift in the SRE but not in the NFAT reporter assay, while the responsiveness of p.Q1011E did not differ from the wild-type. These functional studies therefore do not support the contributions of variant CASR to increasing the risk of pancreatitis. ConclusionsThe three frequent CASR polymorphisms are unlikely to increase the risk for chronic pancreatitis.

Calcium-Sensing Receptor Polymorphisms at rs1801725 Are Associated with Increased Risk of Secondary Malignancies.

Dysregulation of systemic calcium homeostasis during malignancy is common in most patients with high-grade tumors. However, it remains unclear whether single nucleotide polymorphisms (SNPs) that alter the sensitivity of the calcium-sensing receptor (CaSR) to circulating calcium are associated with primary and/or secondary neoplasms at specific pathological sites in patients of European and African ancestry. Multivariable logistic regression models were used to analyze the association of CASR SNPs with circulating calcium, parathyroid hormone, vitamin D, and primary and secondary neoplasms. Circulating calcium is associated with an increased risk for breast, prostate, and skin cancers. In patients of European descent, the rs1801725 CASR SNP is associated with bone-related cancer phenotypes, deficiency of humoral immunity, and a higher risk of secondary neoplasms in the lungs and bone. Interestingly, circulating calcium levels are higher in homozygous patients for the inactivating CASR variant at rs1801725 (TT genotype), and this is associated with a higher risk of secondary malignancies. Our data suggest that expression of CaSR variants at rs1801725 is associated with a higher risk of developing secondary neoplastic lesions in the lungs and bone, due in part to cancer-induced hypercalcemia and/or tumor immune suppression. Screening of patients for CASR variants at this locus may lead to improved management of high calcium associated tumor progression.

Hypoparathyroidism Associated with the DNA Variants in Non-Coding Sequence Region of Calcium-Sensing Receptor

A stable narrow range of extracellular calcium concentration in the blood is essential for life. The calcium-sensing receptor (CaSR), a member of the G protein-coupled receptors family, is required to adjust the set point of blood extracellular calcium concentration, thus regulate parathyroid hormone (PTH) secretion and renal calcium excretion. Loss or gain function of CaSR mutations may result in either hyper- or hypocalcaemia. The CaSR activating mutations increase its sensitivity to extracellular ionized calcium (Ca2+). As consequence, PTH synthesis and secretion are suppressed continuously at normal ionized calcium concentrations. Patients display hypocalcaemia, hyperphosphatemia and lower levels of PTH. Urinary calcium excretion is increased due to the decreased circulating inappropriately PTH level and the activation of the renal tubular CaSR. Therefore, CaSR becomes a good potential clinical therapeutic target for hypoparathyroidism treatment. In order to define new drugs and improve medical management of hypoparathyroidism patients, this study attempts to identify new CaSR variants and analyse in detail the functional change of these CaSR variants, thus better understand the molecular mechanism involved. The study is based on collected hypoparathyroidism patients in our clinical site. In the study we enrolled in 10 patients, obtained all their clinical results and DNA results from seven patients. Our results indicated that the effect of serum intact PTH level correlated to change of serum Ca2+ and phosphate level. The CaSR carrying newly identified DNA variants displayed strong phosphorylation of phospholipase C and mitogen-activated protein kinases. Although the size of clinical cases need to be accumulated, current cases have showed a tendency that the identified multiple DNA variants in CaSR gene revealed an effect on the diagnostic criterion of the hypocalcaemic syndrome. It is undeniable that our research has certain limitations. So far, we tested several DNA variants at the same time, further functional examination for individual DNA variant would largely help to be better understand the mechanisms of CaSR regulation on extracellular calcium concentration.

Heterogeneity of G protein activation by the calcium-sensing receptor.

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G protein subfamilies (Gαq/11, Gαi/o, Gα12/13, Gαs), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messengers or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G protein activation directly, we took advantage of a recently described NanoBiT G protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca2+e stimulation activates all Gαq/11 family and several Gαi/o family proteins, although Gαz was not activated. CaSR stimulated dissociation of Gα12/13 and Gαs from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα11, Gαz, Gαi1 and Gα13 genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα11 and Gαi1 in parathyroids. In kidney and pancreas, the majority of G proteins were similarly expressed, suggesting CaSR may activate multiple G proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.

Functional Analysis of Calcium-Sensing Receptor Variants Identified in Families Provisionally Diagnosed with Familial Hypocalciuric Hypercalcaemia.

Identification of variants in the calcium-sensing receptor (CASR) gene is an important means of distinguishing between familial hypocalciuric hypercalcaemia (FHH) and primary hyperparathyroidism. However, identification and bioinformatics analysis of genetic variants alone is now considered insufficient as definitive proof; additional functional assessment is required to diagnose FHH with certainty. We identified two novel variants, D433Y and C739Y, and one previously reported variant G509R in the CASR of four kindreds provisionally diagnosed with FHH and aimed to functionally characterise these variants to confirm the diagnosis. Variant receptors were cloned as FLAG-tagged constructs into the mammalian expression vector, pcDNA3.1. Wild type and variant receptor constructs were expressed in HEK293 cells and their expression assessed by Western blot analysis and their functionality analysed using an IP-One assay which measures myo-inositol 1-phosphate accumulation following CaSR activation. Western blot analysis showed that the D433Y receptor had diminished mature glycosylated receptor compared with wild type CaSR whereas the G509R receptor had a complete lack of mature receptor. The C739Y receptor was consistently overexpressed. Functional assessment showed the D433Y receptor to be mildly inactivating at physiological calcium concentrations whereas the G509R receptor was inactive at all calcium concentrations. By contrast, the C739Y variant was activating compared to wild type receptor which is inconsistent with it causing FHH. We conclude that functional assessment of CaSR variants using the IP-One assay was useful in the investigation of suspected FHH probands, confirming the D433Y and G509R variants as likely pathogenic/pathogenic, but dismissing the C739Y variant as causing FHH.

Expanding the spectrum of genetic variants in the calcium-sensing receptor (CASR) gene in hypercalcemic individuals.

Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominantly inherited disorder with overlapping biochemistry profile with primary hyperparathyroidism (PHPT), making the correct diagnosis a challenge. The objective of the study was to evaluate the results of the clinical work-up of a large group of hypercalcemic individuals. Cross-sectional study. Patients undergoing clinical work-up of hypercalcemia. Molecular genetic analysis of the CASR gene and exon 2 of the AP2S1 gene. Plasma levels of ionized calcium and PTH as well as calcium creatinine clearance ratio (CCCR). A rare CASR variant was identified in 38 of 624 index patients (6.1%). A total of 18 CASR variants identified in this study were novel. No variants were identified in exon 2 of the AP2S1 gene. The majority of the variants (N=16) were classified as likely pathogenic. The level of plasma calcium, plasma PTH and the CCCR was not affected by the type of variant (ie nonsense vs missense) (all P-values >.05). The CCCR was found to be significantly lower for variants in the transmembrane domain compared with variants located in the extracellular domain (P<.05). Plasma levels of calcium and PTH were not associated with the location of the variant (P>.05). We expanded the spectrum of CASR variants in hypercalcemia with 18 novel variants, and suggest that the location of the CASR variant may affect calcium excretion as determined by the CCCR.

Abstract 5127: Association of inactivating calcium sensing receptor exon 7 SNPs with hypercalcemia-related disease phenotypes

Abstract The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor known to be a major calcium sensor in most tissues and organ systems. It is known to regulate systemic calcium homeostasis by sensing slight increases in systemic calcium and by influencing the secretion of parathyroid hormone by parathyroid chief cells. Unfortunately, the CaSR is known to be invariably mutated and inactivating mutations in particular, are associated with the development of cancer-induced hypercalcemia (CIH). Among the several inactivating CASR polymorphisms described thus far, the rs1801725 (A986S SNP) is common among Caucasian subjects while the rs1801726 (Q1011E SNP) is common among African American subjects. However, whether these mutations contribute to other hypercalcemia-related disease phenotypes remains unclear. In this study, we carried out a phenome-wide association study (PheWAS) using the Synthetic Derivative (SD), a database at VUMC containing de-identified electronic health records (EHRs) linked to patient DNA samples in the BioVU biorepository. Using 39,893 genotyped individuals in the Multi-Ethnic Genotyping Array (MEGA), we observed that SNP rs1801725 was significantly associated with disorders of bone and cartilage (p = 5.93e-6, OR = 1.28) as well as with secondary malignancy of bone (p = 1.46e-5, OR = 1.21); whereas SNP rs1801726 was significantly associated with osteoporosis related phenotypes (p = 5.93e-6, OR = 1.28). Given that breast cancer and other solid tumors frequently metastasize to skeletal tissues, these data suggests that the inactivating rs1801725 and rs1801726 CaSR variants differentially influence hypercalcemia-related comorbidities and cancer metastasis to bone. We will further verify these effects using the associated clinical data and perform genetic risk analyses for the expression of these SNPs and the associated disease phenotypes. Citation Format: Ky'Era Actkins, Heather Beasley, Annika Faucon, Lea Davis, Amos Sakwe. Association of inactivating calcium sensing receptor exon 7 SNPs with hypercalcemia-related disease phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5127.

Abstract 3928: Association of calcium sensing receptor polymorphisms at rs1801725 with circulating calcium in breast cancer patients

Abstract Patients with metastatic or end-stage breast cancer (BC) inevitably develop hypercalcemia, while up to 30% of BC patients develop cancer-induced hypercalcemia (CIH) in the absence of metastases or bone diseases. The commonly diagnosed mild increase in circulating calcium activates the calcium sensing receptor (CaSR) and has been shown to be associated with larger and more aggressive breast tumors in postmenopausal and premenopausal patients respectively. Whether differences in circulating calcium and/or specific inactivating CaSR variants play any role in disparities in BC outcomes remains unclear. DESIGN METHODS: We identified 199 BC cases and 384 age and genetic ancestry-matched controls with calcium assay and genotyping data from the Vanderbilt University DNA biorepository (BioVU) linked to de-identified electronic medical records. The linear mixed effects and codominant models were used to assess the relationship between inactivating CaSR mutations at rs1801725 (codon 986) and rs1801726 (codon 1011) and either circulating calcium levels or risk of high calcium-driven aggressive BC outcomes. RESULTS: We observed that circulating calcium levels were significantly higher in BC cases compared to control subjects (p=0.001) and interestingly, in subjects of African ancestry compared to Caucasians (p=0.001). The A986S mutant CaSR is common among Caucasians while the Q1011E mutant receptor is common among African Americans. However, only inactivating mutations at rs1801725 locus were significantly associated with higher calcium levels (p=0.006) and a higher (69%) risk of high calcium-driven aggressive BC outcomes compared to the wild type receptor. We also demonstrate that invasive BC cells are tolerant to sustained high calcium and that their adaptation to high calcium occurs via up-regulation of calcium-activated early response and malignancy-associated genes. CONCLUSION: These data suggest that inactivating CaSR polymorphisms at rs1801725 predispose BC patients to hypercalcemia and that high circulating calcium-driven aggressive disease outcomes occur via calcium modulated malignancy-associated genes such as MAGEC2/CT10. Citation Format: Diva Whalen, Li Wang, Sarrah Widatalla, Josiah Ochieng, Ann Richmond, Amos Sakwe. Association of calcium sensing receptor polymorphisms at rs1801725 with circulating calcium in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2017-3928

Filamin A is reduced and contributes to the CASR sensitivity in human parathyroid tumors.

Parathyroid tumors display reduced sensitivity to extracellular calcium ([Ca2+]o). [Ca2+]o activates calcium-sensing receptor (CASR), which interacts with the scaffold protein filamin A (FLNA). The study aimed to investigate: (1) the FLNA expression in human parathyroid tumors, (2) its effects on the CASR mRNA and protein expression, and (3) on ERK signaling activation, (4) the effect of the carboxy-terminal CASR variants and (5) of the treatment with the CASR agonist R568 on FLNA-mediated ERK phosphorylation in HEK293 cells. Full-length FLNA immunostaining was variably reduced in parathyroid tumors. Immunofluorescence showed that FLNA localized in membrane and cytoplasm and co-localized with CASR in parathyroid adenomas (PAds)-derived cells. Cleaved C-terminus FLNA fragment could also be detected in PAds nuclear protein fractions. In HEK293 cells transfected with 990R-CASR or 990G-CASR variants, silencing of endogenous FLNA reduced CASR mRNA levels and total and membrane-associated CASR proteins. In agreement, FLNA mRNA levels positively correlated with CASR expression in a series of 74 PAds; however, any significant correlation with primary hyperparathyroidism severity could be detected and FLNA transcript levels did not differ between PAds harboring 990R or 990G CASR variants. R568 treatment was efficient in restoring 990R-CASR and 990G-CASR sensitivity to [Ca2+]o in the absence of FLNA. In conclusion, FLNA is downregulated in parathyroid tumors and parallels the CASR expression levels. Loss of FLNA reduces CASR mRNA and protein expression levels and the CASR-induced ERK phosphorylation. FLNA is involved in receptor expression, membrane localization and ERK signaling activation of both 990R and 990G CASR variants.

Calcium intake, polymorphisms of the calcium-sensing receptor, and recurrent/aggressive prostate cancer.

To assess whether calcium intake and common genetic variants of the calcium-sensing receptor (CASR) are associated with either aggressive prostate cancer (PCa) or disease recurrence after prostatectomy. Calcium intake at diagnosis was assessed, and 65 common single-nucleotide polymorphisms (SNPs) in CASR were genotyped in 886 prostatectomy patients. We investigated the association between calcium intake and CASR variants with both PCa recurrence and aggressiveness (defined as Gleason score ≥4 + 3, stage ≥pT3, or nodal-positive disease). A total of 285 men had aggressive disease and 91 experienced recurrence. A U-shaped relationship between calcium intake and both disease recurrence and aggressiveness was observed. Compared to the middle quintile, the HR for disease recurrence was 3.07 (95% CI 1.41-6.69) for the lowest quintile and 3.21 (95% CI 1.47-7.00) and 2.97 (95% CI 1.37-6.45) for the two upper quintiles, respectively. Compared to the middle quintile, the OR for aggressive disease was 1.80 (95% CI 1.11-2.91) for the lowest quintile and 1.75 (95% CI 1.08-2.85) for the highest quintile of calcium intake. The main effects of CASR variants were not associated with PCa recurrence or aggressiveness. In the subgroup of patients with moderate calcium intake, 31 SNPs in four distinct blocks of high linkage disequilibrium were associated with PCa recurrence. We observed a protective effect of moderate calcium intake for PCa aggressiveness and recurrence. While CASR variants were not associated with these outcomes in the entire cohort, they may be associated with disease recurrence in men with moderate calcium intakes.

Excessive Signal Transduction of Gain-of-Function Variants of the Calcium-Sensing Receptor (CaSR) Are Associated with Increased ER to Cytosol Calcium Gradient

In humans, gain-of-function mutations of the calcium-sensing receptor (CASR) gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA) and reducing expression of Plasma Membrane Calcium-ATPase (PMCA). Wild-type CaSR (hCaSR-wt) and its gain-of-function (hCaSR-R990G; hCaSR-N124K) variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate) receptor inputs to cell function.

Vitamin D and calcium-sensing receptor polymorphisms differentially associate with resting energy expenditure in peripubertal children

Given that calcium metabolism is influenced by genes and is tightly linked to energy-utilizing pathways, this study evaluated the association of single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) and calcium-sensing receptor (CASR) with resting energy expenditure (REE). In 273 boys and girls, 7-12years of age, cross-sectional REE was measured via indirect calorimetry, body composition by DXA, and dietary measures by 24-h recall. SNPs for VDR Cdx-2 (rs11568820) and CASR A986S (rs1801725) were genotyped using the Illumina Golden Gate assay. Multiple linear regression models were used to determine the association between SNPs and REE. African American carriers of the 'A' VDR Cdx2 allele had increased levels of REE in the overall sample, and this association was apparent among participants with an adiposity level of <25% and 30% body fat in males and females, respectively. For CASR, an association between carriers of the 'A' allele and REE was observed only in those in the upper median of calcium intake. VDR and CASR variants are associated with REE in children and are influenced by levels of calcium intake and adiposity. Our results bring awareness to mechanisms underlying the regulation of REE and biological and dietary influential factors.

CASR gene activating mutations in two families with autosomal dominant hypocalcemia

BackgroundAutosomal dominant hypocalcemia (ADH) is an endocrine disorder caused by activating mutations of the calcium-sensing receptor (CASR) gene which plays a major role in maintaining calcium homeostasis. Biochemical features of ADH are hypocalcemia and hypercalciuria with inappropriately low levels of parathyroid hormone (PTH). We report on two four-generation families affected by ADH. AimTo identify mutations of CASR gene in subjects affected by familial idiopathic hypoparathyroidism. To perform functional assays of identified CASR variants by transient transfection on HEK293 cells. ResultsWe identified two CASR variants (Q681R and P221L): the Q681R variant was novel while the P221L had been previously published. Functional assays on the Q681R variant showed that it did not alter the whole expression nor the correct plasmamembrane localization, but enhanced the signaling function, increasing the sensitivity of the receptor as compared to the WT. ConclusionsWe report two activating CASR mutations in two families affected by ADH and the functional assays performed on the novel variant Q681R. Our work enlarged the spectrum of mutations of the CASR and contributed to a better elucidation of the protein function.

Abstract 5313: Sustained hypercalcemia primes non-invasive breast cancer cells for metastasis to high calcium microenvironments

Abstract Breast cancer frequently spreads to calcium-rich organs/metastatic sites such as skeletal tissues but the role of hypercalcemia in breast cancer progression remains poorly understood. While patients with metastatic or end-stage cancer are likely to become hypercalcemic, up to 30% of breast cancer patients develop cancer-induced hypercalcemia (CIH) in the absence of metastases or bone diseases. The resulting high extracellular ionized calcium (Ca2+) activates the calcium sensing receptor (CaSR) that not only promotes the proliferation of breast cancer cells but also the secretion of parathyroid hormone-related protein by the tumor cells. This suggests that CIH may promote both the vicious osteolytic cycle and breast cancer progression. In this study, we examined whether sustained hypercalcemia primes breast cancer cells for metastasis to high Ca2+ microenvironments. We demonstrate that sustained hypercalcemia enables Ca2+-sensitive non invasive breast cancer cells such as MCF-7 to become high Ca2+-adapted and that this adaptation is accompanied by increased motility and more aggressive growth (increased proliferation). Consistent with this observation, invasive and metastatic breast cancer cells are high Ca2+-adapted cells that efficiently grow over a wide range of Ca2+ concentrations (up to 10 mM). Interestingly, we also show that at high Ca2+ (5 mM) breast cancer cells expressing the Q1011E CaSR variant that is common among African Americans proliferate faster than those expressing the A986S CaSR variant that is common among Caucasians. These data suggest that differences in the adaptation of breast cancer cells to hypercalcemia due in part to the expression of distinct CaSR variants underlie the distinct race-related breast cancer outcomes. This work is funded by the following grants made available to JO: DOD-W81XWH-07-1-0254 and 1SC1CA134018-01 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5313. doi:1538-7445.AM2012-5313

Sustained hypercalcemia primes non‐invasive breast cancer cells for metastasis to high calcium microenvironments

Breast cancer frequently spreads to calcium‐rich organs/metastatic sites such as skeletal tissues. While patients with metastatic or end‐stage cancer are likely to become hypercalcemic, up to 30% of breast cancer patients develop cancer‐induced hypercalcemia (CIH) in the absence of metastases or bone diseases. The resulting high extracellular ionized calcium (Ca2+) activates the calcium sensing receptor (CaSR) that promotes cell proliferation and the secretion of parathyroid hormone‐related protein by tumor cells. This suggests that CIH promotes both the vicious osteolytic cycle and breast cancer progression. In this study, we examined whether sustained hypercalcemia primes breast cancer cells for metastasis to high Ca2+ microenvironments. We demonstrate that sustained hypercalcemia enables Ca2+‐sensitive non invasive breast cancer cells such as MCF‐7 to become high Ca2+ adapted and that this adaptation is accompanied by increased motility and more aggressive growth. Moreover, breast cancer cells expressing the Q1011E variant (common among African Americans)grow faster at high Ca2+ than those expressing either the wild type or the A986S variant (common among Caucasians)of the CaSR. These data suggest that differences in the adaptation of breast cancer cells to hypercalcemia due in part to the expression of distinct CaSR variants underlie the distinct race‐related breast cancer outcomes.

Is there a link between the calcium sensing receptor and Hirschsprung's disease? A mutational analysis

IntroductionHirschsprung's disease (HD) is a congenital malformation of the hindgut characterized by the absence of enteric ganglion cells in the submucosal and myenteric plexuses. Hirschsprung's disease is routinely treated by resection of the aganglionic bowel and pull-through procedure of the proximal ganglionated bowel to the anorectal region. Occasionally, after resection of the aganglionic bowel, HD patients still experience persistent disturbances in gut motility. The etiology of HD as well as the underlying pathomechanism for postoperative disturbances in gut motility is unclear. Molecular analysis of putative candidate genes may help to clarify the pathophysiology of these conditions. In the present study, we investigated the correlation between mutations and polymorphisms in the calcium sensing receptor (CaSR) and HD patients. MethodsMutational screening of the CaSR coding sequence was performed via polymerase chain reaction and direct sequencing of the amplified samples from 63 HD patients and 100 controls. ResultsWe identified 3 common polymorphisms (p.A986S, p.G990R, and p.Q1011E) residing in exon 7 and 1 polymorphism in intron 5 (IVS 5-88 T>C) of the CaSR gene. Overall, 16 patients (25%) and 25 controls (25%) were carriers of the p.A986S polymorphism (P = 1). The incidence of p.R990G polymorphism of patients was twice as high as in the control group (P = .17). Furthermore, we verified a 4 times higher incidence of p.Q1011E polymorphism carriers in patients compared to the control group (P = .1). ConclusionWe found a higher incidence of R990G and Q1011E polymorphisms in HD patients compared to controls. However, there was no statistically significant association between HD and the 3 polymorphic variants in the intracellular signaling region of CaSR. It will be important to further investigate genetic variations in CaSR in more patient cohorts to better characterize the function of this gene and to establish a correlation between CaSR variants and HD.