Calcium Sensing Receptor Modulation for Cancer Therapy

In all living cells, calcium is required to maintain the normal structure and function (Barrett et al., 1986). Calcium signaling is also important for cell adhesion and mortality (Capiod et al., 2007). Certainly all events are integral to normal cell function, and disruption in calcium signaling is a well established feature in the generation and progression of cancer (Al-Haddad et al., 1999). The calcium sensing receptors (CaSR) are responsive to the changes in the extracellular Ca2+ concentration (Saidak et al., 2009). The enhanced expression of CaSR expression and altered proliferation of prostate cancer cells in response to increased Ca2+ have also been described(George et al., 2011), in contrast colon and parathyroid cancers often present with reduced or absent CaSR expression, hence decrease in cell proliferation, suggesting a role for the CaSR as a tumor suppressor gene (Saidak et al., 2009). The CaSR are expressed in both normal as well as malignant cells (Saidak et al., 2009), increased expression have been reported in highly metastatic primary tumor contributing to the oncogenic properties of CaSR(Schneider et al., 2005). Thus, the CaSR may play an important role in the development of various benign and malignant tumors. An analysis of intracellular signaling pathways revealed a key role for CaSR-induced phospholipase C activation and the release of intracellular calcium (Tharmalingam et al., 2011). These results demonstrated for the first time that an ion sensing G protein coupled receptor functionally couples to the integrins, and in conjunction with intracellular calcium release, promotes cellular adhesion


Introduction
In all living cells, calcium is required to maintain the normal structure and function (Barrett et al., 1986).Calcium signaling is also important for cell adhesion and mortality (Capiod et al., 2007).Certainly all events are integral to normal cell function, and disruption in calcium signaling is a well established feature in the generation and progression of cancer (Al-Haddad et al., 1999).The calcium sensing receptors (CaSR) are responsive to the changes in the extracellular Ca 2+ concentration (Saidak et al., 2009).The enhanced expression of CaSR expression and altered proliferation of prostate cancer cells in response to increased Ca 2+ have also been described (George et al., 2011), in contrast colon and parathyroid cancers often present with reduced or absent CaSR expression, hence decrease in cell proliferation, suggesting a role for the CaSR as a tumor suppressor gene (Saidak et al., 2009).The CaSR are expressed in both normal as well as malignant cells (Saidak et al., 2009), increased expression have been reported in highly metastatic primary tumor contributing to the oncogenic properties of CaSR (Schneider et al., 2005).Thus, the CaSR may play an important role in the development of various benign and malignant tumors.An analysis of intracellular signaling pathways revealed a key role for CaSR-induced phospholipase C activation and the release of intracellular calcium (Tharmalingam et al., 2011).These results demonstrated for the first time that an ion sensing G protein coupled receptor functionally couples to the integrins, and in conjunction with intracellular calcium release, promotes cellular adhesion

Calcium Sensing Receptor
The calcium sensing receptor (CaSR) is widely expressed homodimeric G protein-coupled receptor (GPCR) that functions as multi-modal nutrient sensor to maintain systemic calcium homeostasis by sensing changes in extracellular Ca 2+ concentration (Ward and Riccardi, 2012).Ca 2+ plays an essential role in numerous physiological processes, including blood clotting (Chattopadhyay et al., 1996), neuromuscular excitability (Chattopadhyay et al., 1996), and maintenance of skeletal integrity (Chattopadhyay et al., 1996).It has been known for years that Ca 2+ selective ion channels enable calcium ion to move across the cell membrane (Brown and MacLeod, 2001).Most of the hormone receptors are activated by nanomolar to picomolar concentration of their agonists (Rodland, 2004).In contrast, the CaSR receptor is sensitive to relatively small changes in Ca 2+ concentration in the extracellular fluid (Rodland, 2004).
In 1993, a membrane-spanning Ca 2+ sensing receptor, containing 1085 amino acids was cloned from bovine parathyroid, and it was shown to belong to the GPCR superfamily (Brown et al., 1993).The structure of CaSR , like of other GPCR superfamily, consists of seven transmembane helices, an extracellular N terminal and an intracellular C terminal (Brown et al., 1993).The CaSR belongs to the family 3 (or C) of the GPCR, which also includes the metabotropic glutamate receptor (mGluR1-8), γ-aminobutyric acid receptor subunits (GABAB1 and GABAB2), sweet and umami taste receptor (T1R1, T1R2, and T1R3), as well as pheromone and seven orphan receptor, and is characterized by a large extracellular N-terminal ligand-binding domain that possesses structural similarity to venus flytrap domain motif of bacterial periplasmic binding protein (Pin et al., 2003).There is evidence that the CaSR may exist as a dimer linked by disulfide bonds on the surface of certain cells, moreover the dimer is more sensitive to changes in calcium and other agonists than the monomeric form (Ray et al., 1999).When the CaR was first cloned from the bovine parathyroid, it was identified as the cation-sensing receptor that is responsible for the sensitivity of the parathyroid chief cells to changes in Ca 2+ (Brown et al., 1993) and it was suggested to be the primary player in Ca 2+ homeostasis (Breitwieser, 2012).However, it has been shown that the CaSR is also expressed by various cells involved in Ca 2+ homeostasis, such as the brain (Ruat et al., 1995), lens epithelial cells (Chattopadhyay et al., 1997), pancreas (Squires et al., 2000) and the antral gastrin-secreting cells of the stomach (Ray et al., 1997).

CaSR and Hematopoietic Stem Cell Niche
Since 1978 when Shofield and colleagues proposed the hematopoietic stem cell (HSCs) reside in a highly specific niche that regulates the overall size of the pool and their development (Holmbeck et al., 2003).Adult hematopoietic stem cell resides within the bone marrow in close association with the endosteal surface of bone (Nilsson et al., 2001).The ability of hematopoietic stem cells (HSCs) to maintain an undifferentiated state and undergo selfrenewal is partly regulated by external signals originating from the stem cell niche (House et al., 1997).One receptor expressed on HSCs that is known to be involved in HSC niche biology is the calcium-sensing receptor (Drueke, 2006).During mammalian hematopoiesis, HSCs shifts from fetal liver to the bone morrow shortly after the onset of bone mineralization (Cumano and Godin, 2007).HSCs obtained from the fetal liver of mice are deficient in CaSR has shown the crucial role of CaSR in HSC lodgment and engraftment in the bone.
Osteopontin, an acidic glycoprotein produced by osteoblasts is highly enriched at the endosteal surface and was also shown necessary for the HSC lodgment (Haylock and Nilsson, 2006;Nilsson et al., 2005).The endosteal niche has uniquely enriched calcium content relative to serum, CaSR expression has been demonstrated on HSCs, including the stem cell-enriched (lin -) Sca-1 + c-Kit + (LSK) population (Adams et al., 2006).Finally, in a series of elegant experiments using CaSR-/-mice, Adams et al demonstrated that it is the mineral content of the niche dictates HSC localization via the CaSR (Adams et al., 2006).These mechanisms by which the CaR dictates preferential localization of HSCs in the bone marrow endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate (Theman and Collins, 2009).

CaSR and Cancer
The CaSR has recently been documented to be expressed in a variety of benign tumor and malignancies, often at expression levels that differ from those in their healthy counterparts like in breast cancer, prostate cancer, as well as in cancers originating from organs involved in Ca 2+ homeostasis, including colorectal cancer and parathyroid adenomas.A recent examination of publicly available gene expression data identified a variety of types of GPCRs (Dores and Trejo, 2012), including protease activated receptor and receptors for various chemokines, adenosine 2B, neuropeptide, metabotropic glutamate, and CaSR that are overexpressed in diverse type of cancer (Rozengurt, 2007) (Figure 1).

CaSR and Metastasis
According to "Seed and Soil" hypothesis formulated by Stephen Paget suggests the high incidence of tumor metastasis in bone, while bone is inhospitable environment for tumor growth (Cumano and Godin, 2007).Bone extracellular matrix (ECM) serves as the reservoir of growth factors such as insulin like growth factor, fibroblastic growth factor, bone morphogenetic proteins, and transforming growth factor β (TGF-β) (Hofer and Brown, 2003).Osteolysis releases growth factors and calcium autocrine signaling for the tumor growth and invasion (Kingsley et al., 2007).
CaSR is expressed on normal mammary epithelial and response to low Ca 2+ by increasing production of PTHrP production (VanHouten et al., 2004).For further investigation Liao et al incubated three prostate cancer cell lines that have different tumorigenic and metastatic potential in low versus high calcium media (Liao et al., 2006).Knockdown of CaSR by RNA interference decreased the tumor metastatic potential (Dvorak-Ewell et al., 2011).CaSR activation increased attachment of cells to extracellular matrix (Sanders et al., 2001).Increased adherence was reduced by the addition of pertussis toxin, a CaSR antagonist (Theman and Collins, 2009).CaSR signals, in part, through MAP kinase to stimulate PTHrP (Chattopadhyay, 2006).Blocking CaSR induced MAP kinase pathway elements inhibited calcium-induced PTHrP release (Chattopadhyay, 2006).TGF-β, one of  the growth factors released from bone matrix, also acts synergistically with CaSR agonists to stimulate PTHrP production (Sanders et al., 2000).Tumor cells express a number of "bone specific" markers including CaSR, osteopontin, osteocalcin and bone sialoprotein (Koeneman et al., 1999;Bellahcene et al., 2008).The chemokine CXCL12/SDF-1, which is highly expressed in bone marrow may also be important (Adams et al., 2006).CaSR-/-HSCs successfully localized to the endosteal niche using a SDF-1 gradient, though they could not lodge, suggesting that CaSR functions downstream of SDF-1 in HSC homing to bone.Blocking of SDF-1 signaling reduced metastasis (Theman and Collins, 2009).

CaSR in Parathyroid Adenoma
The primary site of CaSR regulation is the chief cells of parathyroid gland, where CaSR an increase in blood calcium concentration into decreased parathromone secretion (Rodland, 2004).CaSR protein and mRNA are more abundantly expressed in the parathyroid than in most other tissues (Rodland, 2004).Therefore, it is logical to examine CaSR expression in parathyroid adenomas (Liu et al., 2011).Under normal physiological conditions, one of the functions influenced by CaSR is cellular proliferation (Saidak et al., 2009).Thus CaSR in parathyroid may serve as tumor suppressor gene by virtue of its ability to suppress parathyroid cell growth.Recently, it has been reported about the human CaSR gene, which is encoded by seven exons, was shown to have two promoters and two 5' untranslated exons, and the alternative utilization of exon 1A abd 1B leads to different mRNA (Chikatsu et al., 2000).The expression of exon 1A, contains TATA and CAAT boxes, is reduced in parathyroid adenomas and is expressed at levels only 60% of those in normal glands, whereas of exon 1B is not different between adenomas and normal glands (Garrett et al., 1995;Chikatsu et al., 2000).However, this finding does suggest that CaSR exon 1A is regulated differently than the exon 1B transcripts, and may be correlated with the ability of CaSR to inhibit proliferation in the parathyroid cells.The reduced expression of exon 1A in parathyroid cells demonstrates an alternation that may contribute to tumorigenesis.CaSR expression was decreased primarily in areas of active cell proliferation (Sanders et al., 2001).
The mechanism behind CaSR activation leading to reduced parathyroid cell proliferation is not completely understood and the intracellular pathways involved still remain to be elucidated.One possible candidate in parathyroid cancer that as an oncogene is Cyclin D1, having a role in parathyroid cell proliferation and dysregulated PTH secretion (Motokura et al., 1991).The gene of cyclinD1 is under the influence of regulatory region of PTH gene in parathyroid adenomas, as a result of chromosomal traslocation there is over expression (Arnold et al., 1989).Corbetta et al. have demonstrated that EGF and FGF induced increases in cyclin D1 expression and ERK1/2 phosphorylation were inhibited by CaSR agonists in parathyroid adenomas.This shows that expression of cyclin D1 (Imanishi et al., 2012) of parathyroid cells is modulated by CaSR activation in presence of the growth factors mimicking normal physiological conditions.In absence of growth factors the CaSR activation was not observed.It strongly suggests that there is transactivation between CaSR and the growth factor receptors which is reported in other cell lines (Sanders et al., 2001;Yano et al., 2004).Thus it can be concluded that the activation of CaSR may play an inhibitory role in parathyroid tumorigenesis through cyclin D1 expression.Due to the down regulation of CaSR in many thyroid adenomas, the normal inhibitory effect of parathyroid cells is not able to proceed, leading to detrimental events promoting the development of parathyroid tumors.

CaSR in Colon Cancer
Calcium in case of colon cancer is considered as a chemopreventive agent (Garland et al., 1985;Lipkin, 1999;Kampman et al., 2000;Wargovich et al., 2000).Most epidemiological studies have reported that the colon cancer is inversely related to the dietary Ca 2+ consumption (Garland et al., 1985;Sellers et al., 1998;Kampman et al., 2000;Wu et al., 2002;Lamprecht and Lipkin, 2003).In vitro studies have demonstrated that maintenance of human colon carcinoma cells in Ca 2+ free medium results in increased proliferation of cells that are loosely attached to the substratum.Conversely elevated Ca 2+ decreases the rate of growth, and the cells take on a flattened appearance and behave as a cohesive epithelial unit (Bhagavathula et al., 2005).Studies conducted by Cross et al. have also demonstrated the proliferative potential is inversely proportional to Ca 2+ concentration in case of intestinal Caco-2 cells .The expression of CaSR in colon is not unexpected because numerous other CaSR agonists occur naturally in the colon, such as polyamines, suggestive of a physiological function of the CaSR.In the healthy gut, a gradient of CaSR expression exists in the colonic crypts, and rapidly proliferating epithelial cells at the bottom of the crypt do not express the CaSR, whereas cells in the middle and top of the crypt do express this receptor, with the highest level of expression at the top.Cells of the colonic crypt have been shown to acquire the CaSR expression as they differentiate and move towards the apex of the crypt (Chakrabarty et al., 2005).Thus it is well established that Ca 2+ concentration is a direct modulator of colonocyte proliferation and differentiation.This Ca 2+ concentration gradient was hypothesized to be responsible for the differential CaSR expression, as well as the enhanced differentiation and decreased proliferative activity of the cells from the bottom to top of the crypt.
Chakraborty et al. have shown that Ca 2+ ion acting on the CaSR, promotes the expression of E-cadherin in several human colon carcinoma cell lines, leading to suppression of their malignant properties (Chakrabarty et al., 2003).E-cadherin belongs to the superfamily of intercellular adhesion molecules expressed by the intestinal epithelial cells.Dysregulation of E-cadherin played an important role in the transition from adenoma to carcinoma (Birchmeier and Behrens, 1994;Birchmeier, 1995) leading to increased metastasis and invasiveness (Oka et al., 1993;Pignatelli et al., 1994).In rapidly proliferating epithelial cells the E-cadherin expression is very low, at times of differentiation it is up-regulated (Birchmeier and Behrens, 1994).The tumor-suppressing effects of E-cadherin are believed to be produced through its interaction with β-catenin, a protooncogene (Morin, 1999), which is a member of the Wnt pathway family (Moon et al., 1997).E-cadherin along with β-catenin forms a complex which is then linked to the actin-based cytoskeleton (Brembeck et al., 2006).When it is present as a complex at the cell surface, tissues acts as cohesive unit, otherwise the single cells are able to move and invade surrounding tissues (Kuroda et al., 1998).The activity of β-catenin (Rey et al., 2012) is controlled by E-cadherin by sequestering the protein from cytoplasm to the cell membrane, thus limiting its availability for signaling, hence antagonizing its functions (Fagotto et al., 1996).Activation of CaSR by Ca 2+ leads to decreased binding of β-catenin to TCF4 and suppression of this malignancypromoting pathway (Chakrabarty et al., 2003).These finding suggest that induction of E-cadherin expression and suppression of the β-catenin/TCF4 pathway may contribute to the chemopreventive action of Ca 2+ in colon cancer through activation of the CaSR (Chakrabarty et al., 2003).
A downstream event following the activation β-catenin/TCF4 stimulates the proto-oncogenes, c-myc and cyclin D1 (Tetsu and McCormick, 1999;Wong and Pignatelli, 2002).Bhagavathula et al. (2007) demonstrated relationship between activation of CaSR and cell surface β-catenin localization on one hand and reduced expression of c-myc and cyclin D1 on the other, suggesting that the quiescence induced by Ca 2+ may be produced through inhibition of β-catenin/TCF/c-myc/cyclin D1 sequence of events.Apart from these the association of β-catenin with TCF family of transcription factor leads to increased expression of MMP7 and γ-catenin, which have role in development and progression of colon cancer (Wong and Pignatelli, 2002).Additionally, Ca 2+ has been seen to activate the cyclin-dependent kinase inhibitors p21 and p27, which potently induce differentiation in intestinal epithelial cells (Quaroni et al., 2000;Deschenes et al., 2001).The expression of p21 and p27 is lost in the early stages of colon tumorigenesis is observed along with the reduction of CaSR expression (Polyak et al., 1996).(Figure 2)

CaSR in Prostate and Breast Cancer
Prostate and breast cancer are the two most frequently occurring cancers after lung cancer in men and women respectively, which cause death.Both prostate and breast cancer preferentially metastasize to bone (Coleman, 1997).Approximately 75% of patients who develop advanced breast cancer will have secondary tumors in the bone with the majority being osteolytic, while in case of prostate cancer about 90% of patients die of advanced prostate cancer develop bone metastases (Roodman, 2004).The preferential metastasis of prostate and breast cancer to bone indicates that bone microenvironment provides a favorable niche (Liao et al., 2006).Multiple growth factors favorable chemotactic and growth promoting functions required for tumor localization and owing to bone turnover by remodeling in the skeleton such as the femur, pelvis, and humerus (Eaton and Coleman, 2003;Schneider et al., 2005).
Due to higher remodeling rate trabecular bone are more active than the cortical bone.Ca 2+ is one of the major inorganic factors released as a result of bone remodeling (Parkinson and Fazzalari, 2003).This may lead to numerous adverse effects like sever pain, spinal cord compression, higher risk of fractures and hypercalcemia (Chakravarti et al., 2009).Therefore, CaSR can serve as a major target of Ca 2+ in facilitating the formation and growth of skeletal metastasis of prostate and breast cancer.CaSR are expressed in osteoblasts, osteoclasts (Fukumoto, 1998) and its precursor cells, which can provide groundwork for local cross-talk in event that cancer cells localized in the bone (Sun et al., 2005).Chemoattractant likes TGF-β, IGF-I, IGF-II and platelet derived growth factor are believed to attract cancer cells to their metastatic location and increase their survival and proliferation (Imbriaco et al., 1998;Yin et al., 1999;Greenlee et al., 2001;Morony et al., 2001;Neudert et al., 2003;Yin et al., 2005;Cicek and Oursler, 2006;Le Gall et al., 2007;Zheng et al., 2007).During bone formation these factors are released by bone resorption thereby contributing to the propensity of the prostate and breast cancer to metastasis.
Another important contributor is the PTHrP, which initiates the vicious circle of metastatic breast and prostate cancer in bone (Grill et al., 1991).In normal cells of breast and prostate the secretion of PTHrP is monitored by the CaSR, they are antagonist in nature (Saidak et al., 2009).Recently, it was shown that inhibition and stimulation of PTHrP occurs due to the activation of G-protein by the cancerous cells (Mamillapalli et al., 2008).In the normal condition the CaSR couples to G αi , thus inhibition of cAMP formation and consequently PTHrP secretion, whereas in cancerous cells the CaSR activates G αs thus promotes to PTHrP release (Sanders et al., 2000;Yano et al., 2004).Elevated Ca 2+ level in serum activate the CaSR, produces additional release of PTHrP  doi.org/10.7314/APJCP.2012.13.8.3561The Calcium Sensing Receptor and Cancer Therapy from cancerous cells which stimulates osteoclastogenesis (Sanders et al., 2000).Activated CaSR transactivate the epidermal growth factor receptor, which is then followed by ERK1/2 activation, implying that the transactivation is mediated by the activation of MMP (Prenzel et al., 1999;Gschwind et al., 2001).The mechanism behind activation of TGF-β is unclear but it might be involved in upregulation of CaSR expression or its signaling pathways thereby increasing the amount of PTHrP.PTHrP stimulates osteoclastogenesis by increasing the expression of RANKL on osteoblasts, thereby stimulating the formation of mature osteoclasts (Roodman, 2001).This is how the PTHrP contributes to the increased bone resorption and releases the chemoattractants like TGF-β, IGF-I, IGF-II and platelet derived factor implicating in the induction and development of the vicious circle (Sanders et al., 2000) (Figure 3).

CaSR in Other Cancers
Angiogenesis is one of the six hallmarks of cancer.Leydig tumor cells exhibit high level of angiogenesis (Tfelt-Hansen et al., 2003).CaSR activation enhances new blood vessel formation via the production of angiogenic factor bFGF. Increased Ca 2+ concentration regulates VEGFR which stimulates nitrix oxide systhesis, this promotes neovascularization in tumors (Lala and Chakraborty, 2001).Again it has been seen that high Ca 2+ produced, CaSR mediated stimulation of proliferation as well as resistance to apoptosis.Thus it not only enhances tumor growth but also increases invasiveness and metastasis (Cheng et al., 1998;Thomsen and Miles, 1998).
It has been known that the fibroblastic cells require Ca 2+ rich media for their proliferation while epithelial cells require less Ca 2+ for optimal proliferation and differentiation (Hennings et al., 1980a(Hennings et al., : 1980b)).The surface epithelium of the ovary is contiguous with the mesothelial lining of the peritoneum.Thus the ovarian surface epithelial (OSE) cells are actually derived from a mesodermal lineage (Auersperg et al., 1998;Auersperg et al., 1994).Increasing extracellular calcium from 0.2mM to 5mM has a marked proliferative response in normal ovarian surface epithelial cells (Saxena et al., 2012).CaSR may mediate the growth of human ovarian surface epithelial cells by extracellular calcium (McNeil et al., 1998).Glioma is a collective term for glial cell-derived tumor, including astrocyte, oligodendrocyte and ependymal cell derived tumors.Gliomas are the most common primary neoplasm of the central nervous system (Chattopadhyay et al., 1998).The activation of CaSR via Ca 2+ in turn activates the K + channel which results in cell proliferation and secretion of PTHrP (Chattopadhyay et al., 1999).It generally shows imbalance or disturbance in calcium homeostasis (Bandyopadhyay et al., 2010).The expression of the CaSR in glial cells indicates potential roles in the maintenance of local ionic homeostasis as well as in tumor formation.

Conclusion
CaSR is neither a potent oncogene nor tumor supressor, it does play an important role in Ca 2+ homeostasis, which indirectly maintains a balance between proliferation and differentiation in response to change in extra cellular calcium level.Hence there should be a balanced intake of calcium rich diet, like patients suffering from colon or ovarian cancer should increase the calcium intake, while patients with prostate or breast cancer should decrease the intake of calcium rich food.
CaSR can be considered as a molecule that can either promote or prevent tumor growth depending on the type of cancer.Targeting of receptor based on the cancer may offer a cure in designing new therapies, which might complement the existing therapies.

Figure 1 .
Figure 1.Summary of Various Functions of Casr in Different Types of Cancer Cells.

Figure 2 .
Figure 2. The Role of E-cadherin and the β-catenin / TCF4 Pathway in CaSR Mediated Effects in Colon Cancer

Figure 3 .
Figure 3.The Role of CaSR in Normal As Well As in Cancerous Cells.