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 Ca(2+) entry and MDA-MB-231 cell migration without affecting cell proliferation. Recent studies have reported that Ca(2+) entry through Orai1 channels is required for MDA-MB-231 cell migration. Down-regulation of hEag1 or Orai1 reduced Ca(2+) influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca(2+) 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 Ca(2+) entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development.

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.

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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 ϩ current by 22.6 Ϯ 10.6% (n ϭ 23). The effect of tamoxifen was always obtained in the first minute, peaked at 5.9 Ϯ 2.2 min (n ϭ 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-␤-estradiol (E 2 ) or its membrane-impermeant form, ␤-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 M). At the singlechannel level, tamoxifen significantly increased the open probability of the BK channel by 46.2 Ϯ 10.1% (n ϭ 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 E 2 . The site of action of tamoxifen is probably the channel itself or the auxiliary ␤ subunits.Breast cancer is generally believed to arise when dividing cells undergo mutations, and these genetically damaged cells become susceptible to unrestrained division. Thus, female hormones and other hormones that affect growth of the mammary gland are potential risk factors for breast cancer. In contrast, factors that induce differentiation in the mammary gland, such as pregnancy and lactation, are likely to reduce the risk of breast cancer. In the case of breast cancer, estrogens are still one of the most important risk factors. Indeed, whereas 17-␤-estradiol (E 2 ) is a key growth regulator in the normal mammary gland, clinical and experimental data have clearly established that exposure to this steroid hormone is also the leading cause of sporadic female breast cancer. Thus, the usefulness of antiestrogens and/or chemopreventives is closely associated with antagonizing the activity of E 2 . The fact that estrogens may affect carcinogenesis by acting either as initiators (i.e., directly damage DNA; Liehr and Ricci, 1996) or as promoters (i.e., promoting the growth and/or survival of initiated cells; ESHRE Capri Workshop, 2004;Veronesi et al., 2005) has justified the use of antiestrogenic compounds such as tamoxifen in estrogenic hormone replacement therapy. However, in addition to their key role in female reproductive functions, estrogens have beneficial effects on unrelated tissues, as demonstrated by the effects of hormone replacement therapy on postmenopausal women (Mitlack and Cohen, 1997;Cosman and Lindsay, 1999). Indeed, estrogens can prevent osteoporosis by inhibitin...

DNA methylation at CpG sites is an epigenetic mechanism that regulates cellular gene expression. In cancer cells, aberrant methylation is correlated with the abnormalities in expression of genes that are known to be involved in the particular characteristics of cancer cells such as proliferation, apoptosis, migration or invasion. During the past 30 years, accumulating data have definitely convinced the scientific community that ion channels are involved in cancerogenesis and cancer properties. As they are situated at the cell surface, they might be prime targets in the development of new therapeutic strategies besides their potential use as prognostic factors. Despite the progress in our understanding of the remodeling of ion channels in cancer cells, the molecular mechanisms underlying their over- or down-expression remained enigmatic. In this review, we aimed to summarize the available data on gene promoter methylation of ion channels and to investigate their clinical significance as novel biomarkers in cancer. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

We have investigated the acute effects of 17-b-estradiol (E 2 ) on K + channels in MCF-7 breast epithelial cancer cells. E 2 induced a rapid and irreversible augmentation of the K + current for all membrane potentials superior to À25 mV. The effect of E 2 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 E 2 /BSA. Furthermore, E 2 /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 E 2 that regulates cell proliferation independently of the estrogen receptor.

Activation of the phosphoinositide transduction pathway induces capacitative Ca2+ entry in Xenopus oocytes. This can also be evoked by intracellular injection of Ins(1,4.5)P3, external application of thapsigargin and/or incubation in a Ca2+-free medium. Readmission of Ca2+ to voltage-clamped, thapsigargin-treated Xenopus oocytes triggers Ca2+-dependent Cl- current variations that reflect capacitative Ca2+ entry. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by specific peptides markedly increased the amplitude of the transients, suggesting an involvement of the CaMKII pathway in the regulation of capacitative Ca2+ entry. Biochemical studies provide evidence for the activation of CaMKII in response to the development of capacitative Ca2+ entry. In effect, a CaMKII assay in vivo allows us to postulate that readmission of Ca2+ to thapsigargin-treated oocytes can induce a burst of CaMKII activity. Finally, analysis of the Cl- transient kinetics at high resolution of time suggests that CaMKII inhibition blocks the onset of the inactivation process without affecting the activation rate. We therefore postulate that CaMKII might participate in a negative feedback regulation of store-depletion-evoked Ca2+ entry in Xenopus oocytes.

Inhibition of calmodulin (CaM) sensitizes Ca2+ release mediated by D-myo-inositol (1,4,5)-trisphosphate (InsP3) in Xenoplus 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.