Castration resistance in prostate cancer (PCa) constitutes an advanced, aggressive disease with poor prognosis, associated with uncontrolled cell proliferation, resistance to apoptosis, and enhanced invasive potential. The molecular mechanisms involved in the transition of PCa to castration resistance are obscure. Here, we report that the nonselective cationic channel transient receptor potential vanilloid 2 (TRPV2) is a distinctive feature of castration-resistant PCa. TRPV2 transcript levels were higher in patients with metastatic cancer (stage M1) compared with primary solid tumors (stages T2a and T2b). Previous studies of the TRPV2 channel indicated that it is primarily involved in cancer cell migration and not in cell growth. Introducing TRPV2 into androgen-dependent LNCaP cells enhanced cell migration along with expression of invasion markers matrix metalloproteinase (MMP) 9 and cathepsin B. Consistent with the likelihood that TRPV2 may affect cancer cell aggressiveness by influencing basal intracellular calcium levels, small interfering RNA-mediated silencing of TRPV2 reduced the growth and invasive properties of PC3 prostate tumors established in nude mice xenografts, and diminished expression of invasive enzymes MMP2, MMP9, and cathepsin B. Our findings establish a role for TRPV2 in PCa progression to the aggressive castration-resistant stage, prompting evaluation of TRPV2 as a potential prognostic marker and therapeutic target in the setting of advanced PCa. Cancer Res; 70(3); 1225-35. ©2010 AACR.

The molecular nature of calcium (Ca2+)-dependent mechanisms and the ion channels having a major role in the apoptosis of cancer cells remain a subject of debate. Here, we show that the recently identified Orai1 protein represents the major molecular component of endogenous store-operated Ca2+ entry (SOCE) in human prostate cancer (PCa) cells, and constitutes the principal source of Ca2+ influx used by the cell to trigger apoptosis. The downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways, such as those induced by thapsigargin (Tg), tumor necrosis factor α, and cisplatin/oxaliplatin. The transfection of functional Orai1 mutants, such as R91W, a selectivity mutant, and L273S, a coiled-coil mutant, into the cells significantly decreased both SOCE and the rate of Tg-induced apoptosis. This suggests that the functional coupling of STIM1 to Orai1, as well as Orai1 Ca2+-selectivity as a channel, is required for its pro-apoptotic effects. We have also shown that the apoptosis resistance of androgen-independent PCa cells is associated with the downregulation of Orai1 expression as well as SOCE. Orai1 rescue, following Orai1 transfection of steroid-deprived cells, re-established the store-operated channel current and restored the normal rate of apoptosis. Thus, Orai1 has a pivotal role in the triggering of apoptosis, irrespective of apoptosis-inducing stimuli, and in the establishment of an apoptosis-resistant phenotype in PCa cells.

In recent years, the transient receptor potential melastatin member 8 (TRPM8) channel has emerged as a promising prognostic marker and putative therapeutic target in prostate cancer (PCa). However, the mechanisms of prostate-specific regulation and functional evolution of TRPM8 during PCa progression remain unclear. Here we show, for the first time to our knowledge, that only secretory mature differentiated human prostate primary epithelial (PrPE) luminal cells expressed functional plasma membrane TRPM8 ( PM TRPM8) channels. Moreover, PCa epithelial cells obtained from in situ PCa were characterized by a significantly stronger PM TRPM8-mediated current than that in normal cells. This PM TRPM8 activity was abolished in dedifferentiated PrPE cells that had lost their luminal secretory phenotype. However, we found that in contrast to PM TRPM8, endoplasmic reticulum TRPM8 ( ER TRPM8) retained its function as an ER Ca 2+ release channel, independent of cell differentiation. We hypothesize that the constitutive activity of ER TRPM8 may result from the expression of a truncated TRPM8 splice variant. Our study provides insight into the role of TRPM8 in PCa progression and suggests that TRPM8 is a potentially attractive target for therapeutic intervention: specific inhibition of either ER TRPM8 or PM TRPM8 may be useful, depending on the stage and androgen sensitivity of the targeted PCa.

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I cold/menthol ) that shows inward rectification and high Ca 2؉ selectivity, which are dramatically different properties from "classical" TRPM8-mediated I cold/menthol . Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I cold/menthol and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca 2؉ release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca 2؉ release channel, potentially involved in a number of Ca 2؉ -and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis. Mammalian homologues of the Drosophila transient receptor potential (TRP)7 channel, which initially emerged as a channel specifically linked to phospholipase C-catalyzed inositol phospholipid breakdown signaling pathways, have now grown into a broad family of channelforming proteins displaying extraordinarily diverse activation mechanisms (for reviews, see Refs. 1-5). At present, these channels are grouped into six subfamilies based on structural homology and have been given a standard nomenclature (5).A number of mammalian TRPs show a unique mode of gating, in response to thermal stimuli as well as to the chemical imitators of burning and cooling sensations, capsaicin and menthol, respectively. As such, they represent a group of thermal receptors covering a wide range of physiological temperatures. Most thermal receptors belong to the vanilloid TRP subfamily (TRPV, Ref. 6) including warm-sensitive (Ͻ40°C) TRPV3 (7-9) and heat-and capsaicin-sensitive TRPV1 (Ͼ43°C) (10) and TRPV2 (Ͼ52°C) (11). In contrast, sensitivity to cooling temperatures (Ͻ22°C) and menthol is mediated by a structurally distant thermal receptor, TRPM8, belonging to the melastatine (TRPM) subfamily of TRP channels (12, 13); the ankyrin transmembrane protein 1 (ANKTM1 or TRPA1) is involved in the detection of noxious cold (14).Consistent with their role in the sensation of distinct physiological temperatures, thermal receptors are mostly expressed in subsets of...

The endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca(2+) 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 Ca(2+) homeostasis, and the ER Ca(2+) store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca(2+) leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca(2+) depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca(2+) 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 Ca(2+) permeability and subsequent ER stress can lead to development of a novel therapeutic approach.

Ahidouch, Halima Ouadid, Morad Roudbaraki, Philippe Delcourt, Ahmed Ahidouch, Nathalie Joury, and Natalia Prevarskaya. Functional and molecular identification of intermediate-conductance Ca 2ϩ -activated K ϩ channels in breast cancer cells: association with cell cycle progression. Am J Physiol Cell Physiol 287: C125-C134, 2004. First published February 25, 2004 10.1152/ ajpcell.00488.2003.-We have previously reported that the hEAG K ϩ channels are responsible for the potential membrane hyperpolarization that induces human breast cancer cell progression into the G1 phase of the cell cycle. In the present study, we evaluate the role and functional expression of the intermediate-conductance Ca 2ϩ -activated K ϩ channel, hIK1-like, in controlling cell cycle progression. Our results demonstrate that hIK1 current density increased in cells synchronized at the end of the G1 or S phase compared with those in the early G1 phase. This increased current density paralleled the enhancement in hIK1 mRNA levels and the highly negative membrane potential. Furthermore, in cells synchronized at the end of G1 or S phases, basal cytosolic Ca 2ϩ concentration ([Ca 2ϩ ]i) was also higher than in cells arrested in early G1. Blocking hIK1 channels with a specific blocker, clotrimazole, induced both membrane potential depolarization and a decrease in the [Ca 2ϩ ]i in cells arrested at the end of G1 and S phases but not in cells arrested early in the G1 phase. Blocking hIK1 with clotrimazole also induced cell proliferation inhibition but to a lesser degree than blocking hEAG with astemizole. The two drugs were essentially additive, inhibiting MCF-7 cell proliferation by 82% and arresting Ͼ90% of cells in the G1 phase. Thus, although the progression of MCF-7 cells through the early G1 phase is dependent on the activation of hEAG K ϩ channels, when it comes to G1 and checkpoint G1/S transition, the membrane potential appears to be primarily dependent on the hIK1-activity level. breast cancer; calcium-activated potassium channels; proliferation THERE IS GOOD EVIDENCE from several cell lines that membrane potential in the early G1 phase is depolarized, and the progression through G1 into the S phase is accompanied by a hyperpolarization of the membrane potential. The blockade of K ϩ

One major clinical problem with prostate cancer is the cells' ability to survive and proliferate upon androgen withdrawal. Because Ca 2+ is central to growth control, understanding the mechanisms of Ca 2+ homeostasis involved in prostate cancer cell proliferation is imperative for new therapeutic strategies. Here, we show that agonist-mediated stimulation of A 1 -adrenergic receptors (A 1 -AR) promotes proliferation of the primary human prostate cancer epithelial (hPCE) cells by inducing store-independent Ca 2+ entry and subsequent activation of nuclear factor of activated T cells (NFAT) transcription factor. Such an agonist-induced Ca 2+ entry (ACE) relied mostly on transient receptor potential canonical 6 (TRPC6) channels, whose silencing by antisense hybrid depletion decreased both hPCE cell proliferation and ACE. In contrast, ACE and related growth arrest associated with purinergic receptors (P2Y-R) stimulation involved neither TRPC6 nor NFAT. Our findings show that A 1 -AR signaling requires the coupled activation of TRPC6 channels and NFAT to promote proliferation of hPCE cells and thereby suggest TRPC6 as a novel potential therapeutic target.

Accumulating data point to K þ channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K þ channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IK Ca1 ) in human PCa as well as their involvement in cell proliferation. We showed that IK Ca1 mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IK Ca1 potassium channel suppressed PCa cell proliferation. The activation of IK Ca1 hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IK Ca1 channel is likely to promote carcinogenesis in human prostate tissue.