Natalia Prevarskaya

Calcium in tumour metastasis: new roles for known actors.

In most cases, metastasis, not the primary tumour per se, is the main cause of mortality in cancer patients. In order to effectively escape the tumour, enter the circulation and establish secondary growth in distant organs cancer cells must develop an enhanced propensity to migrate. The ubiquitous second messenger Ca2+ is a crucial regulator of cell migration. Recently, a number of known molecular players in cellular Ca2+ homeostasis, including calcium release-activated calcium channel protein 1 (ORAI1), stromal interaction molecule 1 (STIM1) and transient receptor potential (TRP) channels, have been implicated in tumour cell migration and the metastatic cell phenotype. We discuss how these developments have increased our understanding of the Ca2+ dependence of pro-metastatic behaviours.

Bcl-2-dependent modulation of Ca2+ homeostasis and store-operated channels in prostate cancer cells

Antiapoptotic oncoprotein Bcl-2 has extramitochondrial actions due to its localization on the endoplasmic reticulum (ER); however, the specific mechanisms of such actions remain unclear. Here we show that Bcl-2 overexpression in LNCaP prostate cancer epithelial cells results in downregulation of store-operated Ca(2+) current by decreasing the number of functional channels and inhibiting ER Ca(2+) uptake through a reduction in the expression of calreticulin and SERCA2b, two key proteins controlling ER Ca(2+) content. Furthermore, we demonstrate that Ca(2+) store depletion by itself is not sufficient to induce apoptosis in Bcl-2 overexpressing cells, and that sustained Ca(2+) entry via activated store-operated channels (SOCs) is required as well. Our data therefore suggest the pivotal role of SOCs in apoptosis and cancer progression.

Ion channels and the hallmarks of cancer

Plasma membrane (PM) ion channels contribute to virtually all basic cellular processes and are also involved in the malignant phenotype of cancer cells. Here, we review the role of ion channels in cancer in the context of their involvement in the defined hallmarks of cancer: 1) self-sufficiency in growth signals, 2) insensitivity to antigrowth signals, 3) evasion of programmed cell death (apoptosis), 4) limitless replicative potential, 5) sustained angiogenesis and 6) tissue invasion and metastasis. Recent studies have indicated that the contribution of specific ion channels to these hallmarks varies for different types of cancer. Therefore, to determine the importance of ion channels as targets for cancer diagnosis and treatment their expression, function and regulation must be assessed for each cancer.

TRPV6 channel controls prostate cancer cell proliferation via Ca(2+)/NFAT-dependent pathways.

The transient receptor potential channel, subfamily V, member 6 (TRPV6), is strongly expressed in advanced prostate cancer and significantly correlates with the Gleason >7 grading, being undetectable in healthy and benign prostate tissues. However, the role of TRPV6 as a highly Ca2+-selective channel in prostate carcinogenesis remains poorly understood. Here, we report that TRPV6 is directly involved in the control of prostate cancer cell (LNCaP cell line) proliferation by decreasing: (i) proliferation rate; (ii) cell accumulation in the S-phase of cell cycle and (iii) proliferating cell nuclear antigen (PCNA) expression. We demonstrate that the Ca2+ uptake into LNCaP cells is mediated by TRPV6, with the subsequent downstream activation of the nuclear factor of activated T-cell transcription factor (NFAT). TRPV6-mediated Ca2+ entry is also involved in apoptosis resistance of LNCaP cells. Our results suggest that TRPV6 expression in LNCaP cells is regulated by androgen receptor, however, in a ligand-independent manner. We conclude that the upregulation of TRPV6 Ca2+ channel in prostate cancer cells may represent a mechanism for maintaining a higher proliferation rate, increasing cell survival and apoptosis resistance as well.

Functional implications of calcium permeability of the channel formed by pannexin 1

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.

Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation

Store‐operated calcium entry (SOCE) is the main Ca2+ influx pathway involved in controlling proliferation of the human hepatoma cell lines Huh‐7 and HepG2. However, the molecular nature of the calcium channels involved in this process remains unknown. Huh‐7 and HepG2 cells express transient receptor potential canonical 1 (TRPC1) and TRPC6, as well as STIM1 and Orai1, and these 4 channels are the most likely candidates to account for the SOCE in these cells. We generated stable TRPC6‐overexpressing or TRPC6‐knockdown Huh‐7 clones, in which we investigated correlations between the presence of the protein, the rate of cell proliferation, and SOCE amplitude. TRPC6‐overexpressing Huh‐7 cells proliferated 80% faster than did untransfected cells and their SOCE amplitude was 160% higher. By contrast, proliferation rate was 50% lower and SOCE amplitude 85% lower in TRPC6‐knockdown clones than in untransfected cells. OAG (olyl acetyl glycerol)‐induced calcium entry was similar in all cells, and small interfering RNA (siRNA) against TRPC1 had no effect on SOCE amplitude, highlighting the relationship among SOCE, TRPC6 and cell proliferation in Huh‐7 cells. SOCE amplitude was reduced by STIM1 and Orai1 knockdowns, suggesting possible cooperation between these proteins and TRPC6 in these cells. Endothelial growth factor and hepatocyte growth factor increased TRPC6 expression and SOCE amplitude in Huh‐7 cells, and cyclin D1 expression was decreased by STIM1, Orai1, and TRPC6 knockdowns. Conclusion: TRPC6 was very weakly expressed in isolated hepatocytes from healthy patients and expressed more strongly in tumoral samples from the liver of a cancer patient, strongly supporting a role for these calcium channels in liver oncogenesis. (HEPATOLOGY 2008;47:2068–2077.)

Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function.

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.

Overexpression of an α1H (Cav3.2) T-type Calcium Channel during Neuroendocrine Differentiation of Human Prostate Cancer Cells

Neuroendocrine differentiation of prostate epithelial cells is usually associated with an increased aggressivity and invasiveness of prostate tumors and a poor prognosis. However, the molecular mechanisms involved in this process remain poorly understood. We have investigated the possible expression of voltage-gated calcium channels in human prostate cancer epithelial LNCaP cells and their modulation during neuroendocrine differentiation. A small proportion of undifferentiated LNCaP cells displayed a voltage-dependent calcium current. This proportion and the calcium current density were significantly increased during neuroendocrine differentiation induced by long-term treatments with cyclic AMP permeant analogs or with a steroid-reduced culture medium. Biophysical and pharmacological properties of this calcium current suggest that it is carried by low-voltage activated T-type calcium channels. Reverse transcriptase-PCR experiments demonstrated that only a single type of LVA calcium channel mRNA, an α1H calcium channel mRNA, is expressed in LNCaP cells. Quantitative real-time reverse transcriptase-PCR revealed that α1H mRNA was overexpressed during neuroendocrine differentiation. Finally, we show that this calcium channel promotes basal calcium entry at resting membrane potential and may facilitate neurite lengthening. This voltage-dependent calcium channel could be involved in the stimulation of mitogenic factor secretion and could therefore be a target for future therapeutic strategies.

Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells.

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 (Icold/menthol) that shows inward rectification and high Ca2+ selectivity, which are dramatically different properties from “classical” TRPM8-mediated Icold/menthol. Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both Icold/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 Ca2+ 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 Ca2+ release channel, potentially involved in a number of Ca2+- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.

Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis

1 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. 2 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). 3 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. 4 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). 5 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. 6 The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA‐AM). 7 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.