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

Calcium signalling in the epidermis homeostasis and during aging : implication in cancer

L’épiderme est la partie superficielle de la peau. Ce tissu, parfaitement organisé, est principalement composé de kératinocytes dont la prolifération dans la couche basale garantit le renouvellement constant des cellules supérieures qui suivent le processus de différenciation terminale et viennent desquamer en surface. Cela assure l’homéostasie de l’épiderme. Toute dermatose est liée à une dérégulation de la balance prolifération/différenciation des kératinocytes dont l’incidence, pour certaines, augmente avec l’âge. Or, ces deux processus sont fortement régulés par le calcium qui est présent dans l’épiderme sous forme d’un gradient croissant vers la surface.Nous avons suivi l’expression de 90 gènes, impliqués dans la signalisation calcique, au cours du vieillissement de l’épiderme. Grâce à nos collaborations, nous avons obtenu une vingtaine d’échantillons de peau provenant de sujets sains de 19 à 70 ans. La même a été réalisée à partir de cultures primaires de kératinocytes humains afin de déterminer l’expression spécifique de ces gènes dans la différenciation induite par le calcium ou la sénescence réplicatives des kératinocytes. Ensuite, le rôle du canal Orai1 a plus particulièrement été décortiqué dans la physiologie de l’épiderme et nous avons montré que celui-ci maintient les kératinocytes à l’état indifférencié prolifératif et intervient dans la migration de ces cellules. Enfin, nous avons examiné l’implication des canaux Orai1 et TRPV6 dans la progression d’un cancer épithélial, le cancer de la prostate, que nous pourrions maintenant transposer dans les cancers cutanés baso- et spino-cellulaires.Epidermis is the upper part of the skin. This tissue is highly organized and mainly composed of keratinocytes able to divide in the basal layer assuring the constant cell supply moving upwards while entering the terminal differentiation and finally leaving the skin. The impairment of this tiny balance between proliferation and differentiation will definitely lead to skin disorders. The appearance of most skin disorders increases with age as well as the incidence of skin cancer such as basal and squamous cell carcinomas. Moreover, since both processes are highly dependent on calcium, the existence of calcium gradient has been reported in the skin and the crucial role of calcium was demonstrated in vitro. We have thus studied the expression of 90 genes involved in calcium signalling during epidermis aging in order to identify the prospective target genes. Our collaborations have given us the possibility to obtain a cohort of 20 healthy human subjects from 19 to 70 years old. The appropriate in vitro model was also established using primary human keratinocytes to study the gene expression during calcium-induced differentiation and replicative senescence. Further, the role of Orai1 channel in epidermis physiology has been studied in detail showing its role in maintening keratinocytes under the undifferentiated and proliferative state with its crucial role in migration. Finally, we have demonstrated the involvement of both Orai1 and TRPV6 calcium channels in cancer using a model of such epithelial cancer as prostate adenocarcinoma. This data will be employed in the future study for basal and squamous cell carcinomas

The role of the TRPV6 channel in cancer

International audienceAbstract The TRPV6 channel belongs to the superfamily of transient receptor potential (TRP) channels, subfamily vanilloid, member 6. Its expression in health is mainly confined to epithelial tissue of different organs such as digestive tract, kidney, testis, ovaries and skin. Due to its high calcium selectivity over other TRP channels, this channel was shown to participate in close regulation of calcium homeostasis in the body. In cancer a number of pieces of evidence demonstrate its upregulation and correlation with the advanced stages in prostate, colon, breast, thyroid, and ovarian carcinomas. Little is known about its role in initiation or progression for most of cancers, though in prostate cancer its oncogenic potential in vitro has been suggested. The most probable mechanisms involve calcium signalling in the control of processes such as proliferation and apoptosis resistance, though in some cases first evidence was reported as to its likely protective role in some cancers such as colon cancer. Further studies are needed to confirm whether this channel does really have an oncogenic potential or is just the last hope for transformed cells/tissues to stop cancer

Cytoskeleton Reorganization as an Alternative Mechanism of Store-Operated Calcium Entry Control in Neuroendocrine-Differentiated Cells

International audienceNeuroendocrine differentiation (NED) is a hallmark of advanced androgen-independent prostate cancer, for which no successful therapy exists. NED tumour cells escape apoptotic cell death by alterations of Ca(2+) homeostasis where the store-operated Ca(2+) entry (SOCE) is known to be a key event. We have previously shown that the downregulation of Orai1 protein representing the major molecular component of endogenous SOCE in human prostate cancer cells, and constituting the principal source of Ca(2+) influx used by the cell to trigger apoptosis, contributes to the establishment of an apoptosis-resistant phenotype (Cell Death Dis. 2010 Sep 16;1:e75.). Here, we report for the first time that the decrease of SOCE during NED may be caused by alternative NED-induced mechanism involving cytoskeleton reorganisation. NED induced by androgen deprivation resulted in a decrease of SOCE due to cortical F-actin over-polymerization which inhibits thapsigargin-induced SOCE. The disruption of F-actin polymerization by Cytochalasin D in NED cells restored SOCE, while the induction of F-actin polymerization by jasplakinolide or calyculin A diminished SOCE without changing the expression of key SOCE players: Orai1, STIM1, and TRPC1. Our data suggest that targeting cytoskeleton-induced pathways of malignant cells together with SOCE-involved channels may prove a useful strategy in the treatment of advanced prostate cancer

Cytoskeleton Reorganization as an Alternative Mechanism of Store-Operated Calcium Entry Control in Neuroendocrine-Differentiated Cells

Neuroendocrine differentiation (NED) is a hallmark of advanced androgen-independent prostate cancer, for which no successful therapy exists. NED tumour cells escape apoptotic cell death by alterations of Ca(2+) homeostasis where the store-operated Ca(2+) entry (SOCE) is known to be a key event. We have previously shown that the downregulation of Orai1 protein representing the major molecular component of endogenous SOCE in human prostate cancer cells, and constituting the principal source of Ca(2+) influx used by the cell to trigger apoptosis, contributes to the establishment of an apoptosis-resistant phenotype (Cell Death Dis. 2010 Sep 16;1:e75.). Here, we report for the first time that the decrease of SOCE during NED may be caused by alternative NED-induced mechanism involving cytoskeleton reorganisation. NED induced by androgen deprivation resulted in a decrease of SOCE due to cortical F-actin over-polymerization which inhibits thapsigargin-induced SOCE. The disruption of F-actin polymerization by Cytochalasin D in NED cells restored SOCE, while the induction of F-actin polymerization by jasplakinolide or calyculin A diminished SOCE without changing the expression of key SOCE players: Orai1, STIM1, and TRPC1. Our data suggest that targeting cytoskeleton-induced pathways of malignant cells together with SOCE-involved channels may prove a useful strategy in the treatment of advanced prostate cancer

ORAI1 calcium channel orchestrates skin homeostasis

International audienceTo achieve and maintain skin architecture and homeostasis, keratinocytes must intricately balance growth, differentiation, and polarized motility known to be governed by calcium. Orai1 is a pore subunit of a store-operated Ca(2+) channel that is a major molecular counterpart for Ca(2+) influx in nonexcitable cells. To elucidate the physiological significance of Orai1 in skin, we studied its functions in epidermis of mice, with targeted disruption of the orai1 gene, human skin sections, and primary keratinocytes. We demonstrate that Orai1 protein is mainly confined to the basal layer of epidermis where it plays a critical role to control keratinocyte proliferation and polarized motility. Orai1 loss of function alters keratinocyte differentiation both in vitro and in vivo. Exploring underlying mechanisms, we show that the activation of Orai1-mediated calcium entry leads to enhancing focal adhesion turnover via a PKCβ-Calpain-focal adhesion kinase pathway. Our findings provide insight into the functions of the Orai1 channel in the maintenance of skin homeostasis

F-actin polymerization by calyculin A (CalA) in both control and NED-LNCaP cells.

<p>(<b>A</b>) Representative images of immunofluorescence staining of F-actin with phalloidin-FITC in NED- and NED-CalA (50 nM for 10 min)-LNCaP cells. n = 3. (<b>B</b>) Histograms representing Tg-induced SOCE quantification (Tg 1 µM, 2 mM extracellular Ca²⁺) in CT-, CT-CalA-, NED- and NED-CalA-LNCaP cells. n = 3, N = 30–40 cells, in triplicates. Asterisks denote statistical significance: * p<0.05.</p

Cytochalasine D (CytD) partially restores the store-operated current induced by TG.

<p>(<b>A</b>) The whole-cell electrophysiological recordings of SOCE induced by 1 µM TG in LNCaP cells control (CT), NED (NED), and NED LNCaP cells treated with 3 µM CytD (NED-CytD). (<b>B</b>) Histograms showing the current density quantifications in the above conditions, n = 3, N = 23; in triplicates, ** - p<0.01.</p

Regulation of Ca²⁺ homeostasis and F-actin polymerization in NED-LNCaP cells.

<p>After 4 (<b>A</b>) or one (<b>B</b>) days of culture in a charcoal-stripped culture medium used to induce NED, the capacitative Ca²⁺ entry quantified by Ca²⁺ imaging is induced by the application of 1 µM Tg in the presence of 2 mM extracellulaire CaCl_2. Asterisks denote statistical significance * - p<0.05; ** - p<0.01, n = 3, N = 30–40 cells, in triplicates. The presence of F-actin fibres was observed in both CT-LNCaP (<b>C</b>) and NED-LNCaP (<b>D</b>) using phalloidin-FITC. Scale bar equals 10 µM. n = 3. <b>E</b>, Expression of Orai1, STIM1, Orai2, and Orai3 as compared to β-Actin in LNCaP cells and NED-LNCaP cells using semiquantitative PCR. n = 2. <b>F</b>, Quantitative real-time PCR for Orai1 and STIM1 in LNCaP (CT) versus LNCaP-NED (NED), n = 3, * - P<0.05.</p