Data pertaining to aberrant intracellular calcium handling during androgen deprivation therapy in prostate cancer

The data generated here in relates to the research article “CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer”. A model of prostate cancer (PCa) progression to castration resistance was employed, with untreated androgen sensitive LNCaP cell line alongside two androgen deprived (bicalutamide) sublines, either 10 days (LNCaP-ADT) or 2 years (LNCaP-ABL) treatment, in addition to androgen insensitive PC3. With this PCa model, qPCR was used to examined fold change in markers linked to androgen resistance, androgen receptor (AR) and neuron specific enolase (NSE), observing an increase under androgen deprivation. In addition, the gene expression of a range of calcium channels was measured, with only the L-type Voltage gated calcium channel, CACNA1D, demonstrating an increase during androgen deprivation. With CACNA1D knockdown the channel was found not to influence the gene expression of calcium channels, ORAI1 and STIM1. The calcium channel blocker (CCB), nifedipine, was employed to determine the impact of CaV1.3 on the observed store release and calcium entry measured via Fura-2AM ratiometric dye in our outlined PCa model. In both the presence and absence of androgen deprivation, nifedipine was found to have no impact on store release induced by thapsigargin (Tg) in 0mM Ca(2+) nor store operated calcium entry (SOCE) following the addition of 2mM Ca(2+). However, CACNA1D siRNA knockdown was able to reduce SOCE in PC3 cells. The effect of nifedipine on CaV1.3 in PCa biology was measured through cell proliferation assay, with no observed change in the presence of CCB. While siCACNA1D reduced PC3 cell proliferation. This data can be reused to inform new studies investigating altered calcium handling in androgen resistant prostate cancer. It provides insight into the mechanism of CaV1.3 and its functional properties in altered calcium in cancer, which can be of use to researchers investigating this channel in disease. Furthermore, it could be helpful in interpreting studies investigating CCB's as a therapeutic and in the development of future drugs targeting CaV1.3

The interaction between the Orai3 calcium channel and the reticular protein STIM2 contributes to prostate cancer cell proliferation and survival

Le calcium intracellulaire (Ca2 +) joue un rôle essentiel en tant que second messager pour réguler la transcription génique, la progression du cycle cellulaire, la prolifération et la mort des cellules. Il est maintenant clair que dans le cancer de la prostate (CaP), la signalisation calcique est altérée favorisant ainsi, la croissance et la progression tumorales. Par conséquent, les canaux calciques sont devenus de nouvelles et potentielles cibles thérapeutiques pour le traitement du CaP. Ces canaux comprennent les canaux de type Orai, impliqués dans les entrées indépendantes ou dépendantes des vidanges calciques réticulaires (Store-Operated Ca2+ Entry ou SOCE) ainsi que leurs régulateurs STIM1 et STIM2 (STromal Interaction Molecules isoformes 1 et 2). Nos travaux récents (données non publiées) ont montré que le canal calcique de type Orai3 et la protéine réticulaire STIM2 sont fortement exprimés dans un modèle de CaP de souris Knock-in (KIMAP), et les expériences d'immunohistochimie effectuées sur des tissus prostatiques tumoraux issus de patients, ont montré des résultats similaires. Nous avons donc étudié si Orai3 et STIM2 peuvent interagir dans les cellules cancéreuses pour induire une entrée calcique qui serait impliquée dans la croissance ou la progression du CaP. In vitro, les expériences de co-immunoprécipitation effectuées sur la lignée cancéreuse prostatique PC-3, ont montré une interaction constitutive entre Orai3 et STIM2 quels que soient les composés utilisés pour induire l'influx de Ca2+. Le test de Proximity Ligation Assay (PLA) a révélé que l'interaction Orai3/STIM2 a lieu au niveau de la membrane plasmique, ou à proximité de celle-ci. Nous avons donc étudié s'il existe une entrée calcique associée à cette interaction, en effectuant des expériences d'imagerie calcique sur des PC-3 n’exprimant pas Orai3 ou STIM2. L’invalidation de STIM2 a diminué l’amplitude de l'entrée calcique basale (constitutive) sans affecter SOCE. D'autre part, la suppression d'Orai3 n'a pas affecté l'entrée basale de Ca2+, mais a considérablement augmenté les niveaux d’influx calcique de type SOCE. Nous avons ensuite analysé les effets de telles fluctuations calciques cytosoliques sur le comportement des cellules cancéreuses. L’invalidation d’Orai3 ou de STIM2 a diminué significativement la migration, la viabilité et la prolifération des PC-3. De manière très intéressante, les cellules cancéreuses n’exprimant pas Orai3, ont présenté un changement morphologique et les expériences de cycle cellulaire ont révélé un blocage de ces cellules en phase G2/M. Cet arrêt s'est accompagné d’une accumulation de la forme inactive de CDK1-Cycline B1, le complexe clé qui régule la transition de la phase G2 à la phase M. Bien que l'invalidation de STIM2 n'ait aucun effet significatif sur la progression du cycle cellulaire, les niveaux transcriptionnels de STIM2 étaient significativement plus élevés dans les cellules synchronisées en G2/M. Ces observations nous ont incités à traiter les PC-3 avec du Docetaxel en l'absence d'Orai3 ou de STIM2 pour étudier son effet sur la viabilité cellulaire. Le traitement au Docétaxel a diminué de façon plus prononcée la viabilité des PC-3 en absence des deux protéines d’intérêt, toutefois, cet effet était plus significatif dans les cellules n’exprimant pas STIM2. L’ensemble de ces résultats révèle un rôle important de cette interaction et de son entrée calcique, dans la prolifération et la survie des cellules CaP. Nous proposons l'hypothèse que l'interaction constitutive Orai3/STIM2 pourrait avoir lieu pour éviter une entrée calcique de type SOCE et l'activation des acteurs SOCE-dépendants, impliqués dans la catastrophe mitotique et la mort cellulaire.Mots clés : cancer de la prostate, signalisation calcique, canaux Orai, STIM2, cycle cellulaire, CDK1-cycline B1, catastrophe mitotique, survie.Intracellular calcium (Ca2+) plays a critical role as a second messenger in gene transcription regulation, cell cycle progression, cell proliferation and death. It is now clear that Ca2+ signaling is altered in prostate cancer (PCa), contributing to tumor growth and progression. Therefore, Ca2+ selective ion channels became prime targets for the development of novel therapeutic approaches for the treatment of PCa. These channels include Orai channels, which are involved in store dependent (Store-operated Ca2+ Entry or SOCE) and independent Ca2+ entries, and their regulators STIM1 and STIM2 (STromal Interaction Molecules isoforms 1 and 2). Our recent work (unpublished data) has shown that Orai3 and STIM2 are highly expressed in a Knock-in PCa mouse model (KIMAP), and immunohistochemistry experiments performed on human PCa tissues yielded similar results. We therefore investigated if Orai3 and STIM2 interact in PCa cells, therefore possibly contributing to Ca2+ entry, and tumor growth or progression. In vitro, co-immunoprecipitation experiments in PC-3 cancer cells demonstrated a constitutive interaction between Orai3 and STIM2 regardless of the compounds used to induce Ca2+ influx. Proximity ligation assay revealed that the Orai3/STIM2 interaction occurs at the plasma membrane, or in close proximity to it. We therefore investigated whether there is a possible related Ca2+ entry by performing Ca2+ imaging experiments on PC-3 cells that have their Orai3 or STIM2 protein expressions silenced. STIM2 knock-down decreased the basal (constitutive) Ca2+ entry levels without affecting SOCE. On the other hand, Orai3 knock-down did not affect the basal Ca2+ entry, but significantly increased SOCE levels. We then analyzed the effects of such changes in cytosolic Ca2+ on PC-3 cell behavior. Both protein invalidations caused a decrease in PC-3 cell migration, viability and proliferation. Very interestingly, Orai3 knock-down cancer cells exhibited morphological changes, and cell cycle experiments revealed their arrest at the G2/M phase. This arrest was accompanied by the accumulation of the CDK1-Cyclin B1 complex, the key complex that regulates the transition from the G2 to the M phase. While STIM2 invalidation had no significant effect on cell cycle progression, the transcriptional levels of STIM2 were significantly higher in G2/M synchronized cells. These observations prompted us to treat PC-3 cells with Docetaxel in the absence of Orai3 or STIM2 to investigate its effect on cell death. Docetaxel led to a stronger decrease in cell viability, an effect that was more pronounced in STIM2 knock-down cells. Together, our findings reveal an important role of Orai3/STIM2 interaction and its related Ca2+ entry in PCa cell survival. We propose the hypothesis that the constitutively active Orai3/STIM2 interaction may occur in cancer cells in order to prevent SOCE, hence avoiding activation of SOCE-dependent players that induce a mitotic catastrophe and death.Keywords: Prostate cancer, Ca2+ signaling, Orai channels, STIM2, Cell Cycle, CDK1-cyclinB1, mitotic catastrophe, survival

Crosstalk between Ca(2+) Signaling and Cancer Stemness: The Link to Cisplatin Resistance.

International audienceIn the fight against cancer, therapeutic strategies using cisplatin are severely limited by the appearance of a resistant phenotype. While cisplatin is usually efficient at the beginning of the treatment, several patients endure resistance to this agent and face relapse. One of the reasons for this resistant phenotype is the emergence of a cell subpopulation known as cancer stem cells (CSCs). Due to their quiescent phenotype and self-renewal abilities, these cells have recently been recognized as a crucial field of investigation in cancer and treatment resistance. Changes in intracellular calcium (Ca(2+)) through Ca(2+) channel activity are essential for many cellular processes such as proliferation, migration, differentiation, and survival in various cell types. It is now proved that altered Ca(2+) signaling is a hallmark of cancer, and several Ca(2+) channels have been linked to CSC functions and therapy resistance. Moreover, cisplatin was shown to interfere with Ca(2+) homeostasis; thus, it is considered likely that cisplatin-induced aberrant Ca(2+) signaling is linked to CSCs biology and, therefore, therapy failure. The molecular signature defining the resistant phenotype varies between tumors, and the number of resistance mechanisms activated in response to a range of pressures dictates the global degree of cisplatin resistance. However, if we can understand the molecular mechanisms linking Ca(2+) to cisplatin-induced resistance and CSC behaviors, alternative and novel therapeutic strategies could be considered. In this review, we examine how cisplatin interferes with Ca(2+) homeostasis in tumor cells. We also summarize how cisplatin induces CSC markers in cancer. Finally, we highlight the role of Ca(2+) in cancer stemness and focus on how they are involved in cisplatin-induced resistance through the increase of cancer stem cell populations and via specific pathways

S-acylation of Ca2+ transport proteins : molecular basis and functional consequences

Calcium (Ca2+) regulates a multitude of cellular processes during fertilization and throughout adult life by acting as an intracellular messenger to control effector functions in excitable and non-excitable cells. Changes in intracellular Ca2+ levels are driven by the co-ordinated action of Ca2+ channels, pumps, and exchangers, and the resulting signals are shaped and decoded by Ca2+-binding proteins to drive rapid and long-term cellular processes ranging from neurotransmission and cardiac contraction to gene transcription and cell death. S-acylation, a lipid post-translational modification, is emerging as a critical regulator of several important Ca2+-handling proteins. S-acylation is a reversible and dynamic process involving the attachment of long-chain fatty acids (most commonly palmitate) to cysteine residues of target proteins by a family of 23 proteins acyltransferases (zDHHC, or PATs). S-acylation modifies the conformation of proteins and their interactions with membrane lipids, thereby impacting intra- and intermolecular interactions, protein stability, and subcellular localization. Disruptions of S-acylation can alter Ca2+ signalling and have been implicated in the development of pathologies such as heart disease, neurodegenerative disorders, and cancer. Here, we review the recent literature on the S-acylation of Ca2+ transport proteins of organelles and of the plasma membrane and highlight the molecular basis and functional consequence of their S-acylation as well as the therapeutic potential of targeting this regulation for diseases caused by alterations in cellular Ca2+ fluxes

Lipid metabolism and Calcium signaling in epithelial ovarian cancer

International audienceEpithelial Ovarian cancer (EOC) is the deadliest gynecologic malignancy and represents the fifth leading cause of all cancer-related deaths in women. The majority of patients are diagnosed at an advanced stage of the disease that has spread beyond the ovaries to the peritoneum or to distant organs (stage FIGO III-IV) with a 5-year overall survival of about 29%. Consequently, it is necessary to understand the pathogenesis of this disease. Among the factors that contribute to cancer development, lipids and ion channels have been described to be associated to cancerous diseases particularly in breast, colorectal and prostate cancers. Here, we reviewed the literature data to determine how lipids or lipid metabolites may influence EOC risk or progression. We also highlighted the role and the expression of the calcium (Ca2+) and calcium-activated potassium (KCa) channels in EOC and how lipids might regulate them. Although lipids and some subclasses of nutritional lipids may be associated to EOC risk, lipid metabolism of LPA (lysophosphatidic acid) and AA (arachidonic acid) emerges as an important signaling network in EOC. Clinical data showed that they are found at high concentrations in EOC patients and in vitro and in vivo studies referred to them as triggers of the Ca2+entry in the cancer cells inducing their proliferation, migration or drug resistance. The cross-talk between lipid mediators and Ca2+ and/or KCa channels needs to be elucidated in EOC in order to facilitate the understanding of its outcomes and potentially suggest novel therapeutic strategies including treatment and prevention

Orai3 Calcium Channel Regulates Breast Cancer Cell Migration through Calcium-Dependent and -Independent Mechanisms

International audienceOrai3 calcium (Ca2+) channels are implicated in multiple breast cancer processes, such as proliferation and survival as well as resistance to chemotherapy. However, their involvement in the breast cancer cell migration processes remains vague. In the present study, we exploited MDA-MB-231 and MDA-MB-231 BrM2 basal-like estrogen receptor-negative (ER-) cell lines to assess the direct role of Orai3 in cell migration. We showed that Orai3 regulates MDA-MB-231 and MDA-MB-231 BrM2 cell migration in two distinct ways. First, we showed that Orai3 remodels cell adhesive capacities by modulating the intracellular Ca2+ concentration. Orai3 silencing (siOrai3) decreased calpain activity, cell adhesion and migration in a Ca2+-dependent manner. In addition, Orai3 interacts with focal adhesion kinase (FAK) and regulates the actin cytoskeleton, in a Ca2+-independent way. Thus, siOrai3 modulates cell morphology by altering F-actin polymerization via a loss of interaction between Orai3 and FAK. To summarize, we demonstrated that Orai3 regulates cell migration through a Ca2+-dependent modulation of calpain activity and, in a Ca2+-independent manner, the actin cytoskeleton architecture via FAK

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

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