NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

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.

TRPV6 Calcium Channel Targeting by Antibodies Raised against Extracellular Epitopes Induces Prostate Cancer Cell Apoptosis.

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.

Use of 2,6-diaminopurine as a potent suppressor of UGA premature stop codons in cystic fibrosis.

Nonsense mutations are responsible for around 10% of cases of genetic diseases, including cystic fibrosis. 2,6-diaminopurine (DAP) has recently been shown to promote efficient readthrough of UGA premature stop codons. In this study, we show that DAP can correct a nonsense mutation in the Cftr gene in vivo in a new CF mouse model, in utero, and through breastfeeding, thanks, notably, to adequate pharmacokinetic properties. DAP turns out to be very stable in plasma and is distributed throughout the body. The ability of DAP to correct various endogenous UGA nonsense mutations in the CFTR gene and to restore its function in mice, in organoids derived from murine or patient cells, and in cells from patients with cystic fibrosis reveals the potential of such readthrough-stimulating molecules in developing a therapeutic approach. The fact that correction by DAP of certain nonsense mutations reaches a clinically relevant level, as judged from previous studies, makes the use of this compound all the more attractive.

Allosteric cross-talk between the hydrophobic cleft and the BH4 domain of Bcl-2 in control of inositol 1,4,5-trisphosphate receptor activity.

Aim: Inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitous calcium (Ca2+) channel involved in the regulation of cellular fate and motility. Its modulation by anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) plays an important role in cancer progression. Disrupting this interaction could overcome apoptosis avoidance, one of the hallmarks of cancer, and is, thus, of great interest. Earlier reports have shown the involvement of both the Bcl-2 homology 4 (BH4) and the transmembrane domains (TMDs) of Bcl-2 in regulating IP3R activity, while the Bcl-2 hydrophobic cleft was associated primarily with its anti-apoptotic and IP3R-independent action at the mitochondria (Oncotarget. 2016;7:55704-20. doi: 10.18632/oncotarget.11005). The aim of this study was to investigate how targeting the BH3 hydrophobic cleft of Bcl-2 affects IP3R:Bcl-2 interaction. Methods: Organelle membrane-derived (OMD) patch-clamp and circular dichroism (CD) thermal melting experiments were used to elucidate the effects of the ABT-199 (venetoclax) on the IP3R:Bcl-2 interaction. Molecular dynamics (MD) simulations of free and ABT-199 bound Bcl-2 were used to propose a molecular model of such interaction. Results: It was shown that occlusion of Bcl-2's hydrophobic cleft by the drug ABT-199 finely modulates IP3R gating in the low open probability (Po) regime, characteristic of the basal IP3R activity in non-excited cells. Complementary MD simulations allowed to propose a model of this modulation, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2. Conclusions: Bcl-2 is an important regulator of IP3R activity and, thus of Ca2+ release from internal stores and associated processes, including cellular proliferation and death. The presence of multiple regulatory domains in both proteins suggests a complex interaction. Thus, it was found that the occlusion of the hydrophobic cleft of Bcl-2 by ABT-199 disrupts IP3R activity, leading to Bcl-2 rebinding with smaller affinity and lesser inhibitory effect. MDs simulations of free and ABT-199 bound Bcl-2 propose a molecular model of such disruption, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.

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.

Store operated calcium channels in cancer progression.

In recent decades cancer emerged as one of the leading causes of death in the developed countries, with some types of cancer contributing to the top 10 causes of death on the list of the World Health Organization. Carcinogenesis, a malignant transformation causing formation of tumors in normal tissues, is associated with changes in the cell cycle caused by suppression of signaling pathways leading to cell death and facilitation of those enhancing proliferation. Further progression of cancer, during which benign tumors acquire more aggressive phenotypes, is characterized by metastatic dissemination through the body driven by augmented motility and invasiveness of cancer cells. All these processes are associated with alterations in calcium homeostasis in cancer cells, which promote their proliferation, motility and invasion, and dissuade cell death or cell cycle arrest. Remodeling of store-operated calcium entry (SOCE), one of the major pathways regulating intracellular Ca2+ concentration ([Ca2+]i), manifests a key event in many of these processes. This review systematizes current knowledge on the mechanisms recruiting SOCE-related proteins in carcinogenesis and cancer progression.

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.

Organelle membrane derived patches: reshaping classical methods for new targets.

Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp "golden standard", is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP3R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.

Metabolic Disorders and Cancer: Store-Operated Ca2+ Entry in Cancer: Focus on IP3R-Mediated Ca2+ Release from Intracellular Stores and Its Role in Migration and Invasion.

Store-operated calcium entry (SOCE) plays important roles in a multitude of cellular processes, from muscle contraction to cellular proliferation and migration. Dysregulation of SOCE is responsible for the advancement of multiple diseases, ranging from immune diseases, myopathies, to terminal ones like cancer. Naturally, SOCE has been a focus of many studies and review papers which, however, primarily concentrated on the principal players localized to the plasma membrane and responsible for Ca2+ entry into the cell. Much less has been said about other players participating in the entire SOCE event. This review aims to address this shortcoming by discussing the accumulated scientific knowledge focused on the inositol trisphosphate receptors (IP3Rs), principal player responsible for emptying intracellular Ca2+ stores in a majority of cells, and their involvement in regulation of cell migration and invasion in cancer.

The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP3 receptor inhibition.

The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2's hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2ß, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2's C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.

Role of TRP ion channels in cancer and tumorigenesis.

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner.

Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.

Functional and physiopathological implications of TRP channels.

Transient Receptor Potential (TRP) channel proteins are a diverse family of proteins that are expressed in many organisms, tissues and cell types. TRP channels respond to a variety of stimuli, including light, mechanical or chemical stimuli, temperature, pH or osmolarity. In addition, several TRP family members have been identified as downstream molecules in the G protein-coupled receptor signaling pathway. TRP proteins are involved in a variety of cell functions both in non-excitable and excitable cells due to their diverse permeability to cations and their ability to modulate intracellular Ca2+ signaling. Emerging evidence suggests that TRP channel dysfunction significantly contributes to the physiopathology of a number of diseases, including cardiovascular, neurological, metabolic or neoplastic disorders. This review focuses on the implication of TRP proteins in the pathogenesis of some of the most prevalent disorders in human. We summarize the current findings regarding the role of TRP proteins in the development of cardiovascular disease, diabetes mellitus as well as diabetic complications, and tumorigenesis and present TRP proteins as targets of potential diagnostic and therapeutic strategies.

TRP channel-associated factors are a novel protein family that regulates TRPM8 trafficking and activity.

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.

Opiates modulate thermosensation by internalizing cold receptor TRPM8.

Stimulation of µ-opioid receptors (OPRMs) brings powerful pain relief, but it also leads to the development of tolerance and addiction. Ensuing withdrawal in abstinent patients manifests itself with severe symptoms, including cold hyperalgesia, often preventing addicted patients from successfully completing the rehabilitation. Unsurprisingly, OPRMs have been a central point of many studies. Nonetheless, a satisfactory understanding of the pathways leading to distorted sensory responses during opiate administration and abstinence is far from complete. Here, we present a mechanism that leads to modulation by OPRMs of one of the sensory responses, thermosensation. Activation of OPRM1 leads to internalization of a cold-sensor TRPM8, which can be reversed by a follow-up treatment with the inverse OPRM agonist naloxone. Knockout of TRPM8 protein leads to a decrease in morphine-induced cold analgesia. The proposed pathway represents a universal mechanism that is probably shared by regulatory pathways modulating general pain sensation in response to opioid treatment.

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.

Calcium channels and prostate cancer.

Calcium channels significantly contribute to many cellular processes, including such critical ones as proliferation, differentiation, and apoptosis. In prostate cancer, the influence of calcium channels has been known for over 30 years, with the first observations that calcium channel blockers affect the progression of cancer towards more aggressive phase. Later research identified additional classes of channel proteins having an important regulatory role and affecting malignant transformation. This review discusses the accumulated scientific knowledge focused on calcium channel involvement in regulation of cell fate in prostate tissues as well as recent developments in patents targeted at prostate cancer treatments and involving calcium channel modulators.

Ion channnels and transporters in cancer. 5. Ion channels in control of cancer and cell apoptosis.

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.

TRP channels in cell survival and cell death in normal and transformed cells.

TRP channels form a superfamily of channel proteins exhibiting versatile regulatory characteristics with many channels participating in the regulation of Ca(2+) homeostasis and influencing the cell fate. Multitude of evidence is emerging that the colocalization of TRP channels with Ca(2+)-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 Ca(2+) 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.

Phospholipase A2-derived lysophosphatidylcholine triggers Ca2+ entry in dystrophic skeletal muscle fibers.

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca(2+) entry through sarcolemmal cationic channels. Using the Mn(2+) quench method, we show here that Mn(2+) entry triggered by Ca(2+) store depletion but not basal Mn(2+) entry relies on Ca(2+)-independent PLA(2) (iPLA(2)) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx(5cv) mouse. iPLA(2) was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA(2) lipid product lysophosphatidylcholine was found to trigger Ca(2+) entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA(2) and lysophospholipid production may be of interest to reduce Ca(2+) entry and subsequent degeneration of dystrophic muscle.