Halima Ouadid-Ahidouch

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.

Expression of TRPC6 channels in human epithelial breast cancer cells

BackgroundTRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.MethodsMolecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration).ResultsA non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPC7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours.ConclusionOur results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

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.

The Antiestrogen Tamoxifen Activates BK Channels and Stimulates Proliferation of MCF-7 Breast Cancer Cells

In the present study, we investigated the effect of the antiestrogen compound tamoxifen on BK channels by the use of the patch-clamp technique. The perfusion of 10 nM tamoxifen significantly increased the magnitude of a voltage-dependent K+ current by 22.6 ± 10.6% (n = 23). The effect of tamoxifen was always obtained in the first minute, peaked at 5.9 ± 2.2 min (n = 23), and was abolished by the perfusion of tetraethylammonium (0.5 mM), charybdotoxin (50 nM), or iberiotoxin (100 nM). The stimulatory effect of 10 nM tamoxifen was the same at low (50 nM) and high (700 nM) internal calcium concentration and was not additive to that of 17-β-estradiol (E2) or its membrane-impermeant form, β-estradiol 6-(O-carboxymethyl)oxime:bovine serum albumin. Furthermore, the effect of tamoxifen was still recorded in the presence of the selective estrogen receptor antagonist faslodex (ICI-182,780; 1 μM). At the single-channel level, tamoxifen significantly increased the open probability of the BK channel by 46.2 ± 10.1% (n = 4) without changing its unitary conductance. Moreover, we show here that the stimulation of BK channel activity by tamoxifen is involved in MCF-7 cell proliferation. Taken together, these results permitted us to identify the BK channel as the molecular target of tamoxifen that probably acts at the same extracellular molecular level as E2. The site of action of tamoxifen is probably the channel itself or the auxiliary β subunits.

Inositol 1,4,5-trisphosphate-induced Ca2+ signalling is involved in estradiol-induced breast cancer epithelial cell growth

BackgroundCa2+ is a ubiquitous messenger that has been shown to be responsible for controlling numerous cellular processes including cell growth and cell death. Whereas the involvement of IP3-induced Ca2+ signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. Our purpose was to characterize the role of IICS in the control of growth of the estrogen-dependent human breast cancer epithelial cell line MCF-7 and its potential regulation by 17β-estradiol (E2).ResultsOur results show that the IP3 receptor (IP3R) inhibitors caffeine, 2-APB and xestospongin C (XeC) inhibited the growth of MCF-7 stimulated by 5% foetal calf serum or 10 nM E2. Furthermore, Ca2+ imaging experiments showed that serum and E2 were able to trigger, in a Ca2+-free medium, an elevation of internal Ca2+ in a 2-APB and XeC-sensitive manner. Moreover, the phospholipase C (PLC) inhibitor U-73122 was able to prevent intracellular Ca2+ elevation in response to serum, whereas the inactive analogue U-73343 was ineffective. Western-blotting experiments revealed that the 3 types of IP3Rs are expressed in MCF-7 cells and that a 48 hours treatment with 10 nM E2 elevated IP3R3 protein expression level in an ICI-182,780 (a specific estrogen receptor antagonist)-dependent manner. Furthermore, IP3R3 silencing by the use of specific small interfering RNA was responsible for a drastic modification of the temporal feature of IICS, independently of a modification of the sensitivity of the Ca2+ release process and acted to counteract the proliferative effect of 10 nM E2.ConclusionsAltogether, our results are in favour of a role of IICS in MCF-7 cell growth, and we hypothesize that the regulation of IP3R3 expression by E2 is involved in this effect.

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.

Human ether à‐gogo K+ channel 1 (hEag1) regulates MDA‐MB‐231 breast cancer cell migration through Orai1‐dependent calcium entry

Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data present ether à go‐go (hEag1) K+ channels as relevant player in controlling cell cycle and proliferation of non‐invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA‐MB‐231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA‐MB‐231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca2+ entry and MDA‐MB‐231 cell migration without affecting cell proliferation. Recent studies have reported that Ca2+ entry through Orai1 channels is required for MDA‐MB‐231 cell migration. Down‐regulation of hEag1 or Orai1 reduced Ca2+ influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca2+ influx or cell migration were observed in cells co‐transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM+). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum‐induced migration of BC cells by controlling the Ca2+ entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development. J. Cell. Physiol. 227: 3837–3846, 2012.

TRP channels: diagnostic markers and therapeutic targets for breast cancer?

Breast cancer is the most frequently occurring cancer in women and has the highest rate of mortality. Ion channels such as the transient receptor potential (TRP) channels could play a critical role in the development and progression of cancer. Although these channels are frequently and abundantly expressed in many tumors, their expression, activity, and roles in the context of breast cancer remain poorly understood. This review summarizes our current knowledge regarding TRP channels expressed in human breast tissue, primary human breast epithelial cells, and cell lines, the functional role of TRP channels during breast cancer cell growth and migration, as well as their relationship with clinical and pathological features.