Ahmed Ahidouch

High Expression of Transient Receptor Potential Channels in Human Breast Cancer Epithelial Cells and Tissues: Correlation with Pathological Parameters

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.

K+ channel expression in human breast cancer cells: involvement in cell cycle regulation and carcinogenesis.

K+ channels are a most diverse class of ion channels in the plasma membrane and are distributed widely throughout a variety of cells including cancer cells. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K+ channels and that these K+ channels play important roles in regulating tumor cell proliferation, cell cycle progression and apoptosis. Moreover, a significant increase in K+ channel expression has been correlated with tumorigenesis, suggesting the possibility of using these proteins as transformation markers and perhaps reducing the tumor growth rate by selectively inhibiting their functional activity. Significant progress has been made in defining the properties of breast K+ channels, including their biophysical and pharmacological properties and distribution throughout different phases of the cell cycle in breast cell line MCF-7. This review aims to provide a comprehensive overview of the current state of research into K+ channels/currents in breast cancer cells. The possible mechanisms by which K+ channels affect tumor cell proliferation and cell cycle progression are discussed.

Down-regulation of Orai3 arrests cell-cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells.

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 Ca2+ can result in different physiological changes including cell growth and death. Orai isoforms are highly Ca2+ 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 p21Waf1/Cip1 (a cyclin‐dependent kinase inhibitor) and p53 (a tumor‐suppressing protein). Orai3 was also involved in MCF‐7 cell survival. Furthermore, Orai3 mediated Ca2+ entry and contributed to intracellular calcium concentration ([Ca2+]i). In MCF‐10A cells, silencing Orai3 failed to modify [Ca2+]i, cell proliferation, cell‐cycle progression, cyclins (D1, E), CDKs (4, 2), and p21Waf1/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. J. Cell. Physiol. 226: 542–551, 2011.

Activation of the calcium-sensing receptor by high calcium induced breast cancer cell proliferation and TRPC1 cation channel over-expression potentially through EGFR pathways

The calcium sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium ([Ca(2+)](o)). In MCF-7 human breast cancer cells, we previously reported that treatment with [Ca(2+)](o) for 24h leads to an over-expression of the Transient Receptor Potential Canonical 1 (TRPC1) cation channel and cell proliferation. Both involve the extracellular signal-regulated Kinases 1 & 2 (ERK1/2). MCF-7 also expressed epidermal growth factor receptor (EGFR) which is involved in cell proliferation through ERK1/2. Therefore, we investigated the cross-talk between CaR and EGFR in mediating ERK1/2 phosphorylation, TRPC1 over-expression and cell proliferation. Our data show that both high [Ca(2+)](o) and EGF phosphorylate ERK1/2. Furthermore, inhibition of EGFR kinase and matrix metalloproteinases (MMPs) reduced the overall effects mediated by [Ca(2+)](o) such as activation of ERK1/2, expression of TRPC1 and cell proliferation. They indicate the important role of the CaR-EGFR-ERK axis in transmitting mitogenic signals generated by high [Ca(2+)](o) in MCF-7 cells.

Orai3 Constitutes a Native Store-Operated Calcium Entry That Regulates Non Small Cell Lung Adenocarcinoma Cell Proliferation

Orai channels have been associated with cell proliferation, survival and metastasis in several cancers. The present study investigates the expression and the role of Orai3 in cell proliferation in non-small cell lung cancer (NSCLC). We show that Orai3 is over-expressed in cancer tissues as compared to the non-tumoral ones. Furthermore, Orai3 staining is stronger in high grade tumors. Pharmacological inhibition or knockdown of Orai3 significantly reduced store operated calcium entry (SOCE), inhibited cell proliferation and arrested cells of two NSCLC cell lines in G0/G1 phase. These effects were concomitant with a down-regulation of cyclin D1, cyclin E, CDK4 and CDK2 expression. Moreover, Orai3 silencing decreased Akt phosphorylation levels. In conclusion, Orai3 constitutes a native SOCE pathway in NSCLC that controls cell proliferation and cell cycle progression likely via Akt pathway.

K+ channels and cell cycle progression in tumor cells

K+ ions play a major role in many cellular processes. The deregulation of K+ signaling is associated with a variety of diseases such as hypertension, atherosclerosis, or diabetes. K+ ions are important for setting the membrane potential, the driving force for Ca2+ influx, and regulate volume of growing cells. Moreover, it is increasingly recognized that K+ channels control cell proliferation through a novel signaling mechanisms triggered and modulated independently of ion fluxes. In cancer, aberrant expression, regulation and/or sublocalization of K+ channels can alter the downstream signals that converge on the cell cycle machinery. Various K+ channels are involved in cell cycle progression and are needed only at particular stages of the cell cycle. Consistent with this idea, the expression of Eag1 and HERG channels fluctuate along the cell cycle. Despite of acquired knowledge, our understanding of K+ channels functioning in cancer cells requires further studies. These include identifying the molecular mechanisms controlling the cell cycle machinery. By understanding how K+ channels regulate cell cycle progression in cancer cells, we will gain insights into how cancer cells subvert the need for K+ signal and its downstream targets to proliferate.

Regulation of endogenous Ca2+ channels by cyclic AMP and cyclic GMP-dependent protein kinases in Pleurodeles oocytes.

The effects of cyclic AMP (cAMP) and cyclic GMP (cGMP) on dihydropyridine sensitive Ca2+ channels were investigated under voltage-clamp in defolliculated Pleurodeles oocytes. Intracellular injection of cAMP or extracellular application of the permeable cAMP analogue (8-Bromo cAMP, 8Br-cAMP) decreased the Ba current (IBa). This effect on IBa was blocked by the injection of protein kinase A inhibitor. Similar results were found upon internal application of the catalytic subunit of protein kinase A. In contrast, the injection of cGMP or perfusion of 8Br-cGMP increased IBa amplitude. The increase of IBa by 8Br-cGMP was blocked by the injection of the selective inhibitor of protein kinase G (KT5823).These results support the hypothesis that the basal Ba current amplitude of Pleurodeles oocytes is under the control of Protein Kinases A (PKA) and G (PKG) activity.This regulation of Ca2+ channels by the second messengers, and particularly by cAMP may reflect an important step in the maturation processus of Pleurodeles oocytes.

Orai3 is a predictive marker of metastasis and survival in resectable lung adenocarcinoma

Orai3 channel has emerged as important player in malignant transformation. Indeed, its expression is increased in cancer and favors cell proliferation and survival by permitting calcium influx. In this study, Orai3 was overexpressed in lung adenocarcinoma as compared to their matched non-tumour samples and was associated with tumoural aggressiveness. Moreover, its expression was associated with estrogen receptor alpha (ERα) expression and visceral pleural invasion in multivariate analysis. Furthermore, both the overall survival (OS) median and the metastasis free survival (MFS) median of tumors with high Orai3 expression were lower than in low Orai3 expression regardless of cancer stage (35.01 months vs. 51.11 months for OS and 46.01 months vs. 62.04 months for MFS). In conclusion, Orai3 protein level constitutes an independent prognostic marker in lung adenocarcinoma, and a novel prognostic marker that could help selecting the patients with worst prognosis to be treated with adjuvant chemotherapy in resectable stage.

sp2‐Iminosugar α‐glucosidase inhibitor 1‐C‐octyl‐2‐oxa‐3‐oxocastanospermine specifically affected breast cancer cell migration through Stim1, β1‐integrin, and FAK signaling pathways

Aberrant glycosylation changes on many glycoproteins are often related to cancer progression and metastasis. sp2‐Iminosugar‐type castanospermine analogues, inhibitors of α‐glucosidases, have been reported to exhibit antitumor activity. However, their effects on cell migration and the underlying molecular mechanism are not fully understood. Here, we investigated the effect of the pseudo‐C‐octyl glycoside 2‐oxa‐3‐oxocastanospermine derivatives (CO‐OCS) on breast cancer cells (MCF‐7 and MDA‐MB‐231 cells), and MCF‐10A mammary normal cell lines. We showed that CO‐OCS treatment results in the drastic decrease of breast cancer cell migration without affecting cell proliferation. Furthermore, CO‐OCS significantly reduced both the expression of β1‐integrin, which is a crucial interacting partner of Focal Adhesion Kinase (FAK), and the phosphorylation rates of FAK and ERK1/2. CO‐OCS also drastically reduced Ca2+ entry through Store Operated Channels (SOC). Orai1 and Stim1, two N‐glycosylated proteins, are involved in Store‐Operated Calcium Entry (SOCE), and are essential for breast tumor cell migration. Our results showed that CO‐OCS decreased the expression, at the protein level, of Stim1 without affecting that of Orai1. Moreover, cell migration and SOCE were attenuated by CO‐OCS as well as when Stim1 was silenced. In contrast, in MCF‐10A cells, CO‐OCS slightly reduced cell migration, but was without effect on gene expression of Stim1, Orai1, β1‐integrin, or FAK and ERK1/2 activation. Our results provide strong evidence for a significant effect of CO‐OCS on breast cancer cell migration and support that this effect was associated with β1‐integrin, Stim1, and FAK signaling pathways.

Functional cooperation between KCa3.1 and TRPC1 channels in human breast cancer: Role in cell proliferation and patient prognosis

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.