Ion Channel Profiling in Prostate Cancer: Toward Cell Population-Specific Screening.

In the last three decades, a growing number of studies have implicated ion channels in all essential processes of prostate carcinogenesis, including cell proliferation, apoptosis, migration, and angiogenesis. The changes in the expression of individual ion channels show a specific profile, making these proteins promising clinical biomarkers that may enable better molecular subtyping of the disease and lead to more rapid and accurate clinical decision-making. Expression profiles and channel function are mainly based on the tumoral tissue itself, in this case, the epithelial cancer cell population. To date, little data on the ion channel profile of the cancerous prostate stroma are available, even though tumor interactions with the microenvironment are crucial in carcinogenesis and each distinct population plays a specific role in tumor progression. In this review, we describe ion channel expression profiles specific for the distinct cell population of the tumor microenvironment (stromal, endothelial, neuronal, and neuroendocrine cell populations) and the technical approaches used for efficient separation and screening of these cell populations.

TRPM8 as an Anti-Tumoral Target in Prostate Cancer Growth and Metastasis Dissemination.

In the fight against prostate cancer (PCa), TRPM8 is one of the most promising clinical targets. Indeed, several studies have highlighted that TRPM8 involvement is key in PCa progression because of its impact on cell proliferation, viability, and migration. However, data from the literature are somewhat contradictory regarding the precise role of TRPM8 in prostatic carcinogenesis and are mostly based on in vitro studies. The purpose of this study was to clarify the role played by TRPM8 in PCa progression. We used a prostate orthotopic xenograft mouse model to show that TRPM8 overexpression dramatically limited tumor growth and metastasis dissemination in vivo. Mechanistically, our in vitro data revealed that TRPM8 inhibited tumor growth by affecting the cell proliferation and clonogenic properties of PCa cells. Moreover, TRPM8 impacted metastatic dissemination mainly by impairing cytoskeleton dynamics and focal adhesion formation through the inhibition of the Cdc42, Rac1, ERK, and FAK pathways. Lastly, we proved the in vivo efficiency of a new tool based on lipid nanocapsules containing WS12 in limiting the TRPM8-positive cells' dissemination at metastatic sites. Our work strongly supports the protective role of TRPM8 on PCa progression, providing new insights into the potential application of TRPM8 as a therapeutic target in PCa treatment.

Testosterone-androgen receptor: The steroid link inhibiting TRPM8-mediated cold sensitivity.

Recent studies have revealed gender differences in cold perception, and pointed to a possible direct action of testosterone (TST) on the cold-activated TRPM8 (Transient Receptor Potential Melastatin Member 8) channel. However, the mechanisms by which TST influences TRPM8-mediated sensory functions remain elusive. Here, we show that TST inhibits TRPM8-mediated mild-cold perception through the noncanonical engagement of the Androgen Receptor (AR). Castration of both male rats and mice increases sensitivity to mild cold, and this effect depends on the presence of intact TRPM8 and AR. TST in nanomolar concentrations suppresses whole-cell TRPM8-mediated currents and single-channel activity in native dorsal root ganglion (DRG) neurons and HEK293 cells co-expressing recombinant TRPM8 and AR, but not TRPM8 alone. AR cloned from rat DRGs shows no difference from standard AR. However, biochemical assays and confocal imaging reveal the presence of AR on the cell surface and its interaction with TRPM8 in response to TST, leading to an inhibition of channel activity.

Ca2+ Channel Toolkit in Neuroendocrine Tumors.

Neuroendocrine tumors (NET) constitute a heterogeneous group of malignancies with various clinical presentations and growth rates but a common origin in neuroendocrine cells located all over the body. NET are a relatively low-frequency disease mostly represented by gastroenteropancreatic (GEP) and bronchopulmonary tumors (pNET); on the other hand, an increasing frequency and prevalence have been associated with NET. Despite great efforts in recent years, the management of NET is still a critical unmet need due to the lack of knowledge of the biology of the disease, the lack of adequate biomarkers, late presentation, the relative insensitivity of imaging modalities, and a paucity of predictably effective treatment options. In this context Ca2+ signals, being pivotal molecular devices in sensing and integrating signals from the microenvironment, are emerging to be particularly relevant in cancer, where they mediate interactions between tumor cells and the tumor microenvironment to drive different aspects of neoplastic progression (e.g., cell proliferation and survival, cell invasiveness, and proangiogenetic programs). Indeed, ion channels represent good potential pharmacological targets due to their location on the plasma membrane, where they can be easily accessed by drugs. The present review aims to provide a critical and up-to-date overview of NET development integrating Ca2+ signal involvement. In this perspective, we first give an introduction to NET and Ca2+ channels and then describe the different families of Ca2+ channels implicated in NET, i.e., ionotropic receptors, voltage-dependent Ca2+ channels, and transient receptor potential channels, as well as intracellular Ca2+ channels and their signaling molecules.

4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer.

Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5­triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca2+ release channels.

TRPV1 variants impair intracellular Ca2+ signaling and may confer susceptibility to malignant hyperthermia.

PURPOSE: Malignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete. METHODS: We aimed to identify transient receptor potential vanilloid 1 (TRPV1) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1-/- mice, and a murine model of human MH. RESULTS: We identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1-/- mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model. CONCLUSION: We propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.

Partners in Crime: Towards New Ways of Targeting Calcium Channels.

The characterization of calcium channel interactome in the last decades opened a new way of perceiving ion channel function and regulation. Partner proteins of ion channels can now be considered as major components of the calcium homeostatic mechanisms, while the reinforcement or disruption of their interaction with the channel units now represents an attractive target in research and therapeutics. In this review we will focus on the targeting of calcium channel partner proteins in order to act on the channel activity, and on its consequences for cell and organism physiology. Given the recent advances in the partner proteins' identification, characterization, as well as in the resolution of their interaction domain structures, we will develop the latest findings on the interacting proteins of the following channels: voltage-dependent calcium channels, transient receptor potential and ORAI channels, and inositol 1,4,5-trisphosphate receptor.

TRPM8 and prostate: a cold case?

While originally cloned from the prostate in 2001, transient receptor potential, melastatin member 8 (TRPM8) has since been identified as the cold/menthol receptor in the peripheral nervous system. This discovery has led to hundreds of studies regarding the role of this channel in pain and thermosensation phenomena, while relegating TRPM8 involvement in cancer to a secondary role. Despite these findings, there is growing evidence that TRPM8 should be carefully studied within the frame of carcinogenesis, especially in the prostate, where it is highly expressed and where many teams have confirmed variations in its expression during cancer progression. Its regulation by physiological factors, such as PSA and androgens, has proved that TRPM8 can exhibit an activity beyond that of a cold receptor, thus explaining how the channel can be activated in organs not exposed to temperature variations. With this review, we aim to provide a brief overview of the current knowledge regarding the complex roles of TRPM8 in prostate carcinogenesis and to show that this research path still represents a "hot" topic with potential clinical applications in the short term.

New Insight in Cold Pain: Role of Ion Channels, Modulation, and Clinical Perspectives.

Cold temperature detection involves the process of sensory transduction in cutaneous primary sensory nerve terminals, which converts thermal stimuli into depolarizations of the membrane. This transformation into electrical signals is followed by the subsequent propagation of action potentials in cold-sensitive afferent nerve fibers. A large array of ion channels shapes this process; however, the precise contribution of specific ion channel subtypes to cold perception and cold pain remains elusive. This review aims at giving an update on our current understanding of the role played by TRPs, leak K+ and voltage-gated Na+ and K+ channels in the transduction of cold by nociceptors and in cold-induced pain.

Complex modulation of the cold receptor TRPM8 by volatile anaesthetics and its role in complications of general anaesthesia.

The mechanisms by which volatile general anaesthetics (VAs) produce a depression of central nervous system are beginning to be better understood, but little is known about a number of side effects. Here, we show that the cold receptor transient receptor potential melastatin 8 (TRPM8) undergoes a complex modulation by clinical concentrations of VAs in dorsal root ganglion neurons and HEK-293 cells heterologously expressing TRPM8. VAs produced a transient enhancement of TRPM8 through a depolarizing shift of its activation towards physiological membrane potentials, followed by a sustained TRPM8 inhibition. The stimulatory action of VAs engaged molecular determinants distinct from those used by the TRPM8 agonist. Transient TRPM8 activation by VAs could explain side effects such as inhibition of respiratory drive, shivering and the cooling sensation during the beginning of anaesthesia, whereas the second phase of VA action, that associated with sustained TRPM8 inhibition, might be responsible for hypothermia. Consistent with this, both hypothermia and the inhibition of respiratory drive induced by VAs are partially abolished in Trpm8-knockout animals. Thus, we propose TRPM8 as a new clinical target for diminishing common and serious complications of general anaesthesia.

Environmental footprint of constructed wetlands treating wastewater.

The aim of the study is to determine environmentally friendlier construction materials for constructed wetland facilities treating wastewater. This is done by computing the environmental footprint of the facility based on the methodology of life cycle assessment (LCA). This methodology reveals the dominant aggravating processes during the construction of a constructed wetland (CW) and can help to create alternative environmentally friendlier solutions. This methodology was applied for the determination of the overall environmental profile of a hybrid CW facility. The LCA was applied first to the facility as originally designed, where reinforced concrete was used in some components. Then, alternative construction materials to reinforced concrete were used, such as earth covered with high density polyethylene (HDPE) or clay, and LCA was applied again. Earth structures were found to have reduced environmental impact compared to concrete ones, and clay was found environmentally friendlier compared to HDPE. Furthermore, estimation of the construction costs of the three scenarios indicate that the last scenario is also the least expensive.

Differential sensitivity of prostate tumor derived endothelial cells to sorafenib and sunitinib.

BACKGROUND: Prostate cancer is the second leading cause of male cancer death in developed countries. Although the role of angiogenesis in its progression is well established, the efficacy of anti-angiogenic therapy is not clearly proved. Whether this could depend on differential responses between tumor and normal endothelial cells has not been tested. METHODS: We isolated and characterized three lines of endothelial cells from prostate cancer and we tested the effect of Sunitinib and Sorafenib, and the combined treatment with the anti-androgen Casodex, on their angiogenic functions. RESULTS: Endothelial cells isolated from prostate tumors showed angiogenic properties and expression of androgen and vascular endothelial cell growth factor receptors. Sunitinib affected their proliferation, survival and motility while Sorafenib only showed a minor effect. At variance, Sunitinib and Sorafenib showed similar cytotoxic and anti-angiogenic effects on normal endothelial cells. Sorafenib and Sunitinib inhibited vascular endothelial cell growth factor receptor2 phosphorylation of prostate cancer endothelial cells, while they differentially modulated Akt phosphorylation as no inhibitory effect of Sorafenib was observed on Akt activation. The combined treatment of Casodex reverted the observed resistance to Sorafenib both on cell viability and on Akt activation, whereas it did not modify the response to Sunitinib. CONCLUSIONS: Our study demonstrates a resistant behavior of endothelial cells isolated from prostate cancer to Sorafenib, but not Sunitinib. Moreover, it shows the benefit of a multi-target therapy combining anti-angiogenic and anti-hormonal drugs to overcome resistance.

Construction and operation costs of constructed wetlands treating wastewater.

Design data from nine constructed wetlands (CW) facilities of various capacities (population equivalent (PE)) are used to estimate construction and operation costs, and then to derive empirical equations relating the required facility land area and the construction cost to PE. In addition, comparisons between the costs of CW facilities based on various alternative construction materials, i.e., reinforced concrete and earth structures (covered with either high density polyethylene or clay), are presented in relation to the required area. The results show that earth structures are economically advantageous. The derived equations can be used for providing a preliminary cost estimate of CW facilities for domestic wastewater treatment.

TRP Channels: The Neglected Culprits in Breast Cancer Chemotherapy Resistance?

Breast cancer is a major health concern worldwide, and resistance to therapies remains a significant challenge in treating this disease. In breast cancer, Transient Receptor Potential (TRP) channels are well studied and constitute key players in nearly all carcinogenesis hallmarks. Recently, they have also emerged as important actors in resistance to therapy by modulating the response to various pharmaceutical agents. Targeting TRP channels may represent a promising approach to overcome resistance to therapies in breast cancer patients.

Complex regulation of the TRPM8 cold receptor channel: role of arachidonic acid release following M3 muscarinic receptor stimulation.

Cold/menthol-activated TRPM8 (transient receptor potential channel melastatin member 8) is primarily expressed in sensory neurons, where it constitutes the principal receptor of environmental innocuous cold. TRPM8 has been shown to be regulated by multiple influences such as phosphorylation, pH, Ca(2+), and lipid messengers. One such messenger is arachidonic acid (AA), which has been shown to inhibit TRPM8 channel activity. However, the physiological pathways mediating the inhibitory effect of AA on TRPM8 still remain unknown. Here, we demonstrate that TRPM8 is regulated via M3 muscarinic acetylcholine receptor-coupled signaling cascade based on the activation of cytosolic phospholipase A2 (cPLA2) and cPLA2-catalyzed derivation of AA. Stimulation of M3 receptors heterologously co-expressed with TRPM8 in HEK-293 cells by nonselective muscarinic agonist, oxotremorine methiodide (Oxo-M), caused inhibition of TRPM8-mediated membrane current, which could be mimicked by AA and antagonized by pharmacological or siRNA-mediated cPLA2 silencing. Our results demonstrate the intracellular functional link between M3 receptor and TRPM8 channel via cPLA2/AA and suggest a novel physiological mechanism of arachidonate-mediated regulation of TRPM8 channel activity through muscarinic receptors. We also summarize the existing TRPM8 regulations and discuss their physiological and pathological significance.

TRPM8-Rap1A Interaction Sites as Critical Determinants for Adhesion and Migration of Prostate and Other Epithelial Cancer Cells.

Emerging evidence indicates that the TRPM8 channel plays an important role in prostate cancer (PCa) progression, by impairing the motility of these cancer cells. Here, we reveal a novel facet of PCa motility control via direct protein-protein interaction (PPI) of the channel with the small GTPase Rap1A. The functional interaction of the two proteins was assessed by active Rap1 pull-down assays and live-cell imaging experiments. Molecular modeling analysis allowed the identification of four putative residues involved in TRPM8-Rap1A interaction. Point mutations of these sites impaired PPI as shown by GST-pull-down, co-immunoprecipitation, and PLA experiments and revealed their key functional role in the adhesion and migration of PC3 prostate cancer cells. More precisely, TRPM8 inhibits cell migration and adhesion by trapping Rap1A in its GDP-bound inactive form, thus preventing its activation at the plasma membrane. In particular, residues E207 and Y240 in the sequence of TRPM8 and Y32 in that of Rap1A are critical for the interaction between the two proteins not only in PC3 cells but also in cervical (HeLa) and breast (MCF-7) cancer cells. This study deepens our knowledge of the mechanism through which TRPM8 would exert a protective role in cancer progression and provides new insights into the possible use of TRPM8 as a new therapeutic target in cancer treatment.

TRPC3 shapes the ER-mitochondria Ca2+ transfer characterizing tumour-promoting senescence.

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.

The transient receptor potential channel TRPM8 is inhibited via the alpha 2A adrenoreceptor signaling pathway.

The transient receptor potential channel melastatin member 8 (TRPM8) is expressed in sensory neurons, where it constitutes the main receptor of environmental innocuous cold (10-25 degrees C). Among several types of G protein-coupled receptors expressed in sensory neurons, G(i)-coupled alpha 2A-adrenoreceptor (alpha 2A-AR), is known to be involved in thermoregulation; however, the underlying molecular mechanisms remain poorly understood. Here we demonstrated that stimulation of alpha 2A-AR inhibited TRPM8 in sensory neurons from rat dorsal root ganglia (DRG). In addition, using specific pharmacological and molecular tools combined with patch-clamp current recordings, we found that in heterologously expressed HEK-293 (human embryonic kidney) cells, TRPM8 channel is inhibited by the G(i) protein/adenylate cyclase (AC)/cAMP/protein kinase A (PKA) signaling cascade. We further identified the TRPM8 S9 and T17 as two key PKA phosphorylation sites regulating TRPM8 channel activity. We therefore propose that inhibition of TRPM8 through the alpha 2A-AR signaling cascade could constitute a new mechanism of modulation of thermosensation in both physiological and pathological conditions.

TRP Channels in Brain Tumors.

Malignant glioma including glioblastoma (GBM) is the most common group of primary brain tumors. Despite standard optimized treatment consisting of extensive resection followed by radiotherapy/concomitant and adjuvant therapy, GBM remains one of the most aggressive human cancers. GBM is a typical example of intra-heterogeneity modeled by different micro-environmental situations, one of the main causes of resistance to conventional treatments. The resistance to treatment is associated with angiogenesis, hypoxic and necrotic tumor areas while heterogeneity would accumulate during glioma cell invasion, supporting recurrence. These complex mechanisms require a focus on potential new molecular actors to consider new treatment options for gliomas. Among emerging and underexplored targets, transient receptor potential (TRP) channels belonging to a superfamily of non-selective cation channels which play critical roles in the responses to a number of external stimuli from the external environment were found to be related to cancer development, including glioma. Here, we discuss the potential as biological markers of diagnosis and prognosis of TRPC6, TRPM8, TRPV4, or TRPV1/V2 being associated with glioma patient overall survival. TRPs-inducing common or distinct mechanisms associated with their Ca2+-channel permeability and/or kinase function were detailed as involving miRNA or secondary effector signaling cascades in turn controlling proliferation, cell cycle, apoptotic pathways, DNA repair, resistance to treatment as well as migration/invasion. These recent observations of the key role played by TRPs such as TRPC6 in GBM growth and invasiveness, TRPV2 in proliferation and glioma-stem cell differentiation and TRPM2 as channel carriers of cytotoxic chemotherapy within glioma cells, should offer new directions for innovation in treatment strategies of high-grade glioma as GBM to overcome high resistance and recurrence.

Molecular mechanisms of TRP regulation in tumor growth and metastasis.

Transient Receptor Potential (TRP) channels modulate intracellular Ca2+ concentrations, consequently affecting both cell death and proliferation. It is not, therefore, surprising that the membrane expression of some TRP channels is altered during tumor growth and metastasis. These variations in channel abundance are due to TRP regulation on the transcriptional, translational, and targeting levels. This article mainly reviews the transcriptional mechanisms modulating TRP expression during tumorigenesis, involving hormones, growth factors, and alternative splicing.