ORAI family channels have emerged as important players in malignant transformation, yet the way in which they reprogram cancer cells remains elusive. Here we show that the relative expression levels of ORAI proteins in prostate cancer are different from that in noncancerous tissue. By mimicking ORAI protein remodeling observed in primary tumors, we demonstrate in in vitro models that enhanced ORAI3 expression favors heteromerization with ORAI1 to form a novel channel. These channels support store-independent Ca(2+) entry, thereby promoting cell proliferation and a smaller number of functional homomeric ORAI1-based store-operated channels, which are important in supporting susceptibility to apoptosis. Thus, our findings highlight disrupted dynamic equilibrium of channel-forming proteins as an oncogenic mechanism.

Although human pannexins (PanX) are homologous to gap junction molecules, their physiological function in vertebrates remains poorly understood. Our results demonstrate that overexpression of PanX1 results in the formation of Ca2+-permeable gap junction channels between adjacent cells, thus, allowing direct intercellular Ca2+ diffusion and facilitating intercellular Ca2+ wave propagation. More intriguingly, our results strongly suggest that PanX1 may also form Ca2+-permeable channels in the endoplasmic reticulum (ER). These channels contribute to the ER Ca2+ leak and thereby affect the ER Ca2+ load. Because leakage remains the most enigmatic of those processes involved in intracellular calcium homeostasis, and the molecular nature of the leak channels is as yet unknown, the results of this work provide new insight into calcium signaling mechanisms. These results imply that for vertebrates, a new protein family, referred to as pannexins, may not simply duplicate the connexin function but may also provide additional pathways for intra- and intercellular calcium signaling and homeostasis.

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.

The physiological role, the mechanisms of activation, as well as the endogenous regulators for the non-selective cationic channel TRPV2 are not known so far. In the present work we report that endogenous lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) induce a calcium influx via TRPV2 channel. This activation is dependent on the length of the side-chain and the nature of the lysophospholipid head-group. TRPV2-mediated calcium uptake stimulated by LPC and LPI occurred via Gq/Go-protein and phosphatidylinositol-3,4 kinase (PI3,4K) signalling. We have shown that the mechanism of TRPV2 activation induced by LPC and LPI is due to the TRPV2 channel translocation to the plasma membrane. The activation of TRPV2 channel by LPC and LPI leads to an increase in the cell migration of the prostate cancer cell line PC3. We have demonstrated that TRPV2 is directly involved in both steady-state and lysophospholipid-stimulated cancer cell migration. Thus, for the first time, we have identified one of the natural regulators of TRPV2 channel, one of the mechanisms of TRPV2 activation and regulation, as well as its pathophysiological role in cancer.

Under resting conditions, the endoplasmic reticulum (ER) intraluminal free calcium concentration ([Ca2+]ER) reflects a balance between active uptake by Ca2+-ATPases and passive efflux via `leak channels'. Despite their physiological importance and ubiquitous leak pathway mechanism, very little is known about the molecular nature of these channels. As it has been suggested that the open translocon pore complex of the ER is permeable to ions and neutral molecules, we hypothesized that the ribosome-bound translocon would be permeable to calcium after treatment with puromycin, a translation inhibitor that specifically releases polypeptide chains. At this time, the translocon channel is left open. We measured the fluctuations in cytoplasmic and luminal calcium concentrations using fluorescent dyes (fura-2 and magfura-2, respectively). The calcium release induced by thapsigargin (a Ca2+-ATPase inhibitor) was lower after puromycin treatment. Puromycin also reduced the [Ca2+]ER level when perfused into the medium, but was ineffective after anisomycin pre-treatment (an inhibitor of the peptidyl transferase). Puromycin had a similar effect in the presence of heparin and ryanodine. This puromycin-evoked [Ca2+]ER decrease was specific to the translocon. We conclude that the translocon complex is a major calcium leak channel. This work reveals a new role for the translocon which is involved in the control of the [Ca2+]ER and could therefore supervise many physiological processes, including gene expression and apoptosis.

Neuroendocrine (NE) differentiation is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. NE tumor cells are nonproliferating and escape apoptotic cell death; therefore, an understanding of the apoptotic status of the NE phenotype is imperative for the development of new therapies for prostate cancer. Here, we report for the first time on alterations in intracellular Ca 2 þ homeostasis, which is a key factor in apoptosis, caused by NE differentiation of androgen-dependent prostate cancer epithelial cells. NEdifferentiating regimens, either cAMP elevation or androgen deprivation, resulted in a reduced endoplasmic reticulum Ca 2 þ -store content due to both SERCA 2b Ca 2 þ ATPase and luminal Ca 2 þ binding/storage chaperone calreticulin underexpression, and to a downregulated store-operated Ca 2 þ current. NE-differentiated cells showed enhanced resistance to thapsigargin-and TNF-a-induced apoptosis, unrelated to antiapoptotic Bcl-2 protein overexpression. Our results suggest that targeting the key players determining Ca 2 þ homeostasis in an attempt to enhance the proapoptotic potential of malignant cells may prove to be a useful strategy in the treatment of advanced prostate cancer.

In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+‐ATPase inhibitor thapsigargin (TG), in androgen‐sensitive human prostate cancer LNCaP cells. Exposure of fura‐2‐loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store‐operated Ca2+ channels (SOCs). For the first time we have identified and characterized the SOC‐mediated membrane current (Istore) in prostate cells using whole‐cell, cell‐attached, and perforated patch‐clamp techniques, combined with fura‐2 microspectrofluorimetric and Ca2+‐imaging measurements. I store in LNCaP cells lacked voltage‐dependent gating and displayed an inwardly rectifying current‐voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 ± 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5–3 mM) and La3+ (1 μM). Treatment of LNCaP cells with TG (0.1 μM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG‐induced apoptosis. The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA‐AM). These results indicate that in androgen‐sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store‐activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.

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 Ca 2+