Almost all non-small cell lung cancer (NSCLC) patients initially responding to EGFR tyrosine kinase inhibitors (TKIs) develop acquired resistance. Since K Ca 3.1 channels, expressed in mitochondria and plasma membrane, regulate similar behavioral traits of NSCLC cells as EGFR, we hypothesized that their blockade contributes to overcoming EGFR-TKI resistance. Meta-analysis of microarray data revealed that K Ca 3.1 channel expression in erlotinib-resistant NSCLC cells correlates with that of genes of integrin and apoptosis pathways. Using erlotinib-sensitive and –resistant NSCLC cells we monitored the role of mitochondrial K Ca 3.1 channels in integrin signaling by studying cell-matrix adhesion with single-cell force spectroscopy. Apoptosis was quantified with fluorescence-based assays. The function of mitochondrial K Ca 3.1 channels in these processes was assessed by measuring the mitochondrial membrane potential and by quantifying ROS production. Functional assays were supplemented by biochemical analyses. We show that K Ca 3.1 channel inhibition with senicapoc in erlotinib-resistant NSCLC cells increases cell adhesion by increasing β1-integrin expression, that in turn depends on mitochondrial ROS release. Increased adhesion impairs migration of NSCLC cells in a 3D matrix. At the same time, the senicapoc-dependent ROS production induces cytochrome C release and triggers apoptosis of erlotinib-resistant NSCLC cells. Thus, K Ca 3.1 channel blockade overcomes EGFR-TKI resistance by inhibiting NSCLC motility and inducing apoptosis.

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.

Clathrin-mediated endocytosis (CME) is an essential cell physiological process of broad biomedical relevance. Since the recent introduction of Pitstop-2 as a potent CME inhibitor, we and others have reported on substantial clathrin-independent inhibitory effects. Herein, we developed and experimentally validated a novel fluorescent derivative of Pitstop-2, termed RVD-127, to clarify Pitstop-2 diverse effects. Using RVD-127, we were able to trace additional protein targets of Pitstop-2. Besides inhibiting CME, Pitstop-2 and RVD-127 proved to directly and reversibly bind to at least two members of the small GTPase superfamily Ran and Rac1 with particularly high efficacy. Binding locks the GTPases in a guanosine diphosphate (GDP)-like conformation disabling their interaction with their downstream effectors. Consequently, overall cell motility, mechanics and nucleocytoplasmic transport integrity are rapidly disrupted at inhibitor concentrations well below those required to significantly reduce CME. We conclude that Pitstop-2 is a highly potent, reversible inhibitor of small GTPases. The inhibition of these molecular switches of diverse crucial signaling pathways, including nucleocytoplasmic transport and overall cell dynamics and motility, clarifies the diversity of Pitstop-2 activities. Moreover, considering the fundamental importance and broad implications of small GTPases in physiology, pathophysiology and drug development, Pitstop-2 and RVD-127 open up novel avenues.

The calcium-activated potassium channel 3.1 (KCa 3.1) is overexpressed in many tumor entities and has predictive power concerning disease progression and outcome. Imaging of the KCa 3.1 channel in vivo using a radiotracer for positron emission tomography (PET) could therefore establish a potentially powerful diagnostic tool. Senicapoc shows high affinity and excellent selectivity toward the KCa 3.1 channel. We have successfully pursued the synthesis of the 18 F-labeled derivative [18 F]3 of senicapoc using the prosthetic group approach with 1-azido-2-[18 F]fluoroethane ([18 F]6) in a "click" reaction. The biological activity of the new PET tracer was evaluated in vitro and in vivo. Inhibition of the KCa 3.1 channel by 3 was demonstrated by patch clamp experiments and the binding pose was analyzed by docking studies. In mouse and human serum, [18 F]3 was stable for at least one half-life of [18 F]fluorine. Biodistribution experiments in wild-type mice were promising, showing rapid and predominantly renal excretion. An in vivo study using A549-based tumor-bearing mice was performed. The tumor signal could be delineated and image analysis showed a tumor-to-muscle ratio of 1.47 ± 0.24. The approach using 1-azido-2-[18 F]fluoroethane seems to be a good general strategy to achieve triarylacetamide-based fluorinated PET tracers for imaging of the KCa 3.1 channel in vivo.

Lung cancer is one of the leading causes of cancer-related deaths worldwide. The Ca2+-activated K+ channel KCa3.1 contributes to the progression of non-small cell lung cancer (NSCLC). Recently, KCa3.1 channels were found in the inner membrane of mitochondria in different cancer cells. Mitochondria are the main sources for the generation of reactive oxygen species (ROS) that affect the progression of cancer cells. Here, we combined Western blotting, immunofluorescence, and fluorescent live-cell imaging to investigate the expression and function of KCa3.1 channels in the mitochondria of NSCLC cells. Western blotting revealed KCa3.1 expression in mitochondrial lysates from different NSCLC cells. Using immunofluorescence, we demonstrate a co-localization of KCa3.1 channels with mitochondria of NSCLC cells. Measurements of the mitochondrial membrane potential with TMRM reveal a hyperpolarization following the inhibition of KCa3.1 channels with the cell-permeable blocker senicapoc. This is not the case when cells are treated with the cell-impermeable peptidic toxin maurotoxin. The hyperpolarization of the mitochondrial membrane potential is accompanied by an increased generation of ROS in NSCLC cells. Collectively, our results provide firm evidence for the functional expression of KCa3.1 channels in the inner membrane of mitochondria of NSCLC cells.

Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohisto-chemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.

Expression of the Ca2+ activated potassium channel 3.1 (KCa3.1) channel (also known as the Gàrdos channel) is dysregulated in many tumor entities and has predictive power with respect to patient survival. Therefore, a positron emission tomography (PET) tracer targeting this ion channel could serve as a potential diagnostic tool by imaging the KCa3.1 channel in vivo. It was envisaged to synthesize [18F]senicapoc ([18F]1) since senicapoc (1) shows high affinity and excellent selectivity towards the KCa3.1 channels. Because problems occurred during 18F-fluorination, the [18F]fluoroethoxy senicapoc derivative [18F]28 was synthesized to generate an alternative PET tracer targeting the KCa3.1 channel. Inhibition of the KCa3.1 channel by 28 was confirmed by patch clamp experiments. In vitro stability in mouse and human serum was shown for 28. Furthermore, biodistribution experiments in wild type mice were performed. Since [18F]fluoride was detected in vivo after application of [18F]28, an in vitro metabolism study was conducted. A potential degradation route of fluoroethoxy derivatives in vivo was found which in general should be taken into account when designing new PET tracers for different targets with a [18F]fluoroethoxy moiety as well as when using the popular prosthetic group [18F]fluoroethyl tosylate for the alkylation of phenols.

Ion channels are a major class of membrane proteins that play central roles in signaling within and among cells, as well as in the coupling of extracellular events with cellular responses. Dysregulated ion channel activity plays a causative role in many diseases including cancer. Here, we will review their role in lung cancer. Lung cancer is one of the most frequently diagnosed cancers, and it causes the highest number of deaths of all cancer types. The overall 5-year survival rate of lung cancer patients is only 19% and decreases to 5% when patients are diagnosed with stage IV. Thus, new therapeutical strategies are urgently needed. The important contribution of ion channels to the progression of various types of cancer has been firmly established so that ion channel-based therapeutic concepts are currently developed. Thus far, the knowledge on ion channel function in lung cancer is still relatively limited. However, the published studies clearly show the impact of ion channel inhibitors on a number of cellular mechanisms underlying lung cancer cell aggressiveness such as proliferation, migration, invasion, cell cycle progression, or adhesion. Additionally, in vivo experiments reveal that ion channel inhibitors diminish tumor growth in mice. Furthermore, some studies give evidence that ion channel inhibitors can have an influence on the resistance or sensitivity of lung cancer cells to common chemotherapeutics such as paclitaxel or cisplatin.

Non-small cell lung cancer (NSCLC) has a poor prognosis with a 5 year survival rate of only ~ 10%. Important driver mutations underlying NSCLC affect the epidermal growth factor receptor (EGFR) causing the constitutive activation of its tyrosine kinase domain. There are efficient EGFR tyrosine kinase inhibitors (TKIs), but patients develop inevitably a resistance against these drugs. On the other hand, K Ca 3.1 channels contribute to NSCLC progression so that elevated K Ca 3.1 expression is a strong predictor of poor NSCLC patient prognosis. The present study tests whether blocking K Ca 3.1 channels increases the sensitivity of NSCLC cells towards the EGFR TKI erlotinib and overcomes drug resistance. mRNA expression of K Ca 3.1 channels in erlotinib-sensitive and -resistant NSCLC cells was analysed in datasets from Gene expression omnibus (GEO) and ArrayExpress. We assessed proliferation and migration of NSCLC cells. These (live cell-imaging) experiments were complemented by patch clamp experiments and Western blot analyses. We identified three out of four datasets comparing erlotinib-sensitive and -resistant NSCLC cells which revealed an altered expression of K Ca 3.1 mRNA in erlotinib-resistant NSCLC cells. Therefore, we evaluated the combined effect of erlotinib and the K Ca 3.1 channel inhibition with sencapoc. Erlotinib elicits a dose-dependent inhibition of migration and proliferation of NSCLC cells. The simultaneous application of the K Ca 3.1 channel blocker senicapoc increases the sensitivity towards a low dose of erlotinib (300 nmol/L) which by itself has no effect on migration and proliferation. Partial erlotinib resistance can be overcome by K Ca 3.1 channel blockade. The sensitivity towards erlotinib as well as the potentiating effect of K Ca 3.1 blockade is further increased by mimicking hypoxia. Our results suggest that K Ca 3.1 channel blockade may constitute a therapeutic concept for treating NSCLC and overcome EGFR TKI resistance. We propose that this is due to complementary mechanisms of action of both blockers.

Nuclear pore complexes (NPCs) selectively mediate all nucleocytoplasmic transport and engage in fundamental cell-physiological processes. It is hypothesized that NPCs are critical for malignant transformation and survival of lung cancer cells, and test the hypothesis in lowly and highly metastatic non-small human lung cancer cells (NSCLCs). It is shown that malignant transformation is paralleled by an increased NPCs density, and a balanced pathological weakening of the physiological stringency of the NPC barrier. Pharmacological interference using barrier-breaking compounds collapses the stringency. Concomitantly, it induces drastic overall structural changes of NSCLCs, terminating their migration. Moreover, the degree of malignancy is found to be paralleled by substantially decreased lamin A/C levels. The latter provides crucial structural and mechanical stability to the nucleus, and interacts with NPCs, cytoskeleton, and nucleoskeleton for cell maintenance, survival, and motility. The recent study reveals the physiological importance of the NPC barrier stringency for mechanical and structural resilience of normal cell nuclei. Hence, reduced lamin A/C levels in conjunction with controlled pathological weakening of the NPC barrier stringency may facilitate deformability of NSCLCs during the metastasis steps. Modulation of the NPC barrier presents a potential strategy for suppressing the malignant phenotype or enhancing the effectiveness of currently existing chemotherapeutics.

The importance of the intracellular Ca(2+)concentration ([Ca2+](i)) in neutrophil function has been intensely studied. However, the role of the intracellular Na(+)concentration ([Na+](i)) which is closely linked to the intracellular Ca(2+)regulation has been largely overlooked. The [Na+](i)is regulated by Na(+)transport proteins such as the Na+/Ca2+-exchanger (NCX1), Na+/K+-ATPase, and Na+-permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na+](i). fMLF induces a sustained increase of [Na+](i), surprisingly, reaching higher values in TRPM2(-/-)neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na+](i). The difference in [Na+](i)measured att= 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na(+)concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2(-/-)neutrophils. Increasing the [Na+](i)by inhibiting the Na+/K+-ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na(+)homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra- to intracellular Na(+)concentration gradients. Increasing the [Na+](i)by a few mmol/l may suffice to switch its transport mode from forward (Ca2+-efflux) to reverse (Ca2+-influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis.

Tissue acidosis plays a pivotal role in tumor progression: in particular, interstitial acidosis promotes tumor cell invasion, and is a major contributor to the dysregulation of tumor immunity and tumor stromal cells. The cell membrane and integral membrane proteins commonly act as important sensors and transducers of altered pH. Cell adhesion molecules and cation channels are prominent membrane proteins, the majority of which is regulated by protons. The pathophysiological consequences of proton-sensitive ion channel function in cancer, however, are scarcely considered in the literature. Thus, the main focus of this review is to highlight possible events in tumor progression and tumor immunity where the pH sensitivity of cation channels could be of great importance.

The Ca(2+)activated potassium channel 3.1 (K(Ca)3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of K(Ca)3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes1and2were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the K(Ca)3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of K(Ca)3.1 channels by the probes confirming interaction with the channel. Both probes1and2were able to stain K(Ca)3.1 channels in non-small-cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre-incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes1and2. Staining of the channel with the fluorescently labeled senicapoc derivatives1or2or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained K(Ca)3.1 channels. However, co-staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes1and2label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes1and2bind within the open channel.

•Mechanical stress a is critical mediator of cancer progression.•Mechanosensitive ion channels are functionally up-regulated in cancer.•Ca2+ signaling in cancer is dependent on mechanosensitive ion channels.•Mechanosensitive transporters are functionally cross-linked in a complex manner.

A dysregulated cellular Ca2+ homeostasis is involved in multiple pathologies including cancer. Changes in Ca2+ signaling caused by altered fluxes through ion channels and transporters (the transportome) are involved in all steps of the metastatic cascade. Cancer cells thereby "re-program" and "misuse" the cellular transportome to regulate proliferation, apoptosis, metabolism, growth factor signaling, migration and invasion. Cancer cells use their transportome to cope with diverse environmental challenges during the metastatic cascade, like hypoxic, acidic and mechanical cues. Hence, ion channels and transporters are key modulators of cancer progression. This review focuses on the role of transient receptor potential (TRP) channels in the metastatic cascade. After briefly introducing the role of the transportome in cancer, we discuss TRP channel functions in cancer cell migration. We highlight the role of TRP channels in sensing and transmitting cues from the tumor microenvironment and discuss their role in cancer cell invasion. We identify open questions concerning the role of TRP channels in circulating tumor cells and in the processes of intra- and extravasation of tumor cells. We emphasize the importance of TRP channels in different steps of cancer metastasis and propose cancer-specific TRP channel blockade as a therapeutic option in cancer treatment.

Neutrophil granulocytes are exposed to widely varying microenvironmental conditions when pursuing their physiological or pathophysiological functions such as fighting invading bacteria or infiltrating cancer tissue. Examples for harsh environmental challenges include among others mechanical shear stress during the recruitment from the vasculature or the hypoxic and acidotic conditions within the tumor microenvironment. Chemokine gradients, reactive oxygen species, pressure, matrix elasticity, and temperature can be added to the list of potential challenges. Transient receptor potential (TRP) channels serve as cellular sensors since they respond to many of the abovementioned environmental stimuli. The present review investigates the role of TRP channels in neutrophil granulocytes and their role in regulating and adapting neutrophil function to microenvironmental cues. Following a brief description of neutrophil functions, we provide an overview of the electrophysiological characterization of neutrophilic ion channels. We then summarize the function of individual TRP channels in neutrophil granulocytes with a focus on TRPC6 and TRPM2 channels. We close the review by discussing the impact of the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) on neutrophil granulocytes. Since neutrophil infiltration into PDAC tissue contributes to disease progression, we propose neutrophilic TRP channel blockade as a potential therapeutic option.

Early metastasis leads to poor prognosis of lung cancer patients, whose 5-year survival rate is only 15%. We could recently show that the Ca2+ sensitive K+ channel KCa3.1 promotes aggressive behavior of non-small cell lung cancer (NSCLC) cells and that it can serve as a prognostic marker in NSCLC. Since NSCLC patients die of metastases, we investigated whether KCa3.1 channels contribute to poor patient prognosis by regulating distinct steps of the metastatic cascade. We investigated the extravasation of NSCLC cells and focused on their adhesion to endothelial cells and on transendothelial migration. We quantified the adhesion forces between NSCLC cells and endothelial cells by applying single cell force spectroscopy, and we monitored transendothelial migration using live-cell imaging. Inhibition of KCa3.1 channels with senicapoc or KCa3.1 silencing increases the adhesion force of A549 lung cancer cells to human microvascular endothelial cells (HMEC-1). Western blotting, immunofluorescence staining and biotinylation assays indicate that the elevated adhesion force is due to increased expression of ICAM-1 in both cell lines when KCa3.1 channels are downregulated. Consistent with this interpretation, an anti-ICAM-1 blocking antibody abolishes the KCa3.1-dependent increase in adhesion. Senicapoc inhibits transendothelial migration of A549 cells by 50%. Selectively silencing KCa3.1 channels in either NSCLC or endothelial cells reveals that transendothelial migration depends predominantly on endothelial KCa3.1 channels. In conclusion, our findings disclose a novel function of KCa3.1 channels in cancer. KCa3.1 channels regulate ICAM-1 dependent cell-cell adhesion between endothelial and cancer cells that affects the transmigration step of the metastatic cascade.

Epigenomic changes are an important feature of malignant tumors. How tumor aggressiveness is affected by DNA methylation of specific loci is largely unexplored. In genome‐wide DNA methylation analyses, we identified the KCa3.1 channel gene (KCNN4) promoter to be hypomethylated in an aggressive non–small‐cell lung carcinoma (NSCLC) cell line and in patient samples. Accordingly, KCa3.1 expression was increased in more aggressive NSCLC cells. Both findings were strong predictors for poor prognosis in lung adenocarcinoma. Increased KCa3.1 expression was associated with aggressive features of NSCLC cells. Proliferation and migration of pro‐metastatic NSCLC cells depended on KCa3.1 activity. Mechanistically, elevated KCa3.1 expression hyperpolarized the membrane potential, thereby augmenting the driving force for Ca2+ influx. KCa3.1 blockade strongly reduced the growth of xenografted NSCLC cells in mice as measured by positron emission tomography–computed tomography. Thus, loss of DNA methylation of the KCNN4 promoter and increased KCa3.1 channel expression and function are mechanistically linked to poor survival of NSCLC patients.