Intracellular calcium homeostasis is crucial for a variety of cellular processes and the maintenance of cell survival. Calcium homeostasis is achieved by the concerted actions of various calcium channels, pumps and exchangers. Alteration in the expression and activity of these transporters can result in impaired calcium signals and cellular functions. Deregulation of calcium transporters have been linked to various types of cancer including those of the breast. Modulation of the activity of specific calcium transporters may represent a potential therapeutic approach in breast cancer.
This thesis is centered on the hypothesis that breast cancer is associated with an alteration in the regulation of calcium store channels and regulators. While research has established roles for calcium pumps and calcium channels of the plasma membrane in breast cancer, studies pertaining to calcium store channels and their regulators in breast cancer are lacking.
This thesis assessed the mRNA levels of calcium store related proteins in breast cancer cell lines. The mRNA levels of four calcium store related protein families, namely, inositol 1,4,5-triphosphate receptors (IP3Rs), ryanodine receptors (RyRs), two-pore channels (TPCs) and IP3R-binding protein released with IP3 (IRBIT) were examined in a panel of breast cell lines. IP3Rs and RyRs are calcium store channels with three isoforms each, localized to the endoplasmic reticulum. TPCs are ion channels localized to the endolysosome, with two isoforms identified in humans. IRBIT is a regulator of the IP3R that competes with IP3 for the same binding domain on IP3R. All three isoforms of IP3Rs were present in all cell lines studied. In contrast, only RyR1 was detected in all studied cell lines. RyR3 was absent in all breast cancer-derived cell lines and the normal-like 184A1 cell line. Both isoforms of TPC were detected in all examined breast cell lines. IRBIT mRNA was also detected in all the breast cell lines studied. There was no correlation between tumorigenicity and estrogen, progesterone and HER2 receptor status with IP3Rs, RyRs, TPCs and IRBIT mRNA levels in the breast cell lines assessed.
Since IRBIT and TPCs are largely unexplored in the context of breast cancer, these two classes of protein were further examined to explore the potential roles of calcium store related proteins in breast cancer cells.
This thesis characterized the role of IRBIT in the luminal-like MCF-7 breast cancer cell line using a gene silencing approach. IRBIT did not appear to be a major regulator of IP3-mediated calcium release in MCF-7 cells. A slight reduction in IP3-mediated calcium release was observed in IRBIT silenced cells, while no effect was detected in receptor- and store-operated calcium entry. Although previous studies have shown that IRBIT silencing increases IP3R sensitivity to IP3, the work presented in this thesis suggests that IRBIT may play a different role in some breast cancer cells. The silencing of IRBIT did not significantly alter the proliferation of MCF-7 breast cancer cells.
In this thesis, the roles of TPC1 and TPC2 in breast cancer cells were explored in the basal-like MDA-MB-468 and MDA-MB-231 breast cancer cell lines. TPC1 and TPC2 do not appear to be global regulators of cytosolic free calcium in MDA-MB-468 and MDA-MB-231 cells. To explore the potential role of TPC1 and TPC2 in processes important in metastasis, the consequences of TPC1 and TPC2 silencing in an MDA-MB-468 model of epithelial to mesenchymal transition (EMT) was assessed. The silencing of TPC1 or TPC2 attenuated epidermal growth factor (EGF)-induced protein increases in the EMT marker vimentin without altering vimentin mRNA levels. The level of endogenous vimentin in MDA-MB-231 cells was not affected by TPC1 or TPC2 silencing. In addition, TPC1 or TPC2 siRNA had no effect on the phosphorylation of EGFR and STAT3, and the induction of the EMT related transcriptional factors snai1 and twist. This work suggests that TPC1 and TPC2 are regulators of EGF-induced vimentin protein expression in MDA-MB-468, but this is not the result of the inhibition of EMT.
Studies in this thesis also suggest that TPC isoforms may play a role in modulating cell death pathways in MDA-MB-231 cells. This regulation is dependent on the cell death stimuli, and, in some cases, the TPC isoform. While TPC1 and TPC2 siRNA had no effect on ceramide-induced cell death, both isoforms, moderately, but significantly, augmented ionomycin-induced cell death. TPC1 but not TPC2 silencing attenuated cell death induced by the Bcl-2 inhibitor ABT-263 in MDA-MB-231 cells.
The work presented in this thesis has helped characterize proteins that have been associated with the regulation of calcium store release in breast cancer cells. In contrast to some specific plasma membrane ion channels, there does not appear to be a major remodeling of these proteins in breast cancer. Further work is required to define the potential role of TPC isoforms in the regulation of EGF-induced vimentin expression and the regulation of cell death by specific stimuli.