Background
Breast cancer (BC) is frequently diagnosed as an aggressive disease with poor prognosis especially in younger and women of African ancestry. The underlying mechanisms and factors that promote the aggressive behavior of BC in this subset of patients remain poorly understood. Among the potential factors is the development of cancer-induced hypercalcemia (CIH), an often overlooked metabolic disorder which is inevitable in late-stage, metastatic and aggressive BC [
1,
2]. Available evidence reveals that serum calcium levels are elevated in women with untreated BC [
3], and that high serum calcium levels are associated with aggressive breast tumors among premenopausal and/or overweight women [
4], and larger breast tumors among postmenopausal women [
5]. However, whether these high calcium associated breast cancer outcomes are related to the functional status of the calcium sensing receptor (CaSR) [
6] remains unclear.
As a major component of the calcium homoeostatic system [
7], the CaSR contributes to the development of CIH by promoting the growth and metastatic properties of tumor cells [
8,
9] and/or by promoting the secretion of tumor cell-derived osteolytic factors such as parathyroid hormone-related protein (PTHrP) [
10‐
12]. However,, in bone and mineral ion disorders, the CaSR is invariably mutated into several loss- or gain-of-function variants [
13,
14] and these are respectively associated with hypercalcemia and hypocalcemia [
15,
16]. The CaSR proteins with loss-of-function or inactivating mutations in the coding sequence have been shown to be less sensitive to calcium [
17,
18] and linked with familial hypocalciuric hypercalcemia, more severe primary hyperparathyroidism, and the risk of kidney stones [
13,
15,
19‐
21].
Among the several mutations in the cytosolic domain of the CASR, single nucleotide polymorphisms (SNP) at rs1801725 and rs1801726 in exon 7 are loss-of-function or inactivating mutations. Polymorphisms at these SNPs have not only been shown to lead to reduced sensitivity (right-shifted response) to calcium [
22] but are also important in the development of hypercalcemia in a mouse model of squamous cell lung carcinoma [
16]. Although the CaSR is pivotal in calcium homeostasis, its contribution in the previously reported association of high calcium with larger or more aggressive breast tumors remain unclear. In this study, we investigated whether these CASR SNPs are associated with higher circulating calcium levels in control versus BC Caucasian and African American women. Our data reveal that CASR polymorphisms at rs1801725 but not at rs1801726 SNP are associated with calcium and suggest that polymorphisms at rs1801725 in about 20% of BC cases, underlie, at least in part, the previously reported association of high circulating calcium with BC progression into larger and/or aggressive tumors.
Discussion
Cancer-induced hypercalcemia (CIH) is a metabolic syndrome which inevitably develops in patients with late-stage BC and/or metastasis to skeletal tissues [
11,
28,
29]. On the other hand, in most patients with low grade tumors, CIH is either undetected or diagnosed as mild, non-life threatening increase in circulating calcium. Nevertheless, such mild increases in circulating calcium levels may substantially promote disease progression by activating the CaSR and/or other calcium dependent oncogenic pathways. Our findings that only polymorphisms in the rs1801725 SNP of the receptor are associated with higher calcium levels suggest that mutations in codon 986 in exon 7 of the CASR are associated with BC outcomes driven by higher than normal circulating calcium levels such as larger and more aggressive breast tumors.
High calcium mediated activation of the CaSR not only leads to increased proliferation and migration of BC cells [
8] but also increased secretion of tumor cell-derived PTHrP [
8,
9] which contributes to the vicious osteolytic cycle [
28,
30]). Alteration of the function of the CaSR by pharmacological inhibition of its activity e.g. using calcilytic agents has been shown to inhibit cancer cell proliferation and metastasis [
31]. Although decreased sensitivity of the receptor may be associated with reduced activity at physiologically normal calcium levels, inactivating mutant CaSRs require higher circulating calcium levels to effectively activate downstream effectors. It is possible that a combination of inactivating mutant CaSR expression and progressive increase in circulating cancer cell-derived osteolytic factors contribute to the observed higher circulating calcium in BC cases. Analysis of the distribution of the common CaSR alleles at rs1801725 and rs1801726 SNPs among BC cases confirmed previous reports that the A986S CaSR variant is common among Caucasians while the Q1011E variant is common among African Americans [
17,
32‐
36]. Therefore, in both the control and BC cohorts, polymorphic variants in exon 7 of the CaSR occur with distinct frequencies among African Americans and Caucasians but the implication, if any, of the CaSR variants in the prognosis of BC patients requires further investigation.
Disparities in BC outcomes between Caucasian and African American patients [
37‐
40] as well as the involvement of the CaSR in cancer progression [
41,
42] have been amply reported. As expected and reported previously, [
17,
32‐
36], the magnitude of the differences in circulating calcium observed in this study were modest. Our observation that circulating calcium levels in BC cases were higher than those in control subjects is consistent with the potential increase in the synthesis and release of PTHrP by BC cells and the effects of this PTH-like factor on bone resorption [
29]. Meanwhile, our finding that circulating calcium levels in African American control subjects are higher than those in Caucasians is intriguing but supports the possibility that the aggressive nature of breast carcinoma in some African American patients may be driven at least in part, by high circulating calcium-dependent mechanisms. Surprisingly, the higher calcium levels in African American patients does not seem to be due to the expression of inactivating CaSR variants at the rs1801726 SNP which is more common in these subjects. One possible explanation for the lack of association between circulating calcium and polymorphisms at the CASR 1801726 SNP may be the generally reported smaller numbers of African American cases in the BioVU and other databases [
43]. Overall, this suggests that the high circulating calcium levels in African Americans may be due to other factors that alter systemic calcium homeostasis including the release of calcium stimulated osteolytic factors by normal and/or malignant breast tissues [
29], and active vitamin D. Unfortunately, PTH and PTHrP were not part of routine clinical tests and only a subset of patient serum chemistries included active vitamin D analysis from the control and BC case cohorts with genotyping data. Therefore, the confounding effects of PTHrP [
10] or Vitamin D [
44] as cancer promoting calciotropic hormones could not be evaluated.
It is well established that the CaSR is invariably mutated especially in parathyroid diseases [
13,
14]. Our study focused on rs1801725 and rs1801726 which are well characterized inactivating mutations of the receptor in exon 7 [
34,
35,
45] to either support their association with CIH or high calcium as an underlying factor for the obvious disparities in the progression of BC in Caucasians and African American patients. Interestingly, other SNPs e.g. rs1751221 [
46] and rs112594756 [
47] have been shown to correlate with BC susceptibility and prognosis. Although these intronic polymorphisms may affect the expression levels of the receptor, it is unlikely that they are relevant in the overall sensitivity of the mature receptor to calcium and/or the association of the receptor with CIH. Hypercalcemia in patients with advanced and/or metastatic disease has been reported to be strongly associated with poor prognosis [
48] while inactivating mutations of the CaSR in exon 7 promoted the development of hypercalcemia in a xenograft mouse model of human squamous cell lung carcinoma [
16]. Although the level of serum calcium in low grade BC patients may not be a prognostic indicator for survival, it is possible that the development of hypercalcemia in 10–30% of BC patients without evidence of skeletal metastases [
49,
50] may at least in part be due to the expression of inactivating CaSR mutations especially at the rs1801725. Contrary to previous studies showing that both the A986S and Q1011E variants of the CaSR are associated with calcium [
34,
35], our findings suggest that polymorphisms at the rs1801725 SNP are more important than those at the rs1801726 SNP in the development of CIH and the associated BC outcomes.
Limitations of the study and conclusions
The objective of this study was to determine if differences in circulating calcium and the expression of inactivating CaSR mutants in BC patients could shed more light on the causes of the highly aggressive disease in African American patients. Unfortunately, the fewer African American BC cases with both calcium test and GWAS data in the BioVU databases led to inconclusive interpretation of the relationship between circulating calcium and polymorphisms at the rs1801726 SNP. Vitamin D (1,25-dihydroxy vitamin D) levels were not available for most of the cases and control subjects and therefore, could not be considered as a confounding variable. Also, the documented lab calcium tests used in this study were total calcium rather than ionized calcium, the actual ligand for the CaSR. Consequently, it was not possible to relate the potential CaSR activity to the prevailing ionized calcium levels especially in BC patients. Another interesting question which could not be addressed in this study is the effect of these SNPs on calcium levels in BC patients with. This will require a larger, multi-site study to establish not only a better understanding of the role of high circulating ionized calcium but also the impact of inactivating CaSR mutants in BC cases with poor prognosis versus those with favorable prognosis. Overall, this retrospective case-control study reveals that decreased sensitivity of the CaSR to calcium due to inactivating polymorphisms at rs1801725 may predispose BC patients to high circulating calcium-driven larger or aggressive breast tumors.
Acknowledgments
We thank Dr. Ann Richmond, Department of Cancer Biology, Vanderbilt Ingram Cancer Center for facilitating this study and critical reading of the manuscript; Pengcheng Lu and Dr. Fei Fe, Vanderbilt Center for Quantitative Sciences, Department of Biostatistics, for statistical analysis; Jennifer Madison, Erica A. Bowton, Sarah P. Collier, and Jana Shirey-Rice, Vanderbilt CTSA for help with the extraction of the datasets; and the Clinical Research Education and Career Development (CRECD) Program at MMC for support to AMS.