K2P2.1 channels modulate the pH- and mechanosensitivity of pancreatic stellate cells.

Pancreatic stellate cells (PSCs) are central in the development of acute pancreatitis and tumor fibrosis in pancreatic ductal adenocarcinoma (PDAC). Fibrosis and a unique pH landscape represent characteristic properties of the PDAC microenvironment. Mechanosensitive ion channels are involved in the activation of PSCs. Among these channels, K2P2.1 has not yet been studied in PSCs. K2P2.1 channels are pH- and mechanosensitive. We confirmed K2P2.1 expression in PSCs by RT-qPCR and immunofluorescence. PSCs from K2P2.1+/+ and K2P2.1-/- mice were studied under conditions mimicking properties of the PDAC microenvironment (acidic extracellular pH (pHe), ambient pressure elevated by + 100 mmHg). Migration and the cell area were taken as surrogates for PSC activation and evaluated with live cell imaging. pHe-dependent changes of the membrane potential of PSCs were investigated with DiBAC4(3), a voltage-sensitive fluorescent dye. We observed a correlation between morphological activation and progressive hyperpolarization of the cells in response to changes in pHe and pressure. The effect was in part dependent on the expression of K2P2.1 channels because the membrane potential of K2P2.1+/+ PSCs was always more hyperpolarized than that of K2P2.1-/- PSCs. Cell migration velocity of K2P2.1+/+ cells decreased upon pressure application when cells were kept in an acidic medium (pHe 6.6). This was not the case in K2P2.1-/- PSCs. Taken together, our study highlights the critical role of K2P2.1 channels in the combined sensing of environmental pressure and pHe by PSCs and in coordinating cellular morphology with membrane potential dynamics. Thus, K2P2.1 channels are important mechano-sensors in murine PSCs.

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.

Pancreatic KCa3.1 channels in health and disease.

Ion channels play an important role for regulation of the exocrine and the endocrine pancreas. This review focuses on the Ca2+-regulated K+ channel KCa3.1, encoded by the KCNN4 gene, which is present in both parts of the pancreas. In the islets of Langerhans, KCa3.1 channels are involved in the regulation of membrane potential oscillations characterizing nutrient-stimulated islet activity. Channel upregulation is induced by gluco- or lipotoxic conditions and might contribute to micro-inflammation and impaired insulin release in type 2 diabetes mellitus as well as to diabetes-associated renal and vascular complications. In the exocrine pancreas KCa3.1 channels are expressed in acinar and ductal cells. They are thought to play a role for anion secretion during digestion but their physiological role has not been fully elucidated yet. Pancreatic carcinoma, especially pancreatic ductal adenocarcinoma (PDAC), is associated with drastic overexpression of KCa3.1. For pharmacological targeting of KCa3.1 channels, we are discussing the possible benefits KCa3.1 channel inhibitors might provide in the context of diabetes mellitus and pancreatic cancer, respectively. We are also giving a perspective for the use of a fluorescently labeled derivative of the KCa3.1 blocker senicapoc as a tool to monitor channel distribution in pancreatic tissue. In summary, modulating KCa3.1 channel activity is a useful strategy for exo-and endocrine pancreatic disease but further studies are needed to evaluate its clinical suitability.

Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression.

TRPV6 calcium channel is a prospective target in prostate cancer (PCa) since it is not expressed in healthy prostate while its expression increases during cancer progression. Despite the role of TRPV6 in PCa cell survival and apoptotic resistance has been already established, no reliable tool to target TRPV6 channel in vivo and thus to reduce tumor burden is known to date. Here we report the generation of mouse monoclonal antibody mAb82 raised against extracellular epitope of the pore region of the channel. mAb82 inhibited TRPV6 currents by 90% at 24 µg/ml in a dose-dependent manner while decreasing store-operated calcium entry to 56% at only 2.4 µg/ml. mAb82 decreased PCa survival rate in vitro by 71% at 12 µg/ml via inducing cell death through the apoptosis cascade via activation of the protease calpain, following bax activation, mitochondria enlargement, and loss of cristae, Cyt C release, pro-caspase 9 cleavage with the subsequent activation of caspases 3/7. In vivo, mice bearing either PC3Mtrpv6+/+ or PC3Mtrpv6-/-+pTRPV6 tumors were successfully treated with mAb82 at the dose as low as 100 µg/kg resulting in a significant reduction tumor growth by 31% and 90%, respectively. The survival rate was markedly improved by 3.5 times in mice treated with mAb82 in PC3Mtrpv6+/+ tumor group and completely restored in PC3Mtrpv6-/-+pTRPV6 tumor group. mAb82 showed a TRPV6-expression dependent organ distribution and virtually no toxicity in the same way as mAbAU1, a control antibody of the same Ig2a isotype. Overall, our data demonstrate for the first time the use of an anti-TRPV6 monoclonal antibody in vitro and in vivo in the treatment of the TRPV6-expressing PCa tumors.

TRPV6 Channel Is Involved in Pancreatic Ductal Adenocarcinoma Aggressiveness and Resistance to Chemotherapeutics.

Pancreatic ductal adenocarcinoma (PDAC) stands as a highly aggressive and lethal cancer, characterized by a grim prognosis and scarce treatment alternatives. Within this context, TRPV6, a calcium-permeable channel, emerges as a noteworthy candidate due to its overexpression in various cancers, capable of influencing the cell behavior in different cancer entities. Nonetheless, the exact expression pattern and functional significance of TRPV6 in the context of PDAC remains enigmatic. This study scrutinizes the expression of TRPV6 in tissue specimens obtained from 46 PDAC patients across distinct stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. Subsequently, we analyzed its impact on multiple facets, encompassing Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth, both in vitro and in vivo. Notably, the data indicate a direct correlation between TRPV6 expression levels, tumor stage, and grade, establishing a link between TRPV6 and PDAC proliferation in tissue samples. Decreasing TRPV6 expression via knockdown hampered Ca2+ influx, resulting in diminished proliferation and viability in both cell lines, and cell cycle progression in Panc-1. The knockdown simultaneously led to an increase in apoptotic rates and increased the susceptibility of cells to 5-FU and gemcitabine treatments. Moreover, it accelerated migration and promoted collective movement among Panc-1 cells. Conversely, TRPV6 overexpression yielded opposing outcomes in terms of proliferation in Panc-1 and Capan-1, and the migration of Panc-1 cells. Intriguingly, both TRPV6 knockdown and overexpression diminished the process of tumor formation in vivo. This intricate interplay suggests that PDAC aggressiveness relies on a fine-tuned TRPV6 expression, raising its profile as a putative therapeutic target.