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 Ca 2+ 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 Ca 2+ . We generated isogenic HEK293T and BT-549 cells expressing these CaSR variants and carried out bulk RNA sequencing of high Ca 2+ 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 Ca 2+ (EC 50 ~5.28 mM) compared to the QE-CaSR (EC 50 of ~3.81 mM) and the wild type (WT-CaSR; EC 50 ~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 Ca 2+ , the viability of empty vector control and WT-CaSR transfected cells strongly decreased at Ca 2+ levels beyond 7.5 mM and these cells were mostly necrotic at ≥15 mM Ca 2+ . The QE-CaSR variant transfected cells on the other hand exhibited limited cell death beyond 10 mM Ca 2+ , while the surviving colonies of the AS-CaSR variant were viable at up to 15 mM Ca 2+ with invasive tendencies. Analysis of differentially expressed genes in high Ca 2+ 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 Ca 2+ supported adaptation of TNBC cells at high Ca 2+ , and that high Ca 2+ adaptation of breast cancer cells may be mediated via a panel of high Ca 2+ 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.