Frédéric Hague

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.

Transient receptor potential melastatin-related 7 channel is overexpressed in human pancreatic ductal adenocarcinomas and regulates human pancreatic cancer cell migration

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancer with a tendency to invade surrounding healthy tissues, leading to a largely incurable disease. Despite many advances in modern medicine, there is still a lack of early biomarkers as well as efficient therapeutical strategies. The melastatin‐related transient receptor potential 7 channel (TRPM7) is a nonselective cation channel that is involved in maintaining Ca2+ and Mg2+ homeostasis. It has been recently reported to regulate cell differentiation, proliferation and migration. However, the role of TRPM7 in PDAC progression is far to be understood. In our study, we show that TRPM7 is 13‐fold overexpressed in cancer tissues compared to the healthy ones. Furthermore, TRPM7 staining is stronger in tumors with high grade, suggesting a correlation between TRPM7 expression and PDAC progression. Importantly, TRPM7 expression is inversely related to patient survival. In BxPC‐3 cell line, dialyzing the cytoplasm during the patch‐clamp whole‐cell recording with a 0‐Mg2+ solution activated a nonselective current with a strong outward rectification. This cation current is inhibited by intracellular Mg2+ and by TRPM7 silencing. The downregulation of TRPM7 by small interference RNA dramatically inhibited intracellular Mg2+ fluorescence and cell migration without affecting cell proliferation, suggesting that TRPM7 contributes to Mg2+ entry and cell migration. Moreover, external Mg2+ following TRPM7 silencing fully restored the cell migration. In summary, our results indicate that TRPM7 is involved in the BxPC‐3 cell migration via a Mg2+‐dependent mechanism and may be a potential biomarker of poor prognosis of PDAC.

Extracellular signal-regulated kinases 1 and 2 and TRPC1 channels are required for calcium-sensing receptor-stimulated MCF-7 breast cancer cell proliferation.

The calcium-sensing receptor (CaR), is a G protein-dependent receptor that responds to increments in extracellular Ca²⁺ ([Ca²⁺]_o). We previously reported that an increase in [Ca²⁺]_o induced a release of intracellular calcium and Ca²⁺ entry via store operated channels (SOCs). We also demonstrated that MCF-7 cells express Transient Receptor Potential canonical 1 (TRPC1) channels. Herein, we investigated CaR intracellular signaling pathways and examined the role of TRPC1 in CaR-induced cell proliferation, through the extracellular signal-regulated Kinases 1 & 2 (ERK1/2) pathways. Treatment by [Ca²⁺]_o increased both MCF-7 cell proliferation and TRPC1 expression. Both the [Ca²⁺]_o proliferative effect and TRPC1 protein levels were abolished by the ERK1/2 inhibitors. Moreover, [Ca²⁺]_o failed to increase cell proliferation either in the presence of CaR or TRPC1 siRNAs. Both [Ca²⁺]_o and the selective CaR activator spermine, elicited time and dose-dependent ERK1/2 phosphorylation. ERK1/2 phosphorylation was almost completely inhibited by treatment with the phospholipase C and the protein kinase C inhibitors. Treatment with 2-aminoethoxydiphenyl borate (2-APB), and SKF-96365 or by siTRPC1 diminished both [Ca²⁺]_o- and spermine-stimulated ERK1/2 phosphorylation. Moreover, down-regulation of TRPC1 by siRNA reduced the Ca²⁺ entry induced by CaR activation. We conclude that the CaR activates ERK1/2 via a PLC/PKC-dependent pathway. Moreover, TRPC1 is required for the ERK1/2 phosphorylation, Ca²⁺ entry and the CaR-proliferative effect.

IGF-1 activates hEAG K(+) channels through an Akt-dependent signaling pathway in breast cancer cells: role in cell proliferation.

Previous work from our laboratory has shown that human ether à go‐go (hEAG) K+ channels are crucial for breast cancer cell proliferation and cell cycle progression. In this study, we investigated the regulation of hEAG channels by an insulin‐like growth factor‐1 (IGF‐1), which is known to stimulate cell proliferation. Acute applications of IGF‐1 increased K+ current‐density and hyperpolarized MCF‐7 cells. The effects of IGF‐1 were inhibited by hEAG inhibitors. Moreover, IGF‐1 increased mRNA expression of hEAG in a time‐dependent manner in parallel with an enhancement of cell proliferation. The MCF‐7 cell proliferation induced by IGF‐1 is inhibited pharmacologically by Astemizole or Quinidine or more specifically using siRNA against hEAG channel. Either mitogen‐activated protein kinase (MAPK) or phosphatidylinositol 3‐kinase (PI3K) are known to mediate IGF‐1 cell proliferative signals through the activation of extracellular signal‐regulated kinase 1/2 (Erk 1/2) and Akt, respectively. In MCF‐7 cells, IGF‐1 rapidly stimulated Akt phosphorylation, whereas IGF‐1 had little stimulating effect on Erk 1/2 which seems to be constitutively activated. The application of wortmannin was found to block the effects of IGF‐1 on K+ current. Moreover, the inhibition of Akt phosphorylation by the application of wortmannin or by a specific reduction of Akt kinase activity reduced the hEAG mRNA levels. Taken together, our results show, for the first time, that IGF‐1 increases both the activity and the expression of hEAG channels through an Akt‐dependent pathway. Since a hEAG channel is necessary for cell proliferation, its regulation by IGF‐1 may thus play an important role in IGF‐1 signaling to promote a mitogenic effect in breast cancer cells. J. Cell. Physiol. 212:690–701, 2007.

ORAI3 silencing alters cell proliferation and cell cycle progression via c-myc pathway in breast cancer cells.

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.

17-β-Estradiol activates maxi-K channels through a non-genomic pathway in human breast cancer cells

We have investigated the acute effects of 17‐β‐estradiol (E2) on K+ channels in MCF‐7 breast epithelial cancer cells. E2 induced a rapid and irreversible augmentation of the K+ current for all membrane potentials superior to −25 mV. The effect of E2 was sensitive to Iberiotoxin, Charybdotoxin and TEA and can be elicited in the presence of the anti‐estrogen ICI 182 780 or be mimicked by the membrane impermeant form E2/BSA. Furthermore, E2/BSA was able to stimulate cell proliferation in a maxi‐K inhibitors‐sensitive manner. Thus, these results permit us to identify the maxi‐K channel as the molecular target of E2 that regulates cell proliferation independently of the estrogen receptor.

Regulation of InsP3-mediated Ca2+ release by CaMKII in Xenopus oocytes

Abstract. Inhibition of calmodulin (CaM) sensitizes Ca2+ release mediated by D-myo-inositol (1,4,5)-trisphosphate (InsP3) in Xenopus oocytes, which results in spontaneous Ca2+-dependent Cl– current oscillations or in a shift of the concentration threshold for lysophosphatidic acid (LPA) by a tenfold factor. The oscillatory currents appear at a low initial Ca2+ concentration and without any significant increase in the inositol phosphate (InsPs) concentrations. These data led us to rule out the direct involvement of CaM, as well as the implied involvement of InsP3 3-kinase. The response to intracellular injection of the non-metabolizable InsP3 analog 3-deoxy-3-fluoro InsP3 (InsP3-F) is obviously affected by previous treatment with CaM inhibitory peptide. Furthermore, these effects have been consistently obtained with specific CaMKII inhibitors such as KN-93 and AIP. CaM plays a key role in the Ca2+-dependent inactivation of type I InsP3 receptors. The experiments presented hereby allow us to postulate that CaM could also exert its inhibitory effect through CaMKII in a way that does not involve InsP3 metabolism regulation. It is concluded that CaMKII could participate in Ca2+-evoked inhibition of InsP3-mediated Ca2+ release by inhibiting the InsP3 receptor.

Regulation of IGF-1-dependent cyclin D1 and E expression by hEag1 channels in MCF-7 cells: the critical role of hEag1 channels in G1 phase progression.

Insulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K(+)) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K(+) channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K(+) channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21(WAF1/Cip1) expression with a kinetic similar to that of cyclin D1, however p27(Kip1) expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6-8h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21(WAF1/Cip1) and p27(Kip1) expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K(+) channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

The carcinogen Cd2+ activates InsP3-mediated Ca2+ release through a specific metal ions receptor in Xenopus oocyte

The effects of the carcinogen Cd(2+) on Xenopus oocyte were evaluated by Inositol (1,4,5)-trisphosphate (InsP(3)) assays and electrophysiological experiments. The stimulation of the Ca(2+)-dependent Cl(-) current by Cd(2+) is clearly linked to InsP(3) formation since the effects of the metal are antagonized by neomycin, heparin and caffeine. A similar inhibition of the Cd(2+) effects is observed when the oocytes are pretreated with thapsigargin. Moreover, the use of sulfhydryl groups reductors such as 2-mercaptoethanol or N-ethylmaleimide strongly suggests that the Cd(2+) response is mediated by an extracellular receptor. Finally, measurements of InsP(3) production demonstrate that Cd(2+) superfusion actually leads to a PIP(2) breakdown. We conclude that extracellular Cd(2+) evokes an increase in [Ca(2+)](i) by stimulating the emptying of the InsP(3)-sensitive Ca(2+) stores, and that it may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing oocytes to respond to heavy metals.

Wittig derivatization of sesquiterpenoid polygodial leads to cytostatic agents with activity against drug resistant cancer cells and capable of pyrrolylation of primary amines

Many types of cancer, including glioma, melanoma, non-small cell lung cancer (NSCLC), among others, are resistant to proapoptotic stimuli and thus poorly responsive to current therapies based on the induction of apoptosis in cancer cells. The current investigation describes the synthesis and anticancer evaluation of unique C12-Wittig derivatives of polygodial, a sesquiterpenoid dialdehyde isolated from Persicaria hydropiper (L.) Delabre. These compounds were found to undergo an unprecedented pyrrole formation with primary amines in a chemical model system, a reaction that could be relevant in the biological environment and lead to the pyrrolation of lysine residues in the target proteins. The anticancer evaluation of these compounds revealed their promising activity against cancer cells displaying various forms of drug resistance, including resistance to proapoptotic agents. Mechanistic studies indicated that compared to the parent polygodial, which displays fixative general cytotoxic action against human cells, the C12-Wittig derivatives exerted their antiproliferative action mainly through cytostatic effects explaining their activity against apoptosis-resistant cancer cells. The possibility for an intriguing covalent modification of proteins through a novel pyrrole formation reaction, as well as useful activities against drug resistant cancer cells, make the described polygodial-derived chemical scaffold an interesting new chemotype warranting thorough investigation.