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, enhances 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.     

Reciprocal positive regulation between TRPV6 and NUMB in PTEN-deficient prostate cancer cells

Calcium acts as a second messenger and plays a crucial role in signaling pathways involved in cell proliferation. Recently, calcium channels related to calcium influx into the cytosol of epithelial cells have attracted attention as a cancer therapy target. Of these calcium channels, TRPV6 is overexpressed in prostate cancer and is considered an important molecule in the process of metastasis. However, its exact role and mechanism is unclear.     

Store-operated Ca2+ current and TRPV6 channels in lymph node prostate cancer cells

The contribution of endogenous and recombinant transient receptor potential vanilloid type 6 (TRPV6) channels to Ca2+ entry across the plasma membrane was studied in the human lymph node prostate cancer cell line (LNCaP). LNCaP cells do express the TRPV6 gene, and Ca2+ entry currents in these cells were detected after active and passive Ca2+ store depletion by intracellular application of inositol 1,4,5-trisphosphate, Ca2+ chelators, and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. This store-operated Ca2+ current (ISOC) had biophysical properties similar to those of the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukemia cells such as the activation mechanism, inward rectification, and Ca2+ selectivity. These properties are also shared by the Ca2+-sensing Ca2+ current (ITRPV6) recorded after heterologous expression of TRPV6 cDNA in human embryonic kidney and rat basophilic leukemia cells (B?dding, M., Wissenbach, U., Flockerzi, V. (2002) J. Biol. Chem. 277, 36656-36664). TRPV6 cDNA transfection of LNCaP cells restored recombinant ITRPV6, which can be distinguished from ISOC by the mechanism of activation, the voltage dependence of monovalent currents in the absence of external divalent cations, and the changes in Ca2+ current densities due to different membrane potentials. In addition, ISOC was not affected by antiandrogen or 1,25-dihydroxyvitamin D3 treatment of LNCaP cells, which up-regulates TRPV6 gene expression, or by androgen treatment, which has the opposite effect. Therefore, native channels responsible for ISOC are different from those for recombinant ITRPV6 and do not appear to be affected if one of their assumed subunits, TRPV6, is up- or down-regulated, suggesting a rather rigid subunit composition in vivo.     

Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.     

HES6 promotes prostate cancer aggressiveness independently of Notch signalling

Notch signalling is implicated in the pathogenesis of a variety of cancers, but its role in prostate cancer is poorly understood. However, selected Notch pathway members are overrepresented in high‐grade prostate cancers. We comprehensively profiled Notch pathway components in prostate cells and found prostate cancer‐specific up‐regulation of NOTCH3 and HES6. Their expression was particularly high in androgen responsive lines. Up‐ and down‐regulating Notch in these cells modulated expression of canonical Notch targets, HES1 and HEY1, which could also be induced by androgen. Surprisingly, androgen treatment also suppressed Notch receptor expression, suggesting that androgens can activate Notch target genes in a receptor‐independent manner. Using a Notch‐sensitive Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) reporter assay, we found that basal levels of Notch signalling were significantly lower in prostate cancer cells compared to benign cells. Accordingly pharmacological Notch pathway blockade did not inhibit cancer cell growth or viability. In contrast to canonical Notch targets, HES6, a HES family member known to antagonize Notch signalling, was not regulated by Notch signalling, but relied instead on androgen levels, both in cultured cells and in human cancer tissues. When engineered into prostate cancer cells, reduced levels of HES6 resulted in reduced cancer cell invasion and clonogenic growth. By molecular profiling, we identified potential roles for HES6 in regulating hedgehog signalling, apoptosis and cell migration. Our results did not reveal any cell‐autonomous roles for canonical Notch signalling in prostate cancer. However, the results do implicate HES6 as a promoter of prostate cancer progression.     

Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6

The molecular assembly of the epithelial Ca(2+) channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd(2+) sensitivity and voltage-dependent gating. Immuno precipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca(2+) channels that varied with respect to Ca(2+)-dependent inactivation, Ba(2+) selectivity and pharmacological block. Thus, Ca(2+)-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca(2+) transport kinetics.     

Interleukin-6 and prostate cancer progression

Prostate cancer, while initially dependent on androgens for proliferation, progresses to an androgen-independent state. Evidence has been accumulating that interleukin-6 (IL-6) may contribute to prostate cancer progression. Serum levels of IL-6 correlate with prostate tumor burden and patient morbidity. The prostate tissue itself appears to be a source of IL-6 and its receptor. Furthermore, experimental data suggest that IL-6 is an autocrine and paracrine growth factor for androgen-independent prostate cancer cell lines. For example, inhibition of IL-6, with anti-IL-6 antibody, sensitizes androgen-independent prostate cancer cells to chemotherapeutic agents in vitro. Finally, IL-6 activates a variety of signal transduction cascades, some which stimulate androgen receptor activity, in prostate cancer cells. These data suggest that targeting IL-6 may have multiple benefits in prostate cancer patients.     

TRPV6 mediates capsaicin-induced apoptosis in gastric cancer cells--Mechanisms behind a possible new "hot" cancer treatment

Capsaicin is an organic compound in chili peppers which are consumed by over one quarter of the world's population daily. Studies have shown that capsaicin can induce apoptosis in some cancer cells by unknown mechanisms. In this study, both gastric cancer and normal epithelial cells were treated with capsaicin and examined for apoptosis by Annexin V binding. Our results showed that capsaicin induces apoptosis in both cells, although cancer cells are more susceptible. This susceptibility is dependent on the availability of TRPV6, a calcium-selective channel protein, as overexpression of TRPV6 in normal cells increased capsaicin-induced apoptosis and knockdown of TRPV6 in cancer cells suppressed this action. Our results further demonstrated that capsaicin increases mitochondrial permeability through activation of Bax and p53 in a JNK-dependent manner. Conclusions: (1) TRPV6, rather than TRPV1 (the well-known capsaicin receptor), mediates capsaicin-induced apoptosis in gastric cells; (2) abundance of TRPV6 in gastric cells determines their live or death under capsaicin treatment; and (3) capsaicin induces apoptosis by stabilization of p53 through JNK activation. Together, our data suggest that capsaicin may be a promising dietary candidate for cancer chemoprevention.     

Human TRPV5 and TRPV6: Key players in cadmium and zinc toxicity

TRPV5 and TRPV6 are two major calcium transport pathways in the human body maintaining calcium homeostasis. TRPV5 is mainly expressed in the distal convoluted and connecting tubule where it is the major, regulated pathway for calcium reabsorption. TRPV6 serves as an important calcium entry pathway in the duodenum and the placenta. Previously, we showed that human TRPV6 (hTRPV6) transports several heavy metals. In this study we tested whether human TRPV5 (hTRPV5) also transports cadmium and zinc, and whether hTRPV5 together with hTRPV6 are involved in cadmium and zinc toxicity. The hTRPV5 mRNA and protein were expressed in HEK293 cells transiently transfected with pTagRFP-C1-hTRPV5. The overexpression of the hTRPV5 protein at the plasma membrane was revealed by cell surface biotinylation and immunofluorescence techniques. We observed that both cadmium and zinc permeate hTRPV5 in ion imaging experiments using Fura-2 or Newport Green DCF. Our results were further confirmed using whole-cell patch clamp technique. Transient overexpression of hTRPV5 or hTRPV6 sensitized cells to cadmium and zinc. Toxicity curves of cadmium and zinc were also shifted in hTRPV6 expressing HEK293 cells clones. Our results suggest that TRPV5 and TRPV6 are crucial gates controlling cadmium and zinc levels in the human body especially under low calcium dietary conditions, when these channels are maximally upregulated.     

A cancer chemopreventive agent silibinin, targets mitogenic and survival signaling in prostate cancer

There are many epigenetic variables that affect the biological responses of autocrine, paracrine and endocrine regulatory molecules, which determine the growth and development of different cancers including prostate cancer (PCA). One of the focuses of the current cancer chemoprevention studies is the search for non-toxic chemopreventive agents that inhibit mitogenic and cell survival signaling in cancer cells. In general, advanced stage cancer cells harbor many constitutively active mitogenic signaling and anti-apoptotic mechanisms, which make them less dependent on external growth factors as well as resistant to chemotherapeutic agents. In this regard, silibinin (a naturally occurring flavanone) has shown the pleiotropic anticancer effects in different cancer cells. Our extensive studies with PCA have shown that inhibition of mitogenic and cell survival signaling, such as epidermal growth factor receptor, insulin-like growth factor receptor type I and nuclear factor kappa B signaling are the most likely molecular targets of silibinin's efficacy in PCA. We have observed that silibinin inhibits prostate tumor growth in animal models without any apparent signs of toxicity. At the same time, silibinin is also physiologically available in different organs of the body including plasma and prostate, which is generally required for the pharmacological dosing and translational mechanistic studies of the compound. There are substantial amount of data to support the inhibitory effect of silibinin on mitogenic and cell survival signaling in PCA, which are reviewed in the present communication.     

Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.     

Functional characteristics of TRPV5 and TRPV6 channels in normal and transformed human lymphocytes

Calcium signaling and Ca²⁺-conducting channels participate in development of immune response, cell proliferation, growth, and differentiation of lymphocytes. In this paper, the calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) were studied in the plasma membrane of the T cell line Jurkat and normal human blood lymphocytes (HBLs). The channels were spontaneously activated after removal of Ca²⁺ and Mg²⁺ from the surrounding solution, and were inactivated in the presence of the effective blocker of TRPV5 and TRPV6, ruthenium red. The current-voltage characteristics of the channels demonstrated an inward rectification. The channel activity in Jurkat cells was significantly higher than in normal HBLs. The real-time RT-PCR analysis revealed a higher level of mRNA of the genes encoding channels TRPV5 and TRPV6 in the proliferating Jurkat T-cells as compared with normal lymphocytes. In general, the data have shown that TRPV5 and TRPV6 channels are expressed in blood lymphocytes are functionally active, and their activity is associated with proliferative status of blood cells.     

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.     

TRPV5 and TRPV6 calcium channels in human T cells

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) has provided a molecular basis for studying previously unidentified calcium influx channels in electrically nonexcitable cells. In the present work using RT-PCR, we obtained the endogenous expression of mRNAs of genes trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in normal human T lymphocytes. Additionally, by immunoblotting, the presence of the channel-forming TRPV5 proteins has been shown both in the total lysate and in crude membrane fractions from Jurkat cells and normal T lymphocytes. The use of immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes, this protein was not detected. The expression pattern and the selective Ca²⁺ permeation properties of TRPV5 and TRPV6 channels indicate the important role of these channels in Ca²⁺ homeostasis, as well as most likely in malignant transformation of blood cells.     

Hsp90: Still a viable target in prostate cancer

Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the maturation, activation and stability of critical signaling proteins that drive the development and progression of prostate cancer, including the androgen receptor. Despite robust preclinical data demonstrating anti-tumor activity of first-generation Hsp90 inhibitors in prostate cancer, poor clinical responses initially cast doubt over the clinical utility of this class of agent. Recent advances in compound design and development, use of novel preclinical models and further biological insights into Hsp90 structure and function have now stimulated a resurgence in enthusiasm for these drugs as a therapeutic option. This review highlights how the development of new-generation Hsp90 inhibitors with improved physical and pharmacological properties is unfolding, and discusses the potential contexts for their use either as single agents or in combination, for men with metastatic prostate cancer.     

PP2B/calcineurin-mediated desensitization of TRPV1 does not require AKAP150

Activation of protein kinases and phosphatases at the plasma membrane often initiates agonist-dependent signalling events. In sensory neurons, AKAP150 (A-kinase-anchoring protein 150) orientates PKA (protein kinase A), PKC (protein kinase C) and the Ca2+/calmodulin-dependent PP2B (protein phosphatase 2B, also known as calcineurin) towards membrane-associated substrates. Recent evidence indicates that AKAP150-anchored PKA and PKC phosphorylate and sensitize the TRPV1 (transient receptor potential subfamily V type 1 channel, also known as the capsaicin receptor). In the present study, we explore the hypothesis that an AKAP150-associated pool of PP2B catalyses the dephosphorylation and desensitization of TRPV1. Biochemical, electrophysiological and cell-based experiments indicate that PP2B associates with AKAP150 and TRPV1 in cultured TG (trigeminal ganglia) neurons. Gene silencing of AKAP150 reduces basal phosphorylation of TRPV1. However, functional studies in neurons isolated from AKAP150-/- mice indicate that the anchoring protein is not required for pharmacological desensitization of TRPV1. Behavioural analysis of AKAP150-/- mice further support this notion, demonstrating that agonist-stimulated desensitization of TRPV1 is sensitive to PP2B inhibition and does not rely on AKAP150. These findings allow us to conclude that pharmacological desensitization of TRPV1 by PP2B may involve additional regulatory components.     

Targeting lactate production and efflux in prostate cancer

Prostate cancer (PCa) is the most commonly diagnosed cancer in men worldwide. Screening and management of PCa remain controversial and, therefore, the discovery of novel molecular biomarkers is urgently needed. Alteration in cancer cell metabolism is a recognized hallmark of cancer, whereby cancer cells exhibit high glycolytic rates with subsequent lactate production, regardless of oxygen availability. To maintain the hyperglycolytic phenotype, cancer cells efficiently export lactate through the monocarboxylate transporters MCT1 and MCT4. The impact of inhibiting lactate production/extrusion on PCa cell survival and aggressiveness was investigated in vitro and ex vivo using primary tumor and metastatic PCa cell lines and the chicken embryo chorioallantoic membrane (CAM) model. In this study, we showed the metastatic PCa cell line (DU125) displayed higher expression levels of MCT1/4 isoforms and glycolysis-related markers than the localized prostate tumor-derived cell line (22RV1), indicating these proteins are differentially expressed throughout prostate malignant transformation. Moreover, disruption of lactate export by MCT1/4 silencing resulted in a decrease in PCa cell growth and motility. To support these results, we pharmacological inhibited lactate production (via inhibition of LDH) and release (via inhibition of MCTs) and a reduction in cancer cell growth in vitro and in vivo was observed. In summary, our data provide evidence that MCT1 and MCT4 are important players in prostate neoplastic progression and that inhibition of lactate production/export can be explored as a strategy for PCa treatment.