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Abstract Glioblastoma (GBM) is the most aggressive glial tumor, where ion channels, including KCa1.1, are candidates for new therapeutic options. Since the auxiliary subunits linked to KCa1.1 in GBM are largely unknown we used electrophysiology combined with pharmacology and gene silencing to address the functional expression of KCa1.1/β subunits complexes in both primary tumor cells and in the glioblastoma cell line U-87 MG. The pattern of the sensitivity (activation/inhibition) of the whole-cell currents to paxilline, lithocholic acid, arachidonic acid, and iberiotoxin; the presence of inactivation of the whole-cell current along with the loss of the outward rectification upon exposure to the reducing agent DTT collectively argue that KCa1.1/β3 complex is expressed in U-87 MG. Similar results were found using human primary glioblastoma cells isolated from patient samples. Silencing the β3 subunit expression inhibited carbachol-induced Ca2+ transients in U-87 MG thereby indicating the role of the KCa1.1/β3 in the Ca2+ signaling of glioblastoma cells. Functional expression of the KCa1.1/β3 complex, on the other hand, lacks cell cycle dependence. We suggest that the KCa1.1/β3 complex may have diagnostic and therapeutic potential in glioblastoma in the future.

Pancreatic ductal adenocarcinoma (PDAC) progresses in an organ with a unique pH landscape, where the stroma acidifies after each meal. We hypothesized that disrupting this pH landscape during PDAC progression triggers pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) to induce PDAC fibrosis. We revealed that alkaline environmental pH was sufficient to induce PSC differentiation to a myofibroblastic phenotype. We then mechanistically dissected this finding, focusing on the involvement of the Na+/H+ exchanger NHE1. Perturbing cellular pH homeostasis by inhibiting NHE1 with cariporide partially altered the myofibroblastic PSC phenotype. To show the relevance of this finding in vivo, we targeted NHE1 in murine PDAC (KPfC). Indeed, tumor fibrosis decreased when mice received the NHE1-inhibitor cariporide in addition to gemcitabine treatment. Moreover, the tumor immune infiltrate shifted from granulocyte rich to more lymphocytic. Taken together, our study provides mechanistic evidence on how the pancreatic pH landscape shapes pancreatic cancer through tuning PSC differentiation.

Fluorometry signals indicating conformational change in an ion channel are generated by quenching amino acids and lipid effects during movement of the dye relative to the plane of the membrane.

Abstract Loss of apical-basal polarity and downregulation of cell-cell contacts is a critical step during the pathogenesis of cancer. Both processes are regulated by the scaffolding protein Pals1, however, it is unclear whether the expression of Pals1 is affected in cancer cells and whether Pals1 is implicated in the pathogenesis of the disease. Using mRNA expression data and immunostainings of cancer specimen, we show that Pals1 is frequently downregulated in colorectal cancer, correlating with poorer survival of patients. We further found that Pals1 prevents cancer cell metastasis by controlling Rac1-dependent cell migration through inhibition of Arf6, which is independent of the canonical binding partners of Pals1. Loss of Pals1 in colorectal cancer cells results in increased Arf6 and Rac1 activity, enhanced cell migration and invasion in vitro and increased metastasis of transplanted tumor cells in mice. Thus, our data reveal a new function of Pals1 as a key inhibitor of cell migration and metastasis of colorectal cancer cells. Notably, this new function is independent of the known role of Pals1 in tight junction formation and apical-basal polarity.

Abstract 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 Ca2+ 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 Ca2+ 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 at t = 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.

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.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an acidic and fibrotic stroma. The extracellular matrix (ECM) causing the fibrosis is primarily formed by pancreatic stellate cells (PSCs). The effects of the altered biomechanics and pH landscape in the pathogenesis of PDAC, however, are poorly understood. Mechanotransduction in cells has been linked to the function of mechanosensitive ion channels such as Piezo1. Here, we tested whether this channel plays crucial roles in transducing mechanical signals in the acidic PDAC microenvironment. We performed immunofluorescence, Ca2+ influx and intracellular pH measurements in PSCs and complemented them by live-cell imaging migration experiments in order to assess the function of Piezo1 channels in PSCs. We evaluated whether Piezo1 responds to changes of extracellular and/or intracellular pH in the pathophysiological range (pH 6.6 and pH 6.9, respectively). We validated our results using Piezo1-transfected HEK293 cells as a model system. Indeed, acidification of the intracellular space severely inhibits Piezo1-mediated Ca2+ influx into PSCs. In addition, stimulation of Piezo1 channels with its activator Yoda1 accelerates migration of PSCs on a two-dimensional ECM as well as in a 3D setting. Furthermore, Yoda1-activated PSCs transmit more force to the surrounding ECM under physiological pH, as revealed by measuring the dislocation of microbeads embedded in the surrounding matrix. This is paralleled by an enhanced phosphorylation of myosin light chain isoform 9 after Piezo1 stimulation. Intriguingly, upon acidification, Piezo1 activation leads to the initiation of cell death and disruption of PSC spheroids. In summary, stimulating Piezo1 activates PSCs by inducing Ca2+ influx which in turn alters the cytoskeletal architecture. This results in increased cellular motility and ECM traction, which can be useful for the cells to invade the surroundings and to detach from the tissue. However, in the presence of an acidic extracellular pH, although net Ca2+ influx is reduced, Piezo1 activation leads to severe cell stress also limiting cellular viability. In conclusion, our results indicate a strong interdependence between environmental pH, the mechanical output of PSCs and stromal mechanics, which promotes early local invasion of PDAC cells.