Background: Transient Receptor Potential (TRP) channels are expressed in many solid tumors. However, their expression in breast cancer remains largely unknown. Here, we investigated the profile expression of 13 TRP channels in human breast ductal adenocarcinoma (hBDA) and performed a correlation between their overexpression and pathological parameters. Methods: The TRP channels expression was determined by RT-PCR in hBDA tissue, in human breast cancer epithelial (hBCE) primary culture and in MCF-7 cell line. The TRP protein level was evaluated by immunohistochemistry in hBDA tissue samples of 59 patients. Results: TRPC1, TRPC6, TRPM7, TRPM8, and TRPV6 channels were overexpressed in hBDA compared to the adjacent non-tumoral tissue. Most interestingly, TRPC1, TRPM7 and TRPM8 expression strongly correlated with proliferative parameters (SBR grade, Ki67 proliferation index, and tumor size), and TRPV6 was mainly overexpressed in the invasive breast cancer cells. Using laser capture microdissection, we found that TRPV6 expression was higher in invasive areas, compared to the corresponding non-invasive ones. Moreover, TRPV6 silencing inhibited MDA-MB-231 migration and invasion, and MCF-7 migration. Conclusion: TRP channels are aberrantly expressed in hBDA, hBCE primary cultures, and cell lines, and associated with pathological parameters. The high expression of TRP channels in tumors suggests the potential of these channels for diagnostic, prognosis and/or therapeutic approaches in human breast ductal adenocarcinoma.

Breast cancer (BC) is the leading cancer in the world in terms of incidence and mortality in women. However, the mechanism by which BC develops remains largely unknown. The increase in cytosolic free Ca(2+) can result in different physiological changes including cell growth and death. Orai isoforms are highly Ca(2+) selective channels. In the present study, we analyzed Orai3 expression in normal and cancerous breast tissue samples, and its role in MCF-7 BC and normal MCF-10A mammary epithelial cell lines. We found that the expression of Orai3 mRNAs was higher in BC tissues and MCF-7 cells than in normal tissues and MCF-10A cells. Down-regulation of Orai3 by siRNA inhibited MCF-7 cell proliferation and arrested cell cycle at G1 phase. This phenomenon is associated with a reduction in CDKs 4/2 (cyclin-dependent kinases) and cyclins E and D1 expression and an accumulation of p21(Waf1/Cip1) (a cyclin-dependent kinase inhibitor) and p53 (a tumor-suppressing protein). Orai3 was also involved in MCF-7 cell survival. Furthermore, Orai3 mediated Ca(2+) entry and contributed to intracellular calcium concentration ([Ca(2+)](i)). In MCF-10A cells, silencing Orai3 failed to modify [Ca(2+)](i), cell proliferation, cell-cycle progression, cyclins (D1, E), CDKs (4, 2), and p21(Waf1/Cip1) expression. Our results provide strong evidence for a significant effect of Orai3 on BC cell growth in vitro and show that this effect is associated with the induction of cell cycle and apoptosis resistance. Our study highlights a possible role of Orai3 as therapeutic target in BC therapy.

Background: TRPM6 and TRPM7 combine ion channel and ␣-kinase functions. Results: ATP inhibits TRPM7 but not TRPM6 or heteromeric TRPM6/M7 channels. Disruption of phosphorylation activity of TRPM6 kinase re-establishes ATP sensitivity to heteromeric channels. Conclusion: TRPM6 uncouples heteromeric channels from cellular energy levels. Significance: The altered characteristics of TRPM6/M7 compared with homomeric TRPM7 may enhance Ca 2ϩ and Mg

The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store-operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7-deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild-type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell-cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild-type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store-operated channel itself. Using kinase-deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca concentration in resting cells, and for the refilling of Ca stores after a Ca signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca homeostasis.

Members of the Orai family are highly selective calcium ion channels that play an important role in store-operated calcium entry. Among the three known Orai isoforms, Orai3 has gained increased attention, notably for its emerging role in cancer. We recently demonstrated that Orai3 channels are over-expressed in breast cancer (BC) biopsies, and involved specifically in proliferation, cell cycle progression and survival of MCF-7 BC cells. Here, we investigate the downstream signaling mechanisms affected by Orai3 silencing, leading to the subsequent functional impact specifically seen in MCF-7 cancer cells. We report a correlation between Orai3 and c-myc expression in tumor tissues and in the MCF-7 cancer cell line by demonstrating that Orai3 down-regulation reduces both expression and activity of the proto-oncogene c-myc. This is likely mediated through the MAP Kinase pathway, as we observed decreased pERK1/2 levels and cell-cycle arrest in G1 phase after Orai3 silencing. Our results provide strong evidence that the c-myc proto-oncogene is influenced by the store-operated calcium entry channel Orai3 through the MAP kinase pathway. This connection provides new clues in the downstream mechanism linking Orai3 channels and proliferation, cell cycle progression and survival of MCF-7 BC cells.

Intracellular Ca2+ levels are important regulators of cell cycle and proliferation. We, and others, have previously reported the role of KCa3.1 (KCNN4) channels in regulating the membrane potential and the Ca2+ entry in association with cell proliferation. However, the relevance of KC3.1 channels in cancer prognosis as well as the molecular mechanism of Ca2+ entry triggered by their activation remain undetermined. Here, we show that RNAi-mediated knockdown of KCa3.1 and/or TRPC1 leads to a significant decrease in cell proliferation due to cell cycle arrest in the G1 phase. These results are consistent with the observed upregulation of both channels in synchronized cells at the end of G1 phase. Additionally, knockdown of TRPC1 suppressed the Ca2+ entry induced by 1-EBIO-mediated KCa3.1 activation, suggesting a functional cooperation between TRPC1 and KCa3.1 in the regulation of Ca2+ entry, possibly within lipid raft microdomains where these two channels seem to co-localize. We also show significant correlations between KCa3.1 mRNA expression and poor patient prognosis and unfavorable clinical breast cancer parameters by mining large datasets in the public domain. Together, these results highlight the importance of KCa3.1 in regulating the proliferative mechanisms in breast cancer cells as well as in providing a promising novel target in prognosis and therapy.

TRPM2 is the second member of the transient receptor potential melastatin-related (TRPM) family of cation channels. The protein is widely expressed including in the brain, immune system, endocrine cells, and endothelia. It embodies both ion channel functionality and enzymatic ADP-ribose (ADPr) hydrolase activity. TRPM2 is a Ca(2+)-permeable nonselective cation channel embedded in the plasma membrane and/or lysosomal compartments that is primarily activated in a synergistic fashion by intracellular ADP-ribose (ADPr) and Ca(2+). It is also activated by reactive oxygen and nitrogen species (ROS/NOS) and enhanced by additional factors, such as cyclic ADPr and NAADP, while inhibited by permeating protons (acidic pH) and adenosine monophosphate (AMP). Activation of TRPM2 leads to increases in intracellular Ca(2+) levels, which can serve signaling roles in inflammatory and secretory cells through release of vesicular mediators (e.g., cytokines, neurotransmitters, insulin) and in extreme cases can induce apoptotic and necrotic cell death under oxidative stress.

Intracellular levels of the divalent cations Ca2+ and Mg2+ are important regulators of cell cycle and proliferation. However, the precise mechanisms by which they are regulated in cancer remain incompletely understood. The channel kinases TRPM6 and TRPM7 are gatekeepers of human Ca2+/Mg2+ metabolism. Here, we investigated the human neuroblastoma cell line SHEP-21N in which the MYCN oncogene (encoding N-Myc) can be reversibly expressed under control of an inducible repressor. We report that N-Myc expression increases cell growth and up-regulates both TRPM6 and TRPM7 expression. Membrane current analyses reveal that endogenous TRPM6/TRPM7 currents exhibit reduced Mg·ATP suppression, increased Mg2+ sensitivity, and diminished sensitivity to 2-APB inhibition. These properties are consistent with N-Myc-induced increase of heteromeric TRPM7/TRPM6 channels promoting Ca2+ and Mg2+ uptake. Genetic suppression of TRPM6/TRPM7 through siRNA inhibits cell proliferation, suggesting that N-Myc can promote neuroblastoma cell proliferation through up-regulation of divalent cation-transporting channels.