Calcium-Sensing Receptor Mediated Calcium Signaling in Cancer Bone Metastasis

Calcium-sensing receptor (CaSR) belongs to the family C of G-protein coupled receptor (GPCR), playing an important role in regulating numerous biological processes in response to extracellular calcium levels. CaSR is the key regulator of parathyroid hormone secretion, which in turn influences calcium homeostasis. More notably, CaSR is also expressed in cancer cells upon the initiation of bone metastasis. However, the molecular basis of Ca 2+ /CaSR mediated bone metastasis is not clear. Human prostate tissue microarrays are first used to measure CaSR expression in both normal human prostate and prostate cancer tissue. Our tissue microarray studies reveal that CaSR is expressed in prostate cancers. The CaSR expression level of metastatic prostate cancer tissues found in the bone is higher than that of the primary prostate cancer tissues found in prostate cancer tissue localized in the prostate. This finding led us hypothesize that this increase of CaSR expression is likely a result of the prostate cancer cells adapting to the new, calcium rich environment and, thereby enhances their capacity to colonize in bone. To test this hypothesis, we investigate the role of CaSR in the cellular process of bone metastasis by knocking down CaSR expression in PC-3 prostate cancer cells with shRNA. It is found that PC-3 cells with reduced expression of CaSR exhibit a significant increase in cell apoptosis and cell invasion compared to wild type PC-3 cells. In addition, epithelialmesenchymal transition (EMT) biomarkers, such as vimentin and smooth muscle actin increase, E-cadherin and γ-catenin decrease in PC-3 cells with knocked down CaSR. This suggests that CaSR may inhibit cell apoptosis and cell invasion, while promoting mesenchymal-epithelial transition (MET) when cancer cells begin to colonize the bone. The bone environment with high calcium plays an important role in MET of cancer metastasis due to the prevention of apoptosis via CaSR. Similar results were observed for breast cancer cells MDA-MB-231. We further observed that the CaSR mediated signaling in PC-3 cells, including calcium oscillation, IP3 production, and ERK activation is different from HEK 293 cells transfected with CaSR. Altogether, such biased CaSR signaling pathway in cancer cells is consistent with differentiated expression level and locations of CaSR in PC-3 cells and HEK 293 cells transfected with CaSR. To unwind the origin for the differential Ca2+/CaSR mediated signaling in cancer cells, we performed various experiments using mass spectrometry, RT-PCR, RNA and DNA sequencing, glycosylation and Western blot to explore the possibility of different isoforms, deletion, mutation, or different glycosylation patterns in CaSR. We have found a new form of CaSR as a monomer on the cell membrane in prostate and breast cancer cells. It has a different molecular weight (120KD) instead of expected dimer band with MW of 250KD. This first observation of low molecular weight CaSR isoform with altered signaling pathway in PC-3 cells is possibly shared by other cancers, especially in bone metastasis and likely applicable for other chronic diseases, like FHH and ADH. This reveals its new role in aiding cancer cells in colonizing bone. Bone environment with high calcium is likely to play an important role in MET of cancer metastasis due to the inhibition of apoptosis through CaSR. New type of cancer therapy against bone metastasis can be further developed by reducing CaSR at bone environment as well as by modulating extracellular calcium signaling in bone environment. INDEX WORDS: calcium-sensing receptor; prostate cancer; bone metastasis; oscillation CALCIUM-SENSING RECEPTOR MEDIATED CALCIUM SIGNALING IN CANCER BONE METASTASIS