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Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with limited treatments and poor prognosis. TRPV6, a calcium-permeable channel overexpressed in cancers, holds potential as an influencer of cancer cell behavior. This study investigates TRPV6 expression in PDAC, analyzing 46 patient tissue samples of varying stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. We then revealed the impact of TRPV6 expression on Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth both in vitro and in vivo. Notably, TRPV6 expression correlated with tumor stage and grade, relating it to PDAC proliferation. Knockdown decreased Ca2+ influx. This was paralleled by reduced proliferation, enhanced apoptosis, and sensitization of cells to chemotherapeutic drugs. Conversely, TRPV6 overexpression yielded opposing effects. Strikingly, both knockdown and overexpression curtailed tumor formation in vivo. These findings underscore an intricate role of TRPV6 channels in PDAC progression, highlighting its potential as a therapeutic target. Pancreatic ductal adenocarcinoma (PDAC) stands as a highly aggressive and lethal cancer, characterized by a grim prognosis and scarce treatment alternatives. Within this context, TRPV6, a calcium-permeable channel, emerges as a noteworthy candidate due to its overexpression in various cancers, capable of influencing the cell behavior in different cancer entities. Nonetheless, the exact expression pattern and functional significance of TRPV6 in the context of PDAC remains enigmatic. This study scrutinizes the expression of TRPV6 in tissue specimens obtained from 46 PDAC patients across distinct stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. Subsequently, we analyzed its impact on multiple facets, encompassing Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth, both in vitro and in vivo. Notably, the data indicate a direct correlation between TRPV6 expression levels, tumor stage, and grade, establishing a link between TRPV6 and PDAC proliferation in tissue samples. Decreasing TRPV6 expression via knockdown hampered Ca2+ influx, resulting in diminished proliferation and viability in both cell lines, and cell cycle progression in Panc-1. The knockdown simultaneously led to an increase in apoptotic rates and increased the susceptibility of cells to 5-FU and gemcitabine treatments. Moreover, it accelerated migration and promoted collective movement among Panc-1 cells. Conversely, TRPV6 overexpression yielded opposing outcomes in terms of proliferation in Panc-1 and Capan-1, and the migration of Panc-1 cells. Intriguingly, both TRPV6 knockdown and overexpression diminished the process of tumor formation in vivo. This intricate interplay suggests that PDAC aggressiveness relies on a fine-tuned TRPV6 expression, raising its profile as a putative therapeutic target. [ABSTRACT FROM AUTHOR]

Background: Transient receptor potential vanilloid subfamily member 6 (TRPV6), a highly calcium-selective channel, has been shown to play a significant role in calcium homeostasis and to participate both in vitro and in vivo in growth, cell survival, and drug resistance of prostate cancer. Its role and the corresponding calcium-dependent pathways were mainly studied in hormone-dependent human prostate cancer cell lines, often used as a model of early-stage prostate cancers. The goal of the present study was to describe the TRPV6- specific phenotype and signaling pathways it is involved in, using castrationresistant prostate cancer cell lines. Methods: RNA sequencing (RNA-seq) was used to study the gene expression impacted by TRPV6 using PC3Mtrpv6−/− versus PC3Mtrpv6+/+ and its derivative PC3Mluc-C6trpv6+/+ cell line in its native and TRPV6 overexpressed form. In addition to the whole-cell RNA sequencing, immunoblotting, quantitative PCR, and calcium imaging were used to validate trpv6 gene status and functional consequences, in both trpv6-/- and TRPV6 overexpression cell lines. Results: trpv6-/- status was validated using both immunoblotting and quantitative PCR, and the functional consequences of either trpv6 gene deletion or TRPV6 overexpression were shown using calcium imaging. RNA-seq analysis demonstrated that the calcium channel TRPV6, being a crucial player of calcium signaling, significantly impacts the expression of genes involved in cancer progression, such as cell cycle regulation, chemotaxis, migration, invasion, apoptosis, ferroptosis as well as drug resistance, and extracellular matrix (ECM) re-organization. Conclusion: Our data suggest that the trpv6 gene is involved in and regulates multiple pathways related to tumor progression and drug resistance in castrationresistant prostate cancer cells. [ABSTRACT FROM AUTHOR]

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+/Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN‐mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+/Ca2+ exchangers, SERCA and store‐operated channels. This signaling cascade promotes activity of the NACLN‐colocalized proto‐oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller. Synopsis: The contribution of neuronal signaling and ion transport proteins to cancerogenesis remains unclear. Here, the sodium leak channel NALCN is shown to promote metastasis in prostate cancer, mediating Na+‐dependent Ca2+ oscillations critical for invasion.NALCN is enriched in human prostate cancer cell lines and malignant tissues.NALCN mediates cellular Ca2+ oscillations.NALCN‐mediated Ca2+ dynamics trigger secretory remodeling required for Src‐dependent actin dynamics, vesicular secretion of proteolytic enzymes, and invadopogenesis.NALCN depletion impairs prostate cancer cell invasion in vitro and metastasis in vivo. [ABSTRACT FROM AUTHOR]

Simple Summary: The TRPV6 channel is upregulated in many cancers and is associated with bad prognosis. Despite this fact, no reliable tool to target TRPV6 is known so far. Two prospective antibodies have been generated capable of binding to TRPV6 and thereby transiently activating calcium currents. This transient activation of TRPV6 and increase in intracellular calcium led to the apoptosis induction of prostate cancer cells. Only cells showing no TRPV6 membrane expression avoided cell death. Thus, the activation of the TRPV6 channel per se is extremely efficient and is likely to be more prospective than its inhibition. The TRPV6 calcium channel is known to be up-regulated in various tumors. The efforts to target the TRPV6 channel in vivo are still ongoing to propose an effective therapy against cancer. Here, we report the generation of two antibodies raised against extracellular epitopes corresponding to the extracellular loop between S1 and S2 (rb79) and the pore region (rb82). These antibodies generated a complex biphasic response with the transient activation of the TRPV6 channel. Store-operated calcium entry was consequently potentiated in the prostate cancer cell line LNCaP upon the treatment. Both rb79 and rb82 antibodies significantly decreased cell survival rate in a dose-dependent manner as compared to the control antibodies of the same isotype. This decrease was due to the enhanced cell death via apoptosis revealed using a sub-G1 peak in a cell cycle assay, TUNEL assay, and a Hoechst staining, having no effects in the PC3Mtrpv6−/− cell line. Moreover, all TUNEL-positive cells had TRPV6 membrane staining as compared to the control antibody treatment where TRPV6-positive cells were all TUNEL negative. These data clearly demonstrate that TRPV6 channel targeting using rb79 and rb82 antibodies is fatal and may be successfully used in the anticancer therapies. [ABSTRACT FROM AUTHOR]

Though the first discovery of TRPV6 channel expression in various tissues took place in the early 2000s, reliable tools for its protein detection in various cells and tissues are still missing. Here we show the generation and validation of rabbit polyclonal anti-TRPV6 channel antibodies (rb79–82) against four epitopes of 15 amino acids. Among them, only one antibody, rb79, was capable of detecting the full-length glycosylated form of the TRPV6 channel at around 100 kDa. The generated antibody was shown to be suitable for all in vitro applications, such as immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, etc. One of the most important applications is immunohistochemistry using the paraffin-embedded sections from cancer resection specimens. Using prostate cancer resection specimens, we have confirmed the absence of the TRPV6 protein in both healthy and benign hyperplasia, as well as its expression and correlation to the prostate cancer grades. Thus, the generated rabbit polyclonal anti-TRPV6 channel antibody rb79 is suitable for all in vitro diagnostic applications and particularly for the diagnosis in clinics using paraffin-embedded sections from patients suffering from various diseases and disorders involving the TRPV6 channel. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) represent a rapidly growing pharmaceutical class of protein drugs that becomes an important part of the precision therapy. mAbs are characterized by their high specificity and affinity for the target antigen, which is mostly present on the cell surface. Ion channels are a large family of transmembrane proteins that control ion transport across the cell membrane. They are involved in almost all biological processes in both health and disease and are widely considered as prospective targets. However, no antibody-based drug targeting ion channel has been developed so far that has progressed to clinical use. Thus, we provide a comprehensive review of the elaborated mAbs against ion channels, describe their mechanisms of action, and discuss their therapeutic potential. [ABSTRACT FROM AUTHOR]

• TRPV6 channel is (over)expressed in a number of human malignancies. • TRPV6 expression in cancer cells is regulated by VDR, AR, ER, and p38α. • TRPV6-triggered pathway reported so far is: Ca2+ - calmodulin – calcineurin – NFaT. • TRPV6 is targeted by some compounds but only SOR-13 passed clinical phase I trials. Significant advances have been made during last two decades as to the discovery of TRPV6 channel expression in various tissues and particularly in cancer. Among them are the cancers of the epithelial origin such as of prostate, breast, pancreas, ovaries, endometrium, testicule, colon, and lung. Though its role in cancer cell survival, proliferation, and apoptosis resistance was already established both in vitro and in vivo , much less was done in the studying of the downstream pathways where TRPV6 channel is involved which are restricted so far to the Ca2+ - calmodulin – calcineurin – NFaT pathways. Finally, in addition to the establishment of its role in human pathophysiology, the first pharmacological approaches were undertaken to target this channel and are described here in detail. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) has developed into a new clinical and non-invasive treatment for cancer over the past 30 years. By the combination of three non-toxic partners, i.e. a photosensitizer (PS), molecular oxygen (O2) and light, cytotoxic reactive oxygen species (ROS) are locally produced leading to irreversible vascular and cellular damage. In the present study, we report for the first time that the combination of two photosensitizers (2 PSs: Protoporphyrin IX, PpIX and Hypericin, Hy) loaded in the same lipid nanocapsules (LNCs) leads to enhanced photodynamic therapy efficiency when compared with previously reported systems. The 2 PS-loaded LNCs are shown to increase the in vitro phototoxicity at the nanomolar range (IC50 = 274 and 278 nM on HeLa and MDA-MB-231 cell lines, respectively), whereas the corresponding single PS-loaded LNCs at the same concentration exhibit a phototoxicity two times lower. Intracellular localization in HeLa cells indicates a subcellular asymmetry of PpIX and Hy, in the plasma, ER membranes and round internal structures. The biodistribution of LNCs was studied upon different routes of injection into Swiss nude mice; based on the obtained data, LNCs were injected intratumorally and used to slow the growth of xenograft tumors in mice. The results obtained in this study suggest that the combination of two or more PSs may be a promising strategy to improve the efficacy of conventional photodynamic therapy as well as to reduce dark toxicity. [ABSTRACT FROM AUTHOR]

Aims P2X receptors (P2XRs) mediate sympathetic control and autoregulation of renal circulation triggering preglomerular vasoconstriction, which protects glomeruli from elevated pressures. Although previous studies established a casual link between glomerular susceptibility to hypertensive injury and decreased preglomerular vascular reactivity to P2XR activation, the mechanisms of attenuation of the P2XR signalling in hypertension remained unknown. We aimed to analyse molecular mechanisms of the impairment of P2XR signalling in renal vascular smooth muscle cells (RVSMCs) in genetic hypertension. Methods and results We compared the expression of pertinent genes and P2XR-linked Ca2+ entry and Ca2+ release mechanisms in RVSMCs of spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar Kyoto (WKY) rats. We found that, in SHR RVSMCs, P2XR-linked Ca2+ entry and Ca2+ release from the sarcoplasmic reticulum (SR) are both significantly reduced. The former is due to down-regulation of the P2X1 subunit. The latter is caused by a decrease of the SR Ca2+ load. The SR Ca2+ load reduction is caused by attenuated Ca2+ uptake via down-regulated sarco-/endoplasmic reticulum Ca2+-ATPase 2b and elevated Ca2+ leak from the SR via ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors. Spontaneous activity of these Ca2+-release channels is augmented due to up-regulation of RyR type 2 and elevated IP3 production by up-regulated phospholipase C-β1. Conclusions Our study unravels the cellular and molecular mechanisms of attenuation of P2XR-mediated preglomerular vasoconstriction that elevates glomerular susceptibility to harmful hypertensive pressures. This provides an important impetus towards understanding of the pathology of hypertensive renal injury. [ABSTRACT FROM AUTHOR]

Adrenomedullin (AM) is a 52-amino acid peptide initially isolated from human pheochromocytoma. AM is expressed in a variety of malignant tissues and cancer cell lines and was shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, AM is a survival factor for certain cancer cells. Some data suggest that AM might be involved in the progression cancer metastasis via angiogenesis and cell migration and invasion control. The Transient Receptor Potential channel TRPV2 is known to promote in prostate cancer cell migration and invasive phenotype and is correlated with the stage and grade of bladder cancer. In this work we show that AM induces prostate and urothelial cancer cell migration and invasion through TRPV2 translocation to plasma membrane and the subsequent increase in resting calcium level. [ABSTRACT FROM AUTHOR]

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 Ca2+ homeostasis where the store-operated Ca2+ 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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ion channels contribute to virtually all basic cellular processes, including such crucial ones for maintaining tissue homeostasis as proliferation, differentiation, and apoptosis. The involvement of ion channels in regulation of programmed cell death, or apoptosis, has been known for at least three decades based on observation that classical blockers of ion channels can influence cell death rates, prolonging or shortening cell survival. Identification of the central role of these channels in regulation of cell cycle and apoptosis as well as the recent discovery that the expression of ion channels is not limited solely to the plasma membrane, but may also include membranes of internal compartments, has led researchers to appreciate the pivotal role of ion channels plays in development of cancer. This review focuses on the aspects of programmed cell death influenced by various ion channels and how dysfunctions and misregulations of these channels may affect the development and progression of different cancers. [ABSTRACT FROM AUTHOR]

Abstract: TRP channels form a superfamily of channel proteins exhibiting versatile regulatory characteristics with many channels participating in the regulation of Ca2+ homeostasis and influencing the cell fate. Multitude of evidence is emerging that the colocalization of TRP channels with Ca2+-sensing elements of specific regulatory pathways leading to either proliferation or apoptosis is what makes these channels participate in cell fate regulation and, in turn, determines the final effect of Ca2+ entry via the particular channel. This review focuses on the aspects of TRP channel localization and function that affect the balance between cell survival and death and how various dysregulations of these channels may lead to perturbed balance and onset of cancer. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The calcium-sensing receptor (CaR), is a G protein-dependent receptor that responds to increments in extracellular Ca2+ ([Ca2+]o). We previously reported that an increase in [Ca2+]o induced a release of intracellular calcium and Ca2+ entry via store operated channels (SOCs). We also demonstrated that MCF-7 cells express Transient Receptor Potential canonical 1 (TRPC1) channels. Herein, we investigated CaR intracellular signaling pathways and examined the role of TRPC1 in CaR-induced cell proliferation, through the extracellular signal-regulated Kinases 1 & 2 (ERK1/2) pathways. Treatment by [Ca2+]o increased both MCF-7 cell proliferation and TRPC1 expression. Both the [Ca2+]o proliferative effect and TRPC1 protein levels were abolished by the ERK1/2 inhibitors. Moreover, [Ca2+]o failed to increase cell proliferation either in the presence of CaR or TRPC1 siRNAs. Both [Ca2+]o and the selective CaR activator spermine, elicited time and dose-dependent ERK1/2 phosphorylation. ERK1/2 phosphorylation was almost completely inhibited by treatment with the phospholipase C and the protein kinase C inhibitors. Treatment with 2-aminoethoxydiphenyl borate (2-APB), and SKF-96365 or by siTRPC1 diminished both [Ca2+]o- and spermine-stimulated ERK1/2 phosphorylation. Moreover, down-regulation of TRPC1 by siRNA reduced the Ca2+ entry induced by CaR activation. We conclude that the CaR activates ERK1/2 via a PLC/PKC-dependent pathway. Moreover, TRPC1 is required for the ERK1/2 phosphorylation, Ca2+ entry and the CaR-proliferative effect. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is the most frequently diagnosed malignant tumor and the second leading cause of cancer death in women worldwide. The risk of developing breast cancer is 12.8%, i.e., 1 in 8 people, and a woman's risk of dying is approximately 1 in 39. Calcium signals play an important role in various cancers and transport calcium ions may have altered expression in breast cancer, such as the TRPM7 calcium permeant ion channel, where overexpression may be associated with a poor prognosis. This review focuses on the TRPM7 channel, and the oncogenic roles studied so far in breast cancer. The TRPM7 ion channel is suggested as a potential and prospective target in the diagnosis and treatment of breast cancer. The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a divalent cations permeant channel but also has intrinsic serine/threonine kinase activity. It is ubiquitously expressed in normal tissues and studies have indicated that it participates in important physiological and pharmacological processes through its channel-kinase activity, such as calcium/magnesium homeostasis, phosphorylation of proteins involved in embryogenesis or the cellular process. Accumulating evidence has shown that TRPM7 is overexpressed in human pathologies including breast cancer. Breast cancer is the second leading cause of cancer death in women with an incidence rate increase of around 0.5% per year since 2004. The overexpression of TRPM7 may be associated with a poor prognosis in breast cancer patients, so more efforts are needed to research a new therapeutic target. TRPM7 regulates the levels of Ca2+, which can alter the signaling pathways involved in survival, cell cycle progression, proliferation, growth, migration, invasion, epithelial-mesenchymal transition and thus determines cell behavior, promoting tumor development. This work provides a complete overview of the TRPM7 ion channel and its main involvements in breast cancer. Special consideration is given to the modulation of the channel as a potential target in breast cancer treatment by inhibition of proliferation, migration and invasion. Taken together, these data suggest the potential exploitation of TRPM7 channel-kinase as a therapeutic target and a diagnostic biomarker. [ABSTRACT FROM AUTHOR]

Abstract: Aberrant keratinocyte differentiation is considered to be a key mechanism in the onset of hyperproliferative dermatological diseases, including basal cell carcinoma (BCC). It is, therefore, vital to understand what drives keratinocytes to develop such pathological phenotypes. The role of calcium in keratinocyte differentiation is uncontested but the mechanisms controlling calcium-induced differentiation have yet to be completely elucidated. This study was designed to investigate the role of calcium-permeable TRPC channels in human keratinocyte differentiation and BCC, using a combination of molecular and cell biology approaches, involving electrophysiology and Ca2+-imaging, on the HaCaT cell line, primary cultures of normal human keratinocytes, and BCC cells. We demonstrated that TRPC1/TRPC4 channel expression was important for keratinocyte differentiation, as knocking out these channels (by siRNA strategy) prevented the induction of Ca2+-induced differentiation. TRPC1/TRPC4-mediated calcium entry and endoplasmic reticulum Ca2+ content increased significantly in differentiated keratinocytes. However, the failure of BCC cells to differentiate was related to a lack of TRPC channel expression and calcium entry. In summary, our data demonstrate that TRPC1 and TRPC4 channels are key elements in keratinocyte Ca2+ homeostasis and differentiation and may therefore be responsible for skin pathologies. [Copyright &y& Elsevier]

Ca2+ is an essential factor inducing keratinocyte differentiation due to the natural Ca2+ gradient in the skin. However, the membrane mechanisms that mediate calcium entry and trigger keratinocyte differentiation had not previously been elucidated. In this study we demonstrate that Ca2+-induced differentiation up-regulates both mRNA and protein expression of a transient receptor potential highly Ca2+-selective channel, TRPV6. The latter mediates Ca2+ uptake and accounts for the basal [Ca2+]i in human keratinocytes. Our results show that TRPV6 is a pre-requisite for keratinocyte entry into differentiation, because the silencing of TRPV6 in human primary keratinocytes led to the development of impaired differentiated phenotype triggered by Ca2+. The expression of such differentiation markers as involucrin, transglutaminase-1, and cytokeratin-10 was significantly inhibited by small interfering RNA-TRPV6 as compared with differentiated control cells. TRPV6 silencing affected cell morphology and the development of intercellular contacts, as well as the ability of cells to stratify. 1,25-Dihydroxyvitamin D3, a cofactor of differentiation, dose-dependently increased TRPV6 mRNA and protein expression in human keratinocytes. This TRPV6 up-regulation led to a significant increase in Ca2+ uptake in both undifferentiated and differentiated keratinocytes. We conclude that TRPV6 mediates, at least in part, the pro-differentiating effects of 1,25-dihydroxyvitamin D3 by increasing Ca2+ entry, thereby promoting differentiation. Taken together, these data suggest that the TRPV6 channel is a key element in Ca2+/1,25-dihydroxyvitamin D3-induced differentiation of human keratinocytes. [ABSTRACT FROM AUTHOR]

The transient receptor potential channels (TRPs) have been related to several different physiologies that range from a role in sensory physiology (including thermo- and osmosensation) to a role in some pathologies like cancer. The great diversity of functions performed by these channels is represented by nine sub-families that constitute the TRP channel superfamily. From the mid-2000s, several reports have shown the potential role of the TRP channels in cancers of multiple origin. The pancreatic cancer is one of the deadliest cancers worldwide. Its prevalence is predicted to rise further. Disappointingly, the treatments currently used are ineffective. There is an urgency to find new ways to counter this disease and one of the answers may lie in the ion channels belonging to the superfamily of TRP channels. In this review, we analyse the existing knowledge on the role of TRP channels in the development and progression of pancreatic ductal adenocarcinoma (PDAC). The functions of these channels in other cancers are also considered. This might be of interest for an extrapolation to the pancreatic cancer in an attempt to identify potential therapeutic interventions. [ABSTRACT FROM AUTHOR]

It has been widely established that transient receptor potential vanilloid (TRPV) channels play a crucial role in calcium homeostasis in mammalian cells. Modulation of TRPV channels activity can modify their physiological function leading to some diseases and disorders like neurodegeneration, pain, cancer, skin disorders, etc. It should be noted that, despite TRPV channels importance, our knowledge of the TRPV channels functions in cells is mostly limited to their plasma membrane location. However, some TRPV channels were shown to be expressed in the endoplasmic reticulum where their modulation by activators and/or inhibitors was demonstrated to be crucial for intracellular signaling. In this review, we have intended to summarize the poorly studied roles and functions of these channels in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Discusses research which aimed to explore the role of translocon complex, present on the endoplasmic reticulum membrane involved in protein translocation, in the activation of store operated calcium entry (SOCE). Possible functional link between the passive leakage via the translocon and SOCE stimulation; Effect of the inhibition of the calcium leak via the translocon by anisomycin; Investigation whether calcium passive leakage occurring specifically through the translocon could activate the store operated channels current via passive leakage activation.

Members of the TRPM (“Melastatin”) family fall into the subclass of the TRP channels having varying permeability to Ca2+ and Mg2+, with three members of the TRPM family being chanzymes, which contain C-terminal enzyme domains. The role of different TRPM members has been shown in various cancers such as prostate cancer for mostly TRPM8 and TRPM2, breast cancer for mostly TRPM2 and TRPM7, and pancreatic cancer for TRPM2/7/8 channels. The role of TRPM5 channels has been shown in lung cancer, TRPM1 in melanoma, and TRPM4 channel in prostate cancer as well. Thus, the TRPM family of channels may represent an appealing target for the anticancer therapy. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Though the first discovery of TRPV6 channel expression in various tissues took place in the early 2000s, reliable tools for its protein detection in various cells and tissues are still missing. Here we show the generation and validation of rabbit polyclonal anti-TRPV6 channel antibodies (rb79-82) against four epitopes of 15 amino acids. Among them, only one antibody, rb79, was capable of detecting the full-length glycosylated form of the TRPV6 channel at around 100 kDa. The generated antibody was shown to be suitable for all in vitro applications, such as immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, etc. One of the most important applications is immunohistochemistry using the paraffin-embedded sections from cancer resection specimens. Using prostate cancer resection specimens, we have confirmed the absence of the TRPV6 protein in both healthy and benign hyperplasia, as well as its expression and correlation to the prostate cancer grades. Thus, the generated rabbit polyclonal anti-TRPV6 channel antibody rb79 is suitable for all in vitro diagnostic applications and particularly for the diagnosis in clinics using paraffin-embedded sections from patients suffering from various diseases and disorders involving the TRPV6 channel.

Pancreatic cancer (PC) is a major cause of cancer-associated mortality in Western countries (and estimated to be the second cause of cancer deaths by 2030). The main form of PC is pancreatic adenocarcinoma, which is the fourth most common cause of cancer-related death, and this situation has remained virtually unchanged for several decades. Pancreatic ductal adenocarcinoma (PDAC) is inherently linked to the unique physiology and microenvironment of the exocrine pancreas, such as pH, mechanical stress, and hypoxia. Of them, calcium (Ca 2+ ) signals, being pivotal molecular devices in sensing and integrating signals from the microenvironment, are emerging to be particularly relevant in cancer. Mutations or aberrant expression of key proteins that control Ca 2+ levels can cause deregulation of Ca 2+ -dependent effectors that control signaling pathways determining the cells' behavior in a way that promotes pathophysiological cancer hallmarks, such as enhanced proliferation, survival and invasion. So far, it is essentially unknown how the cancer-associated Ca 2+ signaling is regulated within the characteristic landscape of PDAC. This work provides a complete overview of the Ca 2+ signaling and its main players in PDAC. Special consideration is given to the Ca 2+ signaling as a potential target in PDAC treatment and its role in drug resistance.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editors in Chief. This article was retracted because of inappropriate use of confidential material and text available to one of the authors through the review of “TRPV6 As A Target For Cancer Therapy”, John M Stewart, J. Cancer, online date 2019-5-13; doi:10.7150/jca.31640., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aims: P2X receptors (P2XRs) mediate sympathetic control and autoregulation of renal circulation triggering preglomerular vasoconstriction, which protects glomeruli from elevated pressures. Although previous studies established a casual link between glomerular susceptibility to hypertensive injury and decreased preglomerular vascular reactivity to P2XR activation, the mechanisms of attenuation of the P2XR signalling in hypertension remained unknown. We aimed to analyse molecular mechanisms of the impairment of P2XR signalling in renal vascular smooth muscle cells (RVSMCs) in genetic hypertension., Methods and Results: We compared the expression of pertinent genes and P2XR-linked Ca(2+) entry and Ca(2+) release mechanisms in RVSMCs of spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar Kyoto (WKY) rats. We found that, in SHR RVSMCs, P2XR-linked Ca(2+) entry and Ca(2+) release from the sarcoplasmic reticulum (SR) are both significantly reduced. The former is due to down-regulation of the P2X1 subunit. The latter is caused by a decrease of the SR Ca(2+) load. The SR Ca(2+) load reduction is caused by attenuated Ca(2+) uptake via down-regulated sarco-/endoplasmic reticulum Ca(2+)-ATPase 2b and elevated Ca(2+) leak from the SR via ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors. Spontaneous activity of these Ca(2+)-release channels is augmented due to up-regulation of RyR type 2 and elevated IP3 production by up-regulated phospholipase C-β1., Conclusions: Our study unravels the cellular and molecular mechanisms of attenuation of P2XR-mediated preglomerular vasoconstriction that elevates glomerular susceptibility to harmful hypertensive pressures. This provides an important impetus towards understanding of the pathology of hypertensive renal injury., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Ion channels and notably TRP channels play a crucial role in a variety of physiological functions and in addition these channels have been also shown associated with several diseases including cancer. The process of cancer initiation and progression involves the altered expression of one or more of TRP proteins, depending on the nature of the cancer. The most clearly described role in pathogenesis has been evidenced for TRPM8, TRPV6 and TRPM1 channels. The increased expression of some other channels, such as TRPV1, TRPC1, TRPC6, TRPM4, and TRPM5 has also been demonstrated in some cancers. Further investigations are required to precise the role of TRP channels in cancer development and/or progression and to specifically develop further knowledge of TRP proteins as discriminative markers and prospective targets for pharmaceutical intervention in treating cancer.

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.

The calcium-sensing receptor (CaR), is a G protein-dependent receptor that responds to increments in extracellular Ca(2+) ([Ca(2+)](o)). We previously reported that an increase in [Ca(2+)](o) induced a release of intracellular calcium and Ca(2+) entry via store operated channels (SOCs). We also demonstrated that MCF-7 cells express Transient Receptor Potential canonical 1 (TRPC1) channels. Herein, we investigated CaR intracellular signaling pathways and examined the role of TRPC1 in CaR-induced cell proliferation, through the extracellular signal-regulated Kinases 1 & 2 (ERK1/2) pathways. Treatment by [Ca(2+)](o) increased both MCF-7 cell proliferation and TRPC1 expression. Both the [Ca(2+)](o) proliferative effect and TRPC1 protein levels were abolished by the ERK1/2 inhibitors. Moreover, [Ca(2+)](o) failed to increase cell proliferation either in the presence of CaR or TRPC1 siRNAs. Both [Ca(2+)](o) and the selective CaR activator spermine, elicited time and dose-dependent ERK1/2 phosphorylation. ERK1/2 phosphorylation was almost completely inhibited by treatment with the phospholipase C and the protein kinase C inhibitors. Treatment with 2-aminoethoxydiphenyl borate (2-APB), and SKF-96365 or by siTRPC1 diminished both [Ca(2+)](o)- and spermine-stimulated ERK1/2 phosphorylation. Moreover, down-regulation of TRPC1 by siRNA reduced the Ca(2+) entry induced by CaR activation. We conclude that the CaR activates ERK1/2 via a PLC/PKC-dependent pathway. Moreover, TRPC1 is required for the ERK1/2 phosphorylation, Ca(2+) entry and the CaR-proliferative effect., (Copyright 2009 S. Karger AG, Basel.)

Ca(2+) is an essential factor inducing keratinocyte differentiation due to the natural Ca(2+) gradient in the skin. However, the membrane mechanisms that mediate calcium entry and trigger keratinocyte differentiation had not previously been elucidated. In this study we demonstrate that Ca(2+)-induced differentiation up-regulates both mRNA and protein expression of a transient receptor potential highly Ca(2+)-selective channel, TRPV6. The latter mediates Ca(2+) uptake and accounts for the basal [Ca(2+)](i) in human keratinocytes. Our results show that TRPV6 is a prerequisite for keratinocyte entry into differentiation, because the silencing of TRPV6 in human primary keratinocytes led to the development of impaired differentiated phenotype triggered by Ca(2+). The expression of such differentiation markers as involucrin, transglutaminase-1, and cytokeratin-10 was significantly inhibited by small interfering RNA-TRPV6 as compared with differentiated control cells. TRPV6 silencing affected cell morphology and the development of intercellular contacts, as well as the ability of cells to stratify. 1,25-Dihydroxyvitamin D3, a cofactor of differentiation, dose-dependently increased TRPV6 mRNA and protein expression in human keratinocytes. This TRPV6 up-regulation led to a significant increase in Ca(2+) uptake in both undifferentiated and differentiated keratinocytes. We conclude that TRPV6 mediates, at least in part, the pro-differentiating effects of 1,25-dihydroxyvitamin D3 by increasing Ca(2+) entry, thereby promoting differentiation. Taken together, these data suggest that the TRPV6 channel is a key element in Ca(2+)/1,25-dihydroxyvitamin D3-induced differentiation of human keratinocytes.

Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiopathology. One of the most enigmatic mechanisms responsible for Ca2+ concentration in the ER is passive calcium leak. Previous studies have shown that the translocon complex is permeable to calcium. However, the involvement of the translocon in the passive calcium leak has not been directly demonstrated. Furthermore, the question whether the passive store depletion via the translocon could activate SOC (store operated channels) replenishing the ER, remains still unresolved. In this study, for the first time, we show that thapsigargin and calcium chelators deplete ER via translocon. Indeed, using confocal imaging, we demonstrate that when the number of opened translocons was lowered neither thapsigargin nor calcium chelators could induce ER store depletion. We also demonstrate that calcium leakage occurring via the translocon activates store-operated current, which is, by its kinetic and pharmacology, similar to that evoked by thapsigargin and EGTA (but not IP3), thus highlighting our hypothesis that calcium leakage via the translocon is a first step for activation of the specific iPLA2-regulated SOC. As the translocon is present in yeast and mammalian cells, our findings suggest that translocon-related calcium signaling is a common phenomenon.

One major clinical problem with prostate cancer is the cells' ability to survive and proliferate upon androgen withdrawal. Because Ca2+ is central to growth control, understanding the mechanisms of Ca2+ homeostasis involved in prostate cancer cell proliferation is imperative for new therapeutic strategies. Here, we show that agonist-mediated stimulation of alpha1-adrenergic receptors (alpha1-AR) promotes proliferation of the primary human prostate cancer epithelial (hPCE) cells by inducing store-independent Ca2+ entry and subsequent activation of nuclear factor of activated T cells (NFAT) transcription factor. Such an agonist-induced Ca2+ 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 alpha1-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.