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

Study of ORAI protein remodeling with CRISPR and quantitative microscopy

L’entrée capacitive de calcium (Ca2+), appelée SOCE en anglais (store operated Ca2+ entry) représente une entrée d’ions Ca2+ dans la cellule consécutive à la vidange des stocks calcique réticulaires. Ce processus constitue l’un des mécanismes d’entré majeure de Ca2+ dans les cellules non-excitables. L’importance physiologique de ce processus est soulignée par la gravité des syndromes induits par des mutations des canaux responsables du SOCE : le syndrome sévère d’immunodéficience combinée induit par des mutations de types perte de fonction du SOCE et les syndromes d’agrégation tubulaire myopathique (TAM) et de Stormorken induit pas des mutations de type gain de fonction du SOCE. Le SOCE résulte de l’interaction de deux familles de protéines appelées STIM (Stromal interaction protein, 1,2) localisées dans la membrane du réticulum endoplasmique (RE) et ORAI (1-3) situées dans la membrane plasmique. Le processus classique d’apparition du SOCE dans les cellules peut être décrit de la sorte : La protéine STIM1, qui possède des motifs EF-hand sensible au Ca2+ dans la lumière du RE, détecte une diminution de la concentration en Ca2+. En réponse, la protéine STIM subit un changement conformationnel qui conduit à son oligomérisation et sa translocation au niveau des jonctions membrane plasmique – RE. Les protéines STIM vont ensuite interagir, regrouper et activer les protéines ORAI1 qui forment le canal appelé Ca2+-release activated Ca2+ (CRAC) aboutissant à la production du SOCE. Cependant, de nombreuses publications ont démontré l’implication des autres isoformes des protéines STIM et ORAI dans ce processus. De manière intéressante, l’intervention des protéines STIM2 et ORAI2/3 dans le mécanisme du SOCE permet de moduler le signal produit et ainsi de réguler finement les effets physiologiques de l’entrée de Ca2+ dans les cellules. En particulier, notre laboratoire a démontré que les canaux hétéromériques formés par les protéines ORAI1 et ORAI3 définissent un "interrupteur" oncogénique dans les cellules cancéreuses prostatiques permettant l’apparition d’un phénotype plus agressif. L’étude des mécanismes amenant à la formation de ces canaux hétéromériques est complexe en raison des limites des techniques à disposition. Par exemple, la plupart des moyens d’études reposent sur des systèmes de surexpression ou de sous expression, qui ne permettent pas de supprimer totalement l’expression des protéines endogènes. La présence de ces protéines endogènes rend difficile l’interprétation des résultats obtenus. Le but de cette thèse était donc d’utiliser des techniques de pointe pour étudier les mécanismes d’association des protéines ORAI. Ainsi, nous avons utilisé la technique CRISPR/Cas9 afin de générer des cellules doubles knockout (KO) pour les protéines ORAI1 et ORAI3. Ces cellules, KO pour ORAI1 et ORAI3 ont été utilisées pour réaliser des expériences de microscopie quantitative. Nous avons notamment ré-exprimé des versions fluorescentes des protéines ORAI1 et ORAI3 dans ces cellules avant de réaliser des expériences de mesure de temps de vie de fluorescence via la technique de FLIM-FRET (fluorescence lifetime imaging microscopy - Förster resonance energy transfer). Cette technique nous a permis de suivre l’évolution des interactions entre les protéines ORAI1 et ORAI3 lors de différentes stimulations cellulaires. De la sorte nous avons pu montrer que l’interaction entre les protéines ORAI1 et ORAI3 est dynamique en fonction de la stimulation appliquée. De plus, nous avons tiré profit des cellules KO générées afin d’étudier le rôle des protéines ORAI1/3 et du SOCE dans les lignées HEK-293 et PC3. Nous avons ainsi démontré que, dans les cellules HEK, la protéine ORAI1 et le SOCE jouent un rôle limité dans le maintien de leur physiologie, alors que dans les cellules PC3, ORAI1 et ORAI3 sont importantes pour le maintien du phénotype migratoire de ces cellules.The store operated calcium (Ca2+) entry (SOCE) represents the entry of Ca2+ through the cell’s plasma membrane consecutive to an endoplasmic reticulum (ER) Ca2+ store depletion. This process is described as one of the main calcium (Ca2+) pathway in the cells. Its importance is highlighted by the severe syndromes induced by loss or gain of function mutation of its constituent named severe combined immunodeficiency (SCID) and tubular aggregate myopathy (TAM)/Stormorken syndrome (STRMK) respectively. SOCE is the result of interaction between two families of proteins, the ER residing protein family Stromal interaction molecule (STIM 1&2) and the plasmalemmal proteins called ORAI (1-3). The classic molecular choreography of SOCE activation is described as follow: a drop in ER-Ca2+ content is detected by the EF-hand domain present in the STIM protein. Following ER Ca2+ depletion, STIM protein oligomerize and translocate to ER- plasma membrane (PM) junctions where they bind and activate ORAI1 composed channel called Ca2+ release activated Ca2+ channel providing SOCE. Interestingly, it appears that this choreography is much more complex than initially thought with the involvement of other STIM and ORAI isoforms (STIM2 and ORAI2,3). The involvement of these isoforms affects the properties of SOCE by modulating its Ca2+ signature resulting in different cellular answers. Especially, it was shown that heteromeric channels composed of ORAI1 and ORAI3 protein are defining an oncogenic switch in prostate cancer cell lines that lead to the more aggressive phenotype. However, the study of the mechanisms leading to the creation of ORAI1/3 channels at the expanse of ORAI1-only channels is problematic due to technical limitations. For example, most of investigations are performed in overexpression or downregulation system where endogenous protein are still present and might blur the results of experiments. The goal of this PhD was to use high end techniques in order to study the mechanisms of ORAI1/3 interactions without facing the limitations mentioned above. Specifically, we implemented and used CRISPR/Cas9 technique to generate double knockout (KO) cell lines for ORAI1 and 3 proteins. We used these “ORAI1/3 free cells” to perform quantitative microscopy experiments. Specifically, we expressed fluorescently tagged ORAI1 and ORAI3 proteins and performed FLIM-FRET (fluorescence lifetime imaging microscopy - Förster resonance energy transfer) experiments enabling us to follow their association process in answer to different stimulations. We thus demonstrated that the association between ORAI1 and ORAI3 is a dynamic process in the cells. Additionally, we took advantage of these KO cell lines to study the role of ORAI1/3 protein and SOCE in HEK-293 and PC3 cells physiology. We thus demonstrated that ORAI1 protein and SOCE only presented a limited role in the maintenance of HEK-293 physiology, while ORAI1 and ORAI3 are important to maintain migrative properties of the cancerous PC3 cells

Etude du remodelage des protéines ORAI grâce à l'utilisation de la technique CRISPR/Cas9 et de la microscopie quantitative

The store operated calcium (Ca2+) entry (SOCE) represents the entry of Ca2+ through the cell’s plasma membrane consecutive to an endoplasmic reticulum (ER) Ca2+ store depletion. This process is described as one of the main calcium (Ca2+) pathway in the cells. Its importance is highlighted by the severe syndromes induced by loss or gain of function mutation of its constituent named severe combined immunodeficiency (SCID) and tubular aggregate myopathy (TAM)/Stormorken syndrome (STRMK) respectively. SOCE is the result of interaction between two families of proteins, the ER residing protein family Stromal interaction molecule (STIM 1&2) and the plasmalemmal proteins called ORAI (1-3). The classic molecular choreography of SOCE activation is described as follow: a drop in ER-Ca2+ content is detected by the EF-hand domain present in the STIM protein. Following ER Ca2+ depletion, STIM protein oligomerize and translocate to ER- plasma membrane (PM) junctions where they bind and activate ORAI1 composed channel called Ca2+ release activated Ca2+ channel providing SOCE. Interestingly, it appears that this choreography is much more complex than initially thought with the involvement of other STIM and ORAI isoforms (STIM2 and ORAI2,3). The involvement of these isoforms affects the properties of SOCE by modulating its Ca2+ signature resulting in different cellular answers. Especially, it was shown that heteromeric channels composed of ORAI1 and ORAI3 protein are defining an oncogenic switch in prostate cancer cell lines that lead to the more aggressive phenotype. However, the study of the mechanisms leading to the creation of ORAI1/3 channels at the expanse of ORAI1-only channels is problematic due to technical limitations. For example, most of investigations are performed in overexpression or downregulation system where endogenous protein are still present and might blur the results of experiments. The goal of this PhD was to use high end techniques in order to study the mechanisms of ORAI1/3 interactions without facing the limitations mentioned above. Specifically, we implemented and used CRISPR/Cas9 technique to generate double knockout (KO) cell lines for ORAI1 and 3 proteins. We used these “ORAI1/3 free cells” to perform quantitative microscopy experiments. Specifically, we expressed fluorescently tagged ORAI1 and ORAI3 proteins and performed FLIM-FRET (fluorescence lifetime imaging microscopy - Förster resonance energy transfer) experiments enabling us to follow their association process in answer to different stimulations. We thus demonstrated that the association between ORAI1 and ORAI3 is a dynamic process in the cells. Additionally, we took advantage of these KO cell lines to study the role of ORAI1/3 protein and SOCE in HEK-293 and PC3 cells physiology. We thus demonstrated that ORAI1 protein and SOCE only presented a limited role in the maintenance of HEK-293 physiology, while ORAI1 and ORAI3 are important to maintain migrative properties of the cancerous PC3 cells.L’entrée capacitive de calcium (Ca2+), appelée SOCE en anglais (store operated Ca2+ entry) représente une entrée d’ions Ca2+ dans la cellule consécutive à la vidange des stocks calcique réticulaires. Ce processus constitue l’un des mécanismes d’entré majeure de Ca2+ dans les cellules non-excitables. L’importance physiologique de ce processus est soulignée par la gravité des syndromes induits par des mutations des canaux responsables du SOCE : le syndrome sévère d’immunodéficience combinée induit par des mutations de types perte de fonction du SOCE et les syndromes d’agrégation tubulaire myopathique (TAM) et de Stormorken induit pas des mutations de type gain de fonction du SOCE. Le SOCE résulte de l’interaction de deux familles de protéines appelées STIM (Stromal interaction protein, 1,2) localisées dans la membrane du réticulum endoplasmique (RE) et ORAI (1-3) situées dans la membrane plasmique. Le processus classique d’apparition du SOCE dans les cellules peut être décrit de la sorte : La protéine STIM1, qui possède des motifs EF-hand sensible au Ca2+ dans la lumière du RE, détecte une diminution de la concentration en Ca2+. En réponse, la protéine STIM subit un changement conformationnel qui conduit à son oligomérisation et sa translocation au niveau des jonctions membrane plasmique – RE. Les protéines STIM vont ensuite interagir, regrouper et activer les protéines ORAI1 qui forment le canal appelé Ca2+-release activated Ca2+ (CRAC) aboutissant à la production du SOCE. Cependant, de nombreuses publications ont démontré l’implication des autres isoformes des protéines STIM et ORAI dans ce processus. De manière intéressante, l’intervention des protéines STIM2 et ORAI2/3 dans le mécanisme du SOCE permet de moduler le signal produit et ainsi de réguler finement les effets physiologiques de l’entrée de Ca2+ dans les cellules. En particulier, notre laboratoire a démontré que les canaux hétéromériques formés par les protéines ORAI1 et ORAI3 définissent un "interrupteur" oncogénique dans les cellules cancéreuses prostatiques permettant l’apparition d’un phénotype plus agressif. L’étude des mécanismes amenant à la formation de ces canaux hétéromériques est complexe en raison des limites des techniques à disposition. Par exemple, la plupart des moyens d’études reposent sur des systèmes de surexpression ou de sous expression, qui ne permettent pas de supprimer totalement l’expression des protéines endogènes. La présence de ces protéines endogènes rend difficile l’interprétation des résultats obtenus. Le but de cette thèse était donc d’utiliser des techniques de pointe pour étudier les mécanismes d’association des protéines ORAI. Ainsi, nous avons utilisé la technique CRISPR/Cas9 afin de générer des cellules doubles knockout (KO) pour les protéines ORAI1 et ORAI3. Ces cellules, KO pour ORAI1 et ORAI3 ont été utilisées pour réaliser des expériences de microscopie quantitative. Nous avons notamment ré-exprimé des versions fluorescentes des protéines ORAI1 et ORAI3 dans ces cellules avant de réaliser des expériences de mesure de temps de vie de fluorescence via la technique de FLIM-FRET (fluorescence lifetime imaging microscopy - Förster resonance energy transfer). Cette technique nous a permis de suivre l’évolution des interactions entre les protéines ORAI1 et ORAI3 lors de différentes stimulations cellulaires. De la sorte nous avons pu montrer que l’interaction entre les protéines ORAI1 et ORAI3 est dynamique en fonction de la stimulation appliquée. De plus, nous avons tiré profit des cellules KO générées afin d’étudier le rôle des protéines ORAI1/3 et du SOCE dans les lignées HEK-293 et PC3. Nous avons ainsi démontré que, dans les cellules HEK, la protéine ORAI1 et le SOCE jouent un rôle limité dans le maintien de leur physiologie, alors que dans les cellules PC3, ORAI1 et ORAI3 sont importantes pour le maintien du phénotype migratoire de ces cellules

TRPV2 Mediates Adrenomedullin Stimulation of Prostate and Urothelial Cancer Cell Adhesion, Migration and Invasion

International audienceAdrenomedullin (AM) is a 52-amino acid peptide initially isolated from human pheochromocytoma. AM is expressed in a variety of malignant tissues and cancer cell lines and was shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, AM is a survival factor for certain cancer cells. Some data suggest that AM might be involved in the progression cancer metastasis via angiogenesis and cell migration and invasion control. The Transient Receptor Potential channel TRPV2 is known to promote in prostate cancer cell migration and invasive phenotype and is correlated with the stage and grade of bladder cancer. In this work we show that AM induces prostate and urothelial cancer cell migration and invasion through TRPV2 translocation to plasma membrane and the subsequent increase in resting calcium level

Opiates Modulate Thermosensation by Internalizing Cold Receptor TRPM8

Stimulation of μ-opioid receptors (OPRMs) brings powerful pain relief, but it also leads to the development of tolerance and addiction. Ensuing withdrawal in abstinent patients manifests itself with severe symptoms, including cold hyperalgesia, often preventing addicted patients from successfully completing the rehabilitation. Unsurprisingly, OPRMs have been a central point of many studies. Nonetheless, a satisfactory understanding of the pathways leading to distorted sensory responses during opiate administration and abstinence is far from complete. Here, we present a mechanism that leads to modulation by OPRMs of one of the sensory responses, thermosensation. Activation of OPRM1 leads to internalization of a cold-sensor TRPM8, which can be reversed by a follow-up treatment with the inverse OPRM agonist naloxone. Knockout of TRPM8 protein leads to a decrease in morphine-induced cold analgesia. The proposed pathway represents a universal mechanism that is probably shared by regulatory pathways modulating general pain sensation in response to opioid treatment

Adrenomedullin induces TRPV2 translocation to plasma membrane.

<p>(A) The effect of AM (200 nM, 45 min) and TRPV2 silencing (siTRPV2, 50 nM, 48 h) on basal cytosolic calcium of PC-3 and T24-83 cells was studied by calcium imaging. (n = 120 cells, N = 4, *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM). (B) TRPV2 presence at the plasma membrane was examined by biotinylation on T24/83 cells control or either treated with AM (200 nM, 45 min) or AM and PI3K inhibitor LY294.002 (10 µM, added 5 min before AM). (C) Effect of LY294.002 on PC-3 and T24/83 cell migration examined by transwell assay after 8 h incubation with or without AM (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM). (D) Effect of LY294.002 on AM-induced migration of PC-3 and T24/83 TRPV2-silenced cells examined by transwell assay after 8 h incubation with or without AM (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM).</p

Adrenomedullin increases PC-3 and T24/83 cell adhesion, migration and invasion.

<p>(A) RT-PCR experiment showing RAMP2, RAMP3 and CLR expression in PC-3 and T24/83 cells. (B) PC-3 (left panel) and T24/83 (right panel) cell adhesion was examined by seeding 3*10⁴ and 1.5*10⁴ cells respectively per well in 96-well plates pre-coated with fibronectin, and incubated for 45 min with or without AM (200 nM) (N = 3, *P<0.05 compared with control cells). β1 integrin phosphorylation was studied by western-blotting on total proteins extracted from PC-3 and T24/83 cells seeded on fibronectin coated plates and treated with or without AM. (C) PC-3 and T24/83 cell migration was studied by Transwell assay after 8 h of treatment (N = 3, *P<0.05 compared with control cells). FAK phosphorylation was studied by western-blotting on total proteins extracted from PC-3 and T24/83 cells treated with or without AM. (D) For invasion assay, transwell membrane was pre-coated with 50 µg Matrigel, and PC-3 and T24/83 cells were let to invade for 24 h (N = 3, *P<0.05 compared with control cells).</p

Adrenomedullin effect is mediated by TRPV2.

<p>(A) Western-blotting analysis of TRPV2 protein level in PC-3 and T24/83 cells treated with either siCTL or siTRPV2 (50 nM, 48 h). Effect of TRPV2 silencing (siTRPV2, 50 nM, 48 h) (B) on PC-3 and T24/83 cell adhesion to fibroncectin incubated or not with AM (200 nM, 45 min) (N = 3, *P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM); (C) on PC-3 and T24/83 cell migration examined by transwell assay after 8 h incubation with or without AM (N = 3 *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM); (D) on PC-3 and T24/83 cell invasion through matrigel (AM 200 nM, 24 h) (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM).</p

TRPV6 calcium channel translocates to the plasma membrane via Orai1-mediated mechanism and controls cancer cell survival

Transient receptor potential vanilloid subfamily member 6 (TRPV6) is a highly selective calcium channel that has been considered as a part of store-operated calcium entry (SOCE). Despite its first discovery in the early 2000s, the role of this channel in prostate cancer (PCa) remained, until now, obscure. Here we show that TRPV6 mediates calcium entry, which is highly increased in PCa due to the remodeling mechanism involving the translocation of the TRPV6 channel to the plasma membrane via the Orai1/TRPC1-mediated Ca2+/Annexin I/S100A11 pathway, partially contributing to SOCE. The TRPV6 calcium channel is expressed de novo by the PCa cell to increase its survival by enhancing proliferation and conferring apoptosis resistance. Xenografts in nude mice and bone metastasis models confirmed the remarkable aggressiveness of TRPV6-overexpressing tumors. Immunohistochemical analysis of these demonstrated the increased expression of clinical markers such as Ki-67, prostate specific antigen, synaptophysin, CD31, and CD56, which are strongly associated with a poor prognosis. Thus, the TRPV6 channel acquires its oncogenic potential in PCa due to the remodeling mechanism via the Orai1-mediated Ca2+/Annexin I/S100A11 pathway.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

International audienceTRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity