Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF)

Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells and cancer-associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer.

peer reviewe

Implication des canaux ioniques dans l'impact des facteurs environnementaux sur la progression des cancers prostatiques

Les facteurs environnementaux appelés perturbateurs endocriniens (PE) semblent avoir un rôle dans le développement et la progression des cancers de la prostate (PCa). Il est bien établi que l'homéostasie calcique et l'activité des canaux ioniques sont impliquées dans la cancérogénèse prostatique et il est possible que ces PE favorisent la cancérogénèse en modulant la signalisation calcique. Dans ce contexte, nous avons étudié l effet du bisphénol A (BPA) et du Triclosan (TCS) sur la signalisation calcique des cellules cancéreuses prostatiques humaines. Nous avons ainsi montré que le BPA pouvait induire la migration des cellules cancéreuses épithéliales prostatiques, par le biais de la modulation de l'expression des canaux ioniques impliqués dans le phénomène de Store-Operated Calcium Entry, dont le canal calcique Orai1. Au niveau des cellules épithéliales cancéreuses, nous avons aussi mis en évidence un remodelage de l'expression des canaux ioniques et une résistance à l'apoptose de ces cellules sous l effet du TCS. Ensuite, nous avons montré dans les cellules stromales de PCa que le TCS induisait une entrée de calcium importante via le canal TRPA1, ce canal étant préférentiellement exprimé dans les cellules stromales de PCa. Par ailleurs, l'augmentation de calcium induite par le TCS est corrélée avec une sécrétion plus importante de VEGF, un facteur mitogène qui pourrait favoriser la croissance des cellules épithéliales ou endothéliales. Ces résultats pourraient permettre d envisager de nouvelles thérapies ou thérapies complémentaires ciblant les canaux et de mettre en place des mesures préventives dans le traitement des PCa qui prennent en compte l'impact de ces PE.Environmental factors called "endocrine disruptors (ED)" appear to have a potential role in the development and progression of cancer of the prostate (PCa). It is well established that calcium homeostasis and the activity of ion channels are involved in prostate carcinogenesis and it is possible that these ED promote carcinogenesis by modulating the calcium signalling. In this context, we studied the effect of bisphenol A (BPA) and Triclosan (TCS) on calcium signaling in human PCa cells. We have thus shown that BPA could induce the migration of prostate epithelial cancer cells through the modulation of ion channels expression which are involved in the phenomenon of Store-Operated Calcium Entry, including the calcium channel Orai1. At the level of cancer epithelial cells, we have also highlighted a remodeling of ion channels expression and a resistance to apoptosis of these cells under the influence of TCS. Then, we showed in stromal cells of human PCa that TCS induces an important calcium entry through the TRPA1 channel, this channel being preferentially expressed in stromal cells of PCa. Moreover, the TCS-induced calcium increase is correlated with a greater secretion of VEGF, a mitogenic factor that could promote the growth of epithelial or endothelial cells. These results allow to consider new therapies or complementary therapies targeting ion channels, and/or to implement preventive measures in the treatment of PCa taking into account the impact of these PE.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Expression and Role of T-type Calcium Channels during Neuroendocrine Differentiation

Neuroendocrine cells release their secretion into the extracellular environment through calcium-dependentsignalling pathways. These cells share common morphological and molecular features such as the expression ofspecific biomarkers, neurite outgrowth and dense core secretory granules. In order to elucidate the signallingpathways leading from undifferentiated to differentiated neuroendocrine cells, the role of voltage-dependent calciumchannels, central actors in the excitation-secretion coupling, has been comprehensively investigated. T-type calciumchannels appear to belong to the most important ion channel family involved in the neuroendocrine differentiationprocess. They could also participate in the development of neuroendocrine tumours

Expression and Role of T-type Calcium Channels during Neuroendocrine Differentiation

International audienceNeuroendocrine cells release their secretion into the extracellular environment through calcium-dependentsignalling pathways. These cells share common morphological and molecular features such as the expression ofspecific biomarkers, neurite outgrowth and dense core secretory granules. In order to elucidate the signallingpathways leading from undifferentiated to differentiated neuroendocrine cells, the role of voltage-dependent calciumchannels, central actors in the excitation-secretion coupling, has been comprehensively investigated. T-type calciumchannels appear to belong to the most important ion channel family involved in the neuroendocrine differentiationprocess. They could also participate in the development of neuroendocrine tumours

Rôle du canal calcique de type T, Cav3.2 et de ses protéines partenaires dans la tumorogenèse prostatique

La progression du cancer de la prostate s accompagne d une augmentation du nombre de cellules neuroendocrines, cellules qui expriment fortement les canaux calciques voltage-dépendants de type T Cav3.2. Ces canaux favorisent la sécrétion de phosphatase acide prostatique, marqueur des cellules épithéliales prostatiques, et d autres facteurs mitogènes potentiellement responsables de la prolifération des cellules avoisinantes. L'objectif de cette thèse était de déterminer les protéines partenaires du canal Cav3.2 et leur(s) rôle(s) dans la tumorogenèse prostatique. Nous montrons que les canaux Cav3.2 sont couplés aux canaux potassiques BK dans les cellules cancéreuses prostatiques. Ces 2 types de canaux interviennent dans la prolifération cellulaire. De plus, nous montrons que la sous-unité accessoire a2 2 des canaux calciques voltage-dépendants est exprimée plus fréquemment dans les tissus cancéreux prostatiques par rapport aux tissus sains, et que son expression augmente avec le grade du cancer. Par des études in vitro et in vivo, nous mettons en évidence son rôle promoteur de la croissance tumorale et de l angiogenèse. Par ailleurs, nous montrons que Cav3.2 et a2 2 peuvent s associer dans un même complexe protéique et qu a2 2 est capable de moduler l activité de ce canal. Cependant, nos travaux suggèrent également que le rôle de cette sous-unité dans la prolifération prostatique pourrait être indépendant de son association avec Cav3.2. En conclusion, ce travail amène à une meilleure compréhension de l implication des canaux calciques de type T et de ses protéines associées dans le cancer de la prostate.Prostate cancer progression leads to an androgen-refractory aggressive stage. This is associated with an increased number of neuroendocrine cells overexpressing Cav3.2 T-type calcium channels. Cav3.2 channels promote the secretion of prostatic acid phosphatase and other mitogenic factors responsible for the proliferation of epithelial cells. In order to determine the role of T-type channels and to understand their regulation during cancer progression, the aim of this work was to identify proteins that interact with Cav3.2 channels and their role in physiopathology. First, we demonstrate that Cav3.2 channels are coupled with BK channels in prostate cancer cells. We show that both channels are involved in proliferation. Moreover, we show that accessory a2 2, g4 and b4 subunits, putative partners of voltage-gated calcium channels, are expressed in prostate cancer cell lines and prostatic tissues. We show that the a2 2 subunit is more frequently expressed in cancerous tissues than in healthy ones, and that its expression increases with cancer grade. We propose that the a2 2 subunit may be used as a biomarker for prostate cancer diagnosis. Furthermore, using in vitro and in vivo studies, we highlight the promoting role of a2 2 on tumor growth and angiogenesis. In addition, we show that Cav3.2 and a2 2 can associate in a protein complex and that a2 2 can modulate the activity of Cav3.2 channels. However, our study also suggests that the role of the a2 2 subunit in prostate cell proliferation could be independent of its association with Cav3.2. In conclusion, this work leads to a better understanding of the role of T-type calcium channel and its partners in prostate cancer.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Implication du canal potassique calcium dépendant à conductance intermédiaire IKca1 dans la cancerogenèse humaine

Des études récentes montrent que l'homéostasie calcique intracellulaire, ainsi que l'expression et l'activité de canaux ioniques jouent un rôle essentiel dans le contrôle de la prolifération cellulaire aussi bien dans un contexte physiologique que dans certains cancers. Cependant, aucune approche proposant les canaux ioniques comme cible thérapeutique n'est actuellement envisagée dans le cadre des traitements des cancers de la prostate. Dans ce travail nous avons mis en évidence l'expression, la fonctionnalité et l'implication des canaux potassiques calcium-activés (IKca1) dans la prolifération des lignées cellulaires cancéreuses de la prostate humaine. Ces études ont également montré que l'activation du canal lKca1 favorise l'entrée de calcium via un canal calcique de la famille des TRP, le canal TRPV6, impliqué dans l'entrée passive de calcium dans les cellules cancéreuses prostatiques. De plus, par des études de co-immunoprécipitations, nous avons montré que le canal IKca1 et le canal calcique TRPV6 sont associés formant ainsi un complexe moléculaire fonctionnel permettant l'entrée de calcium et la prolifération des cellules cancéreuses prostatiques humaines. Par ailleurs, une suppression du canal IKcat induit la différenciation neuroendocrine des cellules cancéreuses prostatiques humaines. Ceci suggère un rôle essentiel joué par le canal IKca1 pour favoriser la croissance ou induire la différenciation cellulaire. Nos études ont également mis en évidence une surexpression de l'ARNm et de la protéine d'IKca1 dans les tissus prostatiques atteints d'un cancer de la prostate humaine. Ces données permettraient de proposer ces canaux ioniques comme marqueurs de cancer et/ou comme cibles thérapeutiques potentielles dans le traitement des cancers de la prostate humaine.Recent studies show that the intracellular calcium homeostasis, as well as expression and the activity of ionic channels play an essential role in the control of cell proliferation as weIl in a physiological context as in certain cancers. However, no approaches offering ionic channels as therapeutic target is nowadays envisaged as part of the treatments of the cancers of the prostate. ln the present work, we showed the expression, functionality and involvement of calcium-activated potassium channels (IKCa1) in the proliferation of the human prostate cancer cells. These studies also showed that the activation of the IKca1 channel favours the calcium entry via a member of the TRP family of calcium channels, TRPV6, in the prostate cancer cells. Besides, by studies of co-immunoprécipitations, we showed that the IKca1 potassium channel and the TRPV6 calcium channel form a functional molecular complex allowing the calcium entry and the proliferation of the prostate cancer cells. Moreover, a down-regulation of the 1Kca1 channel leads to the neuroendocrine differentiation of the human prostate cancer cells. This suggests an essential role played by the 1Kca1 channel to favour growth or lead to cell differentiation. Our immunohistotluorescence studies also showed an overexpression of IKca1 mRNA and protein in human prostate cancer compared to non-tumor tissues. These data would allow to propose these ion channels as markers and/or as potential therapeutic targets in the treatment of the human prostate cancers.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma.

Aberrant keratinocyte differentiation is considered to be a key mechanism in the onset of hyperproliferative dermatological diseases, including basal cell carcinoma (BCC). It is, therefore, vital to understand what drives keratinocytes to develop such pathological phenotypes. The role of calcium in keratinocyte differentiation is uncontested but the mechanisms controlling calcium-induced differentiation have yet to be completely elucidated. This study was designed to investigate the role of calcium-permeable TRPC channels in human keratinocyte differentiation and BCC, using a combination of molecular and cell biology approaches, involving electrophysiology and Ca(2+)-imaging, on the HaCaT cell line, primary cultures of normal human keratinocytes, and BCC cells. We demonstrated that TRPC1/TRPC4 channel expression was important for keratinocyte differentiation, as knocking out these channels (by siRNA strategy) prevented the induction of Ca(2+)-induced differentiation. TRPC1/TRPC4-mediated calcium entry and endoplasmic reticulum Ca(2+) content increased significantly in differentiated keratinocytes. However, the failure of BCC cells to differentiate was related to a lack of TRPC channel expression and calcium entry. In summary, our data demonstrate that TRPC1 and TRPC4 channels are key elements in keratinocyte Ca(2+) homeostasis and differentiation and may therefore be responsible for skin pathologies.Journal Articleinfo:eu-repo/semantics/publishe

Evolution of the human cold/menthol receptor, TRPM8

International audienceGenes showing versatile functions or subjected to fast expansion and contraction during the adaptation of species to specific ecological conditions, like sensory receptors for odors, pheromones and tastes, are characterized by a great plasticity through evolution. One of the most fascinating sensory receptors in the family of TRP channels, the cold and menthol receptor TRPM8, has received significant attention in the literature. Recent studies have reported the existence of TRPM8 channel isoforms encoded by alternative mRNAs transcribed from alternative promoters and processed by alternative splicing. Since the first draft of the human genome was accomplished in 2000, alternative transcription, alternative splicing and alternative translation have appeared as major sources of gene product diversity and are thought to participate in the generation of complexity in higher organisms. In this study, we investigate whether alternative transcription has been a driving force in the evolution of the human forms of the cold receptor TRPM8. We identified 33 TRPM8 alternative mRNAs (24 new sequences) and their associated protein isoforms in human tissues. Using comparative genomics, we described the evolution of the human TRPM8 sequences in eight ancestors since the origin of Amniota, and estimated in which ancestors the new TRPM8 variants originated. In order to validate the estimated origins of this receptor, we performed experimental validations of predicted exons in mouse tissues. Our results suggest a first diversification event of the cold receptor in the Boreoeutheria ancestor, and a subsequent divergence at the origin of Simiiformes