Targeting KCa3.1 channels to overcome erlotinib resistance in non-small cell lung cancer cells.

Almost all non-small cell lung cancer (NSCLC) patients initially responding to EGFR tyrosine kinase inhibitors (TKIs) develop acquired resistance. Since KCa3.1 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 KCa3.1 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 KCa3.1 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 KCa3.1 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 KCa3.1 channel inhibition with senicapoc in erlotinib-resistant NSCLC cells increases cell adhesion by increasing ß1-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, KCa3.1 channel blockade overcomes EGFR-TKI resistance by inhibiting NSCLC motility and inducing apoptosis.

The context-dependent role of the Na+/Ca2+-exchanger (NCX) in pancreatic stellate cell migration.

Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger Ca2+ signaling and cellular Na+ loading. The Na+/Ca2+ exchanger (NCX) connects the cellular Ca2+ and Na+ homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca2+ against 3 Na+ ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca2+]i, [Na+]i, and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC4(3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca2+]i, [Na+]i, and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and - 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca2+ contributes to a slow mode of migration of PSCs.

Imaging of KCa 3.1 Channels in Tumor Cells with PET and Small-Molecule Fluorescent Probes.

The Ca2+ activated K+ channel KCa 3.1 is overexpressed in several human tumor cell lines, e. g. clear cell renal carcinoma, prostate cancer, non-small cell lung cancer. Highly aggressive cancer cells use this ion channel for key processes of the metastatic cascade such as migration, extravasation and invasion. Therefore, small molecules, which are able to image this KCa 3.1 channel in vitro and in vivo represent valuable diagnostic and prognostic tool compounds. The [18 F]fluoroethyltriazolyl substituted senicapoc was used as positron emission tomography (PET) tracer and showed promising properties for imaging of KCa 3.1 channels in lung adenocarcinoma cells in mice. The novel senicapoc BODIPY conjugates with two F-atoms (9 a) and with a F-atom and a methoxy moiety (9 b) at the B-atom led to the characteristic punctate staining pattern resulting from labeling of single KCa 3.1 channels in A549-3R cells. This punctate pattern was completely removed by preincubation with an excess of senicapoc confirming the high specificity of KCa 3.1 labeling. Due to the methoxy moiety at the B-atom and the additional oxyethylene unit in the spacer, 9 b exhibits higher polarity, which improves solubility and handling without reduction of fluorescence quantum yield. Docking studies using a cryo-electron microscopy (EM) structure of the KCa 3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.

Functional expression of mitochondrial KCa3.1 channels in non-small cell lung cancer cells.

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.

A redox-dependent thiol-switch and a Ca2+ binding site within the hinge region hierarchically depend on each other in α7ß1 integrin regulation.

Integrin-mediated cell contacts with the extracellular matrix (ECM) are essential for cellular adhesion, force transmission, and migration. Several effectors, such as divalent cations and redox-active compounds, regulate ligand binding activities of integrins and influence their cellular functions. To study the role of the Ca2+ binding site within the hinge region of the integrin α7 subunit, we genetically abrogated it in the α7hiΔCa mutant. This mutant folded correctly, associated with the ß1 subunit and was exposed on the cell surface, but showed reduced ligand binding and weaker cell adhesion to laminin-111. Thus, it resembles the α7hiΔSS mutant, in which the redox-regulated pair of cysteines, closeby to the Ca2+ binding site within the hinge, was abrogated. Comparing both mutants in adhesion strength and cell migration revealed that both Ca2+ complexation and redox-regulation within the hinge interdepend on each other. Moreover, protein-chemical analyses of soluble integrin ectodomains containing the same α7 hinge mutations suggest that integrin activation via the subunit α hinge is primed by the formation of the cysteine pair-based crosslinkage. Then, this allows Ca2+ complexation within the hinge, which is another essential step for integrin activation and ligand binding. Thus, the α hinge is an allosteric integrin regulation site, in which both effectors, Ca2+ and redox-active compounds, synergistically and hierarchically induce far-ranging conformational changes, such as the extension of the integrin ectodomain, resulting in integrin activation of ECM ligand binding and altered integrin-mediated cell functions.

Orai3 Calcium Channel Regulates Breast Cancer Cell Migration through Calcium-Dependent and -Independent Mechanisms.

Orai3 calcium (Ca2+) channels are implicated in multiple breast cancer processes, such as proliferation and survival as well as resistance to chemotherapy. However, their involvement in the breast cancer cell migration processes remains vague. In the present study, we exploited MDA-MB-231 and MDA-MB-231 BrM2 basal-like estrogen receptor-negative (ER-) cell lines to assess the direct role of Orai3 in cell migration. We showed that Orai3 regulates MDA-MB-231 and MDA-MB-231 BrM2 cell migration in two distinct ways. First, we showed that Orai3 remodels cell adhesive capacities by modulating the intracellular Ca2+ concentration. Orai3 silencing (siOrai3) decreased calpain activity, cell adhesion and migration in a Ca2+-dependent manner. In addition, Orai3 interacts with focal adhesion kinase (FAK) and regulates the actin cytoskeleton, in a Ca2+-independent way. Thus, siOrai3 modulates cell morphology by altering F-actin polymerization via a loss of interaction between Orai3 and FAK. To summarize, we demonstrated that Orai3 regulates cell migration through a Ca2+-dependent modulation of calpain activity and, in a Ca2+-independent manner, the actin cytoskeleton architecture via FAK.

KCa channel blockers increase effectiveness of the EGF receptor TK inhibitor erlotinib in non-small cell lung cancer cells (A549).

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, KCa3.1 channels contribute to NSCLC progression so that elevated KCa3.1 expression is a strong predictor of poor NSCLC patient prognosis. The present study tests whether blocking KCa3.1 channels increases the sensitivity of NSCLC cells towards the EGFR TKI erlotinib and overcomes drug resistance. mRNA expression of KCa3.1 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 KCa3.1 mRNA in erlotinib-resistant NSCLC cells. Therefore, we evaluated the combined effect of erlotinib and the KCa3.1 channel inhibition with sencapoc. Erlotinib elicits a dose-dependent inhibition of migration and proliferation of NSCLC cells. The simultaneous application of the KCa3.1 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 KCa3.1 channel blockade. The sensitivity towards erlotinib as well as the potentiating effect of KCa3.1 blockade is further increased by mimicking hypoxia. Our results suggest that KCa3.1 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.

Targeting Kca3.1 Channels in Cancer.

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

Ion Channels in Lung Cancer.

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.

pH-Channeling in Cancer: How pH-Dependence of Cation Channels Shapes Cancer Pathophysiology.

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.