Estrogen regulation of TRPM8 expression in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>The calcium-permeable cation channel TRPM8 (melastatin-related transient receptor potential member 8) is over-expressed in several cancers. The present study aimed at investigating the expression, function and potential regulation of TRPM8 channels by ER alpha (estrogen receptor alpha) in breast cancer.</p> <p>Methods</p> <p>RT-PCR, Western blot, immuno-histochemical, and siRNA techniques were used to investigate TRPM8 expression, its regulation by estrogen receptors, and its expression in breast tissue. To investigate the channel activity in MCF-7 cells, we used the whole cell patch clamp and the calcium imaging techniques.</p> <p>Results</p> <p>TRPM8 channels are expressed at both mRNA and protein levels in the breast cancer cell line MCF-7. Bath application of the potent TRPM8 agonist Icilin (20 μM) induced a strong outwardly rectifying current at depolarizing potentials, which is associated with an elevation of cytosolic calcium concentration, consistent with established TRPM8 channel properties. RT-PCR experiments revealed a decrease in TRPM8 mRNA expression following steroid deprivation for 48 and 72 hours. In steroid deprived medium, addition of 17-beta-estradiol (E₂, 10 nM) increased both TRPM8 mRNA expression and the number of cells which respond to Icilin, but failed to affect the Ca²⁺entry amplitude. Moreover, silencing ERα mRNA expression with small interfering RNA reduced the expression of TRPM8. Immuno-histochemical examination of the expression of TRPM8 channels in human breast tissues revealed an over-expression of TRPM8 in breast adenocarcinomas, which is correlated with estrogen receptor positive (ER⁺) status of the tumours.</p> <p>Conclusion</p> <p>Taken together, these results show that TRPM8 channels are expressed and functional in breast cancer and that their expression is regulated by ER alpha.</p

Acid Adaptation Promotes TRPC1 Plasma Membrane Localization Leading to Pancreatic Ductal Adenocarcinoma Cell Proliferation and Migration through Ca(2+) Entry and Interaction with PI3K/CaM.

International audiencePancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with a low overall survival rate of less than 10% and limited therapeutic options. Fluctuations in tumor microenvironment pH are a hallmark of PDAC development and progression. Many ion channels are bona fide cellular sensors of changes in pH. Yet, the interplay between the acidic tumor microenvironment and ion channel regulation in PDAC is poorly understood. In this study, we show that acid adaption increases PANC-1 cell migration but attenuates proliferation and spheroid growth, which are restored upon recovery. Moreover, acid adaptation and recovery conditions favor the plasma membrane localization of the pH-sensitive calcium (Ca(2+)) channel transient receptor potential C1 (TRPC1), TRPC1-mediated Ca(2+) influx, channel interaction with the PI3K p85α subunit and calmodulin (CaM), and AKT and ERK1/2 activation. Knockdown (KD) of TRPC1 suppresses cell migration, proliferation, and spheroid growth, notably in acid-recovered cells. KD of TRPC1 causes the accumulation of cells in G0/G1 and G2/M phases, along with reduced expression of CDK6, -2, and -1, and cyclin A, and increased expression of p21(CIP1). TRPC1 silencing decreases the basal Ca(2+) influx in acid-adapted and -recovered cells, but not in normal pH conditions, and Ca(2+) chelation reduces cell migration and proliferation solely in acid adaptation and recovery conditions. In conclusion, acid adaptation and recovery reinforce the involvement of TRPC1 in migration, proliferation, and cell cycle progression by permitting Ca(2+) entry and forming a complex with the PI3K p85α subunit and CaM

Acid Adaptation Promotes TRPC1 Plasma Membrane Localization Leading to Pancreatic Ductal Adenocarcinoma Cell Proliferation and Migration through Ca(2+) Entry and Interaction with PI3K/CaM.

International audiencePancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with a low overall survival rate of less than 10% and limited therapeutic options. Fluctuations in tumor microenvironment pH are a hallmark of PDAC development and progression. Many ion channels are bona fide cellular sensors of changes in pH. Yet, the interplay between the acidic tumor microenvironment and ion channel regulation in PDAC is poorly understood. In this study, we show that acid adaption increases PANC-1 cell migration but attenuates proliferation and spheroid growth, which are restored upon recovery. Moreover, acid adaptation and recovery conditions favor the plasma membrane localization of the pH-sensitive calcium (Ca(2+)) channel transient receptor potential C1 (TRPC1), TRPC1-mediated Ca(2+) influx, channel interaction with the PI3K p85α subunit and calmodulin (CaM), and AKT and ERK1/2 activation. Knockdown (KD) of TRPC1 suppresses cell migration, proliferation, and spheroid growth, notably in acid-recovered cells. KD of TRPC1 causes the accumulation of cells in G0/G1 and G2/M phases, along with reduced expression of CDK6, -2, and -1, and cyclin A, and increased expression of p21(CIP1). TRPC1 silencing decreases the basal Ca(2+) influx in acid-adapted and -recovered cells, but not in normal pH conditions, and Ca(2+) chelation reduces cell migration and proliferation solely in acid adaptation and recovery conditions. In conclusion, acid adaptation and recovery reinforce the involvement of TRPC1 in migration, proliferation, and cell cycle progression by permitting Ca(2+) entry and forming a complex with the PI3K p85α subunit and CaM

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. sp-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 Ca 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.This study was also supported by the Spanish Ministerio de Economía y Competitividad (contract numbers SAF2016‐76083‐R and CTQ2015‐64425‐C2‐1‐R), the Junta de Andalucía (contract number FQM2012‐1467) and the European Regional Development Funds (FEDER and FSE). The European Union Seventh Framework Programme (FP7‐People‐2012‐CIG), grant agreement number 333594 (to E. M. S.‐F., Marie Curie Reintegration Grant) is also acknowledged. MINECO/ICTI2013-2016/SAF2016‐76083‐R MINECO/ICTI2013-2016/CTQ2015‐64425‐C2‐1‐

Orai3-Mediates Cisplatin-Resistance in Non-Small Cell Lung Cancer Cells by Enriching Cancer Stem Cell Population through PI3K/AKT Pathway

International audienceSimple Summary Lung cancer is recognized for having a very poor prognosis with an overall survival rate of 5-years not exceeding 15%. Platinum-doublet therapy is the most current chemotherapeutic treatment used to treat lung tumors. However, resistance to such drugs evolves rapidly in patients with non-small cell lung cancer (NSCLC) and is one of the major reasons behind therapy failure. Tumor recurrence due to chemoresistance is mainly attributed to the presence of cancer stem cells (CSCs) subpopulations. Thus, the identification of resistance actors and markers is necessary. The Orai3 channel has been recently identified as a predictive marker of metastasis and survival in resectable NSCLC tumors. Our results show, for the first time, that the Orai3 channel is able to induce chemoresistance by enriching CSCs population. Our findings present Orai3 as a promising predictive biomarker which could help with selecting chemotherapeutic drugs. The development of the resistance to platinum salts is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Among the reasons underlying this resistance is the enrichment of cancer stem cells (CSCs) populations. Several studies have reported the involvement of calcium channels in chemoresistance. The Orai3 channel is overexpressed and constitutes a predictive marker of metastasis in NSCLC tumors. Here, we investigated its role in CSCs populations induced by Cisplatin (CDDP) in two NSCLC cell lines. We found that CDDP treatment increased Orai3 expression, but not Orai1 or STIM1 expression, as well as an enhancement of CSCs markers. Moreover, Orai3 silencing or the reduction of extracellular calcium concentration sensitized the cells to CDDP and led to a reduction in the expression of Nanog and SOX-2. Orai3 contributed to SOCE (Store-operated Calcium entry) in both CDDP-treated and CD133(+) subpopulation cells that overexpress Nanog and SOX-2. Interestingly, the ectopic overexpression of Orai3, in the two NSCLC cell lines, lead to an increase of SOCE and expression of CSCs markers. Furthermore, CD133(+) cells were unable to overexpress neither Nanog nor SOX-2 when incubated with PI3K inhibitor. Finally, Orai3 silencing reduced Akt phosphorylation. Our work reveals a link between Orai3, CSCs and resistance to CDDP in NSCLC cells

Mammary SLAMF3 Regulates Store-Operated Ca2+ Entry and Migration Through STIM1 in Breast Cancer Cells and Cell Lines

International audienceStore Operated Calcium Entry (SOCE) is the main route for calcium entry in breast cells. After it’s activation by STromal Interaction Molecule (STIM) during endoplasmic reticulum store depletion, membrane channels ORAI are the main actors of this cell calcium entry. STIM, ORAI and SOCE alterations might contribute to Breast Cancer (BC) carcinogenesis. Recently, we reported the tumor suppressor role of Signaling Lymphocytic Activation Molecule Family member 3 (SLAMF3) on HepatoCellular Carcinoma (HCC) progression. SLAMF3 has been shown to regulate the activity of immune cells by modulating the calcium influx. In this report, we aimed at exploring the role of SLAMF3 in regulating SOCE and migration of BC cells. We quantified and compared the expression of SLAMF3 and STIM1 by quantitative RT-PCR in tumor and healthy resections of 14 patients followed at the University Hospital of Amiens. The expressions of SLAMF3 and STIM1 were also quantified and compared in non-invasive T47D and invasive MDA-MB-231 cell lines by quantitative RT-PCR, Western blot and flow cytometry. We determined the Ca2+ basal entry as well as SOCE by Mn2+ quenching and calcium imaging, respectively, in T47D and MDA-MB-231 cells overexpressing SLAMF3 ectopically. The cell proliferation and migration/invasion were investigated by MTT, wound healing assay and Boyden chambers tests, respectively. First, we report the expression of SLAMF3 in mammary epithelial cells. We highlight the complete loss of SLAMF3 expression in invasive BC cell lines compared to non-invasive cells. In addition, we show that the forced expression of SLAMF3 in invasive cells down-regulate specifically the STIM1 expression in invasive compared to non-invasive mammary cell lines. Interestingly, an inverse correlation is observed between the low expression of SLAMF3 and the high expression of STIM1 in primary human BC tissues. Our results indicate that SLAMF3 reduces SOCE and therefore restricts BC cell migration by decreasing STIM1 expression. Therefore, SLAMF3 might be used as a predictive marker of BC evolution and aggressiveness

Assembly of a TRPC1/PI3K/CaM complex regulates proliferation in acid adapted pancreatic ductal adenocarcinoma cells

International audienc

The TRPC1 Channel Forms a PI3K/CaM Complex and Regulates Pancreatic Ductal Adenocarcinoma Cell Proliferation in a Ca(2+)-Independent Manner.

International audienceDysregulation of the transient receptor canonical ion channel (TRPC1) has been found in several cancer types, yet the underlying molecular mechanisms through which TRPC1 impacts pancreatic ductal adenocarcinoma (PDAC) cell proliferation are incompletely understood. Here, we found that TRPC1 is upregulated in human PDAC tissue compared to adjacent pancreatic tissue and this higher expression correlates with low overall survival. TRPC1 is, as well, upregulated in the aggressive PDAC cell line PANC-1, compared to a duct-like cell line, and its knockdown (KD) reduced cell proliferation along with PANC-1 3D spheroid growth by arresting cells in the G1/S phase whilst decreasing cyclin A, CDK2, CDK6, and increasing p21(CIP1) expression. In addition, the KD of TRPC1 neither affected Ca(2+) influx nor store-operated Ca(2+) entry (SOCE) and reduced cell proliferation independently of extracellular calcium. Interestingly, TRPC1 interacted with the PI3K-p85α subunit and calmodulin (CaM); both the CaM protein level and AKT phosphorylation were reduced upon TRPC1 KD. In conclusion, our results show that TRPC1 regulates PDAC cell proliferation and cell cycle progression by interacting with PI3K-p85α and CaM through a Ca(2+)-independent pathway

Les paysages sonores

« Si l'homme ne retient pas les sons dans sa mémoire, ils périssent, car ils ne peuvent être écrits », déplore Isidore de Séville. Par définition, les bruits et les sons s'envolent, et de fait, à première écoute, les sociétés anciennes – le monde d'avant Edison – apparaissent désespérément silencieuses. Comment étudier les bruits, les rumeurs, les clameurs qui animent le monde médiéval ? Peut-on même se représenter ces centaines de cloches qui, dans une ville comme Paris, sonnent à la volée les heures de la journée ? Peut-on imaginer les embarras de rues étroites où se côtoient hommes et bêtes, où hurlent du matin au soir crieurs et colporteurs ? C'est cet ensemble qui constitue un paysage sonore. Depuis les travaux pionniers de R. Murray Schafer et d'Alain Corbin, l'histoire du sensible a suscité un intérêt qui ne s'est jamais démenti, et qui a donné lieu à un grand nombre de travaux pour les xixe et xxe siècles. Or, le Moyen Âge est moins silencieux qu'on pourrait le croire, et une partie au moins des sons quotidiens et familiers se retrouve transcrite dans des chroniques, des chansonniers, des romans, voire dans des actes judiciaires. Il s'agit de les retrouver, de les analyser et de les donner à entendre de nouveau, dans une sorte d'extraordinaire essai d'archéologie sonore. Partageant le même intérêt pour la perception sensible de l'univers médiéval, Laurent Vissière et Laurent Hablot se sont attachés à réunir autour du thème des paysages sonores des historiens, des musicologues et des littéraires dans une perspective résolument interdisciplinaire. Rassemblant leurs savoirs, ces spécialistes du Moyen Âge et de la Renaissance nous font entendre ici le bruissement oublié du quotidien des femmes et des hommes de jadis