TRPV6 Determines the Effect of Vitamin D3 on Prostate Cancer Cell Growth

Despite remarkable advances in the therapy and prevention of prostate cancer it is still the second cause of death from cancer in industrialized countries. Many therapies initially shown to be beneficial for the patients were abandoned due to the high drug resistance and the evolution rate of the tumors. One of the prospective therapeutical agents even used in the first stage clinical trials, 1,25-dihydroxyvitamin D3, was shown to be either unpredictable or inefficient in many cases. We have already shown that TRPV6 calcium channel, which is the direct target of 1,25-dihydroxyvitamin D3 receptor, positively controls prostate cancer proliferation and apoptosis resistance (Lehen'kyi et al., Oncogene, 2007). However, how the known 1,25-dihydroxyvitamin D3 antiproliferative effects may be compatible with the upregulation of pro-oncogenic TRPV6 channel remains a mystery. Here we demonstrate that in low steroid conditions 1,25-dihydroxyvitamin D3 upregulates the expression of TRPV6, enchances the proliferation by increasing the number of cells entering into S-phase. We show that these pro-proliferative effects of 1,25-dihydroxyvitamin D3 are directly mediated via the overexpression of TRPV6 channel which increases calcium uptake into LNCaP cells. The apoptosis resistance of androgen-dependent LNCaP cells conferred by TRPV6 channel is drastically inversed when 1,25-dihydroxyvitamin D3 effects were combined with the successful TRPV6 knockdown. In addition, the use of androgen-deficient DU-145 and androgen-insensitive LNCaP C4-2 cell lines allowed to suggest that the ability of 1,25-dihydroxyvitamin D3 to induce the expression of TRPV6 channel is a crucial determinant of the success or failure of 1,25-dihydroxyvitamin D3-based therapies

Role of calcium and TRP channels in prostate and bladder cancer cells growth, migration and response to reticular stress

Les cellules cancéreuses acquièrent lors de la cancérogenèse des caractéristiques telles qu’une insensibilité aux signaux antiprolifératifs, une résistance à l‘apoptose et au stress réticulaire, l’acquisition d’un pouvoir invasif. De nombreux travaux montrent l’implication du calcium dans ces différentes caractéristiques. Selon des données récentes, les canaux de la famille TRP (Transient Receptor Potential) sont des acteurs clefs dans l'homéostasie calcique. Plusieurs travaux ont montré que les changements d’expression de certains canaux TRP pouvaient contribuer à la cancérisation, entre autres TRPV6 et TRPV2 dans les cancers de prostate et de vessie. Nous montrons ici que la vitamine D3, étudiée depuis longtemps dans le cadre de thérapie anticancéreuse pour ses pouvoirs anti-prolifératifs, peut aussi stimuler la prolifération des cellules prostatiques cancéreuses, en augmentant l’expression de TRPV6 et le Ca2+ entrant par ce canal. Ainsi, nos résultats remettent en cause l’utilisation de la vitamine D3 en thérapie. Nous montrons également que l’adrénomédulline (AM), connue pour être impliquée dans la progression tumorale vers les métastases, stimule la migration et l’invasion des cellules cancéreuses prostatiques et urothéliales. Cet effet nécessite la présence du canal TRPV2; l’AM agit en induisant la translocation à la membrane plasmique du canal TRPV2 et de ce fait en augmentant la [Ca2+]i. Enfin, nous mettons en évidence que dans des conditions de stress réticulaire dans les cellules prostatiques cancéreuses, le translocon est le canal de fuite calcique majoritaire du réticulum endoplasmique menant à la vidange des stocks réticulaires et à la mort cellulaire.Cancer cells acquire during carcinogenesis some characteristics such as insensitivity to the antiproliferative signals, resistance to apoptosis and reticular stress, the acquisition of invasive capacity. Numerous studies show involvement of calcium (Ca2+) in these different characteristics. According to recent data, the TRP channels (Transient Receptor Potential) are key players in calcium homeostasis. Several recent works have shown that changes of expression of some TRP channels could contribute to carcinogenesis, among other TRPV6 and TRPV2 in prostate and bladder cancers. Here we show that vitamin D3, studied for many years in cancer therapy for its anti-proliferative capacity, could also stimulates prostate cancer cells proliferation, this effect being associated with overexpression of TRPV6 and the subsequent increase in the Ca2+ entering through this channel. Thus, our results challenge the use of vitamin D3 in prostate cancer therapy. We also show that adrenomedullin (AM), known to be involved in tumor progression to metastasis, stimulates migration and invasion of prostate and urothelial cancer cells. This effect requires the presence of TRPV2; AM acts by inducing TRPV2 translocation to plasmamembrane and the subsequent increase of [Ca2+]i. Finally, we highlight that under conditions of reticular stress in prostate cancer cells, the translocon represents the endoplasmic reticulum calcium leak channel leading to the emptying of reticular stocks and cell death

Rôles du calcium et des canaux TRP dans la croissance, la migration et la réponse au stress réticulaire des cellules cancéreuses prostatiques et urothéliales

Les cellules cancéreuses acquièrent lors de la cancérogenèse des caractéristiques telles qu une insensibilité aux signaux antiprolifératifs, une résistance à l apoptose et au stress réticulaire, l acquisition d un pouvoir invasif. De nombreux travaux montrent l implication du calcium dans ces différentes caractéristiques. Selon des données récentes, les canaux de la famille TRP (Transient Receptor Potential) sont des acteurs clefs dans l'homéostasie calcique. Plusieurs travaux ont montré que les changements d expression de certains canaux TRP pouvaient contribuer à la cancérisation, entre autres TRPV6 et TRPV2 dans les cancers de prostate et de vessie. Nous montrons ici que la vitamine D3, étudiée depuis longtemps dans le cadre de thérapie anticancéreuse pour ses pouvoirs anti-prolifératifs, peut aussi stimuler la prolifération des cellules prostatiques cancéreuses, en augmentant l expression de TRPV6 et le Ca2+ entrant par ce canal. Ainsi, nos résultats remettent en cause l utilisation de la vitamine D3 en thérapie. Nous montrons également que l adrénomédulline (AM), connue pour être impliquée dans la progression tumorale vers les métastases, stimule la migration et l invasion des cellules cancéreuses prostatiques et urothéliales. Cet effet nécessite la présence du canal TRPV2; l AM agit en induisant la translocation à la membrane plasmique du canal TRPV2 et de ce fait en augmentant la [Ca2+]i. Enfin, nous mettons en évidence que dans des conditions de stress réticulaire dans les cellules prostatiques cancéreuses, le translocon est le canal de fuite calcique majoritaire du réticulum endoplasmique menant à la vidange des stocks réticulaires et à la mort cellulaire.Cancer cells acquire during carcinogenesis some characteristics such as insensitivity to the antiproliferative signals, resistance to apoptosis and reticular stress, the acquisition of invasive capacity. Numerous studies show involvement of calcium (Ca2+) in these different characteristics. According to recent data, the TRP channels (Transient Receptor Potential) are key players in calcium homeostasis. Several recent works have shown that changes of expression of some TRP channels could contribute to carcinogenesis, among other TRPV6 and TRPV2 in prostate and bladder cancers. Here we show that vitamin D3, studied for many years in cancer therapy for its anti-proliferative capacity, could also stimulates prostate cancer cells proliferation, this effect being associated with overexpression of TRPV6 and the subsequent increase in the Ca2+ entering through this channel. Thus, our results challenge the use of vitamin D3 in prostate cancer therapy. We also show that adrenomedullin (AM), known to be involved in tumor progression to metastasis, stimulates migration and invasion of prostate and urothelial cancer cells. This effect requires the presence of TRPV2; AM acts by inducing TRPV2 translocation to plasmamembrane and the subsequent increase of [Ca2+]i. Finally, we highlight that under conditions of reticular stress in prostate cancer cells, the translocon represents the endoplasmic reticulum calcium leak channel leading to the emptying of reticular stocks and cell death.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

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

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

Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78

International audienceThe endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca2+ homeostasis. The ability of cells to respond to the ER stress is critical for cell survival, and disruption in such regulation can lead to apoptosis. ER stress is accompanied by alterations in Ca2+ homeostasis, and the ER Ca2+ store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca2+ leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca2+ depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca2+ leak that can be prevented by translocon inhibitor, anisomycin. Expression of GRP78, an ER stress marker, increased following treatment with puromycin (a translocon opener) and was suppressed by anisomycin, confirming a primary role of translocon in ER stress induction. Inhibition of ER store depletion by anisomycin significantly reduces apoptosis stimulated by the ER stress inducers. We suggest that translocon opening is physiologically modulated by GRP78, particularly during the ER stress. The ability to modulate the ER Ca2+ permeability and subsequent ER stress can lead to development of a novel therapeutic approach

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