Channels In Cancer Cells Research Articles

The influence of naturally occurring flavonoids, chalcones and statins on the activity of voltage-gated potassium channels Kv1.3 and viability of Kv1.3 channel-expressing cancer cells

Voltage-gated potassium channels Kv1.3 are widely expressed in many cell types, both in the plasma membrane and in the inner mitochondrial membrane (mito Kv1.3 channels). An inhibition of the channels may putatively find clinical application in therapy of some cancers characterised by an over-expression of Kv1.3 channels, such as melanoma, pancreatic ductal adenocarcinoma (PDAC), multiple myeloma and B-type chronic lymphocytic leukaemia (B-CLL). An ability to inhibit Kv1.3 channels in cancer cells is shared by some plant-derived polycyclic compounds from the groups of flavonoids, chalcones and statins. The inhibitory effect on the channels may be significantly augmented when the flavonoids and the chalcones are co-applied with the statins: simvastatin and mevastatin. The inhibition of Kv1.3 channels, presumably mito Kv1.3 channels, may be involved in a pro-apoptotic activity of these compounds on Kv1.3 channel-expressing cancer cells. This pro-apoptotic activity may significantly be improved when the selected flavonoids and chalcones are co-applied with the statins. Therefore, these naturally occurring compounds may putatively find clinical application in therapy of above-mentioned cancers, especially when applied in a combination.

Novel pathophysiological functions of Na+,K+-ATPases and Cl- channels in cancer cells

Na+,K+-ATPases are essential for maintaining the membrane potential in almost all cells, and their catalytic subunits have four isoforms (α1-α4). Volume-regulated anion channel (VRAC) plays an important role in the cell death signaling pathway in addition to its fundamental role in cell volume maintenance. First, we introduce that disruption of actin filaments cause the dysfunction of VRAC, which elicits resistance to cisplatin in the cancer cells. Next, we summarize the cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na+,K+-ATPase α1-isoform (α1NaK) and VRAC in the membrane microdomain of the cancer cells. In this mechanism, sub-micromolar concentrations of cardiac glycosides bind to the receptor-type α1NaK, and generate VRAC activities concomitantly with a deceleration of cancer cell proliferation. Finally, we summarize the pathophysiological function of α3NaK, which is abnormally expressed in the intracellular vesicles of cancer cells. The cancer cell can survive even under loss of anchorage because they have the avoidance mechanism for anoikis. On cancer cell detachment, we found that intracellular α3NaK is translocated to the plasma membrane and this event contributes to the survival of the cells. Interestingly, cardiac glycosides inhibited the α3NaK translocation and cell survival. Our findings may open up new opportunities for the development of cancer medicines.

Molecular Basis for Mambalgin-2 Interaction with Heterotrimeric α-ENaC/ASIC1a/γ-ENaC Channels in Cancer Cells.

Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for cancer therapy. Mambalgin-2 from the Dendroaspis polylepis venom inhibits the α-ENaC/ASIC1a/γ-ENaC heterotrimer more effectively than the homotrimeric ASIC1a channel, initially proposed as the target of mambalgin-2. Although the molecular basis of such mambalgin selectivity remained unclear. Here, we built the models of the complexes of mambalgin-2 with the α-ENaC/ASIC1a/γ-ENaC and ASIC1a channels, performed MD and predicted the difference in the binding modes. The importance of the 'head' loop region of mambalgin-2 for the interaction with the hetero-, but not with the homotrimeric channel was confirmed by site-directed mutagenesis and electrophysiology. A new mode of allosteric regulation of the ENaC channels by linking the thumb domain of the ASIC1a subunit with the palm domain of the γ-ENaC subunit was proposed. The data obtained provide new insights into the regulation of various types of acid-sensing ion channels and the development of new strategies for cancer treatment.

Screening Ion Channels in Cancer Cells.

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

Abstract 6267: The potassium channel SK1, a new target of EWS-FLI1, drives Ewing sarcoma cell proliferation

Abstract Ewing sarcoma (ES) is the second most common primary bone tumor among children and teenagers. The survival rate for patients is about 70%, but drops dramatically to less than 30% when metastases are present at diagnostic. The current treatment consists of polychemotherapy, followed by a surgical resection of the tumor. ES is characterized by a chromosomic translocation, leading, in 85% of the cases, to the formation of the EWS-Fli1 fusion protein, which acts like an oncogene. In order to improve the current treatments and increase the survival rate, our goal is to better understand the ES biology, by studying the role of ionic channels in this tumor. Indeed, studies are emerging on their role in tumorigenesis, showing an abnormal expression of potassium channels in cancer cells, where their functions have been highjacked to promote the tumor development. Among these channels, there are different subfamilies, including the calcium-activated potassium ones, classified according to their conductance. One of them, SK3, has already been described as highly expressed in bone metastases from breast or prostate cancer. RNA-seq analyses from 117 patients‘ biopsies and 6 ES cell lines show a high expression of SK1, an other SKCa channel. These data led us to wonder about the role of SK1 in ES development. Thanks to different ES cell lines, and using different molecular approaches and RNA-interference methods, our work aims to study i) SK1 expression in ES cell lines, ii) its regulation by EWS-Fli1, iii) its involvement in one of the key role of tumorigenesis: the cell proliferation. The RNA-Seq results have been confirmed by RT-qPCR on different ES cell lines. We were also able to show a higher expression of SK1 gene in ES cell lines compared to multiple osteosarcoma cell lines. Then, using luciferase assays and ChIP-Seq analyses, we were able to prove that its expression is directly regulated by EWS-Fli1. Moreover, with inducible shRNA and siRNA, we have shown SK1 involvement in proliferation and clonogenicity of different ES cell lines. In order to understand the molecular mechanisms allowing SK1 channel to be part of the regulation of ES cells proliferation, we studied the effects of its extinction on calcium signaling. These experiments indicate a significant reduced Store Operative Calcium Entry (SOCE) and constitutive calcium entry, both linked with the knockdown of SK1 gene. On the other hand and as expected, the drug activation of SK1 channel has an opposite effect on ES cells, promoting these two calcium entries. These results indicate that the regulation of cell proliferation by this SKCa depends on the intracellular calcium signaling. Overall, our results indicate that SK1 is highly expressed in ES cells and that it is involved in ES development. All of these results seem to suggest SK1 as a potential therapeutic target. This project is supported by INCA, Ligue contre le cancer, Cancéropôle Grand Ouest, and Fondation ARC. Citation Format: Maryne Dupuy, Maxime Gueguinou, Mathilde Mullard, Robel Tesfaye, Jerome Amiaud, Sarah Morice, Benjamin Ory, Francoise Redini, Christophe Vandier, Franck Verrecchia. The potassium channel SK1, a new target of EWS-FLI1, drives Ewing sarcoma cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6267.

Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin.

Simple SummaryIn metastasis, cancer cells migrate away from the tissue where they first were generated and spread in other body compartments. During this process, stationary epithelial cells undergo multiple biochemical changes to become mesenchymal cells that present enhanced migratory abilities. Among numerous factors controlling the mesenchymal phenotype, TGFβ is one of the most important. Nevertheless, little is known about the mechanisms inhibiting the TGFβ biochemical pathways and metastasis remain the main reason of cancer mortality. We discovered that the protein called Kv11.1 potassium channel plays a major role in motility of colon cancer cells. We found that stimulating Kv11.1 activity with specific activator molecules produces a shutdown of the TGFβ pathway at its early stages. Consequently, cancer cell motility is suppressed by a reprogramming of the mesenchymal into epithelial phenotype. This research opens the possibility of using Kv11.1 activator molecules as a potential therapeutic strategy against metastatic colon cancer. Control of ionic gradients is critical to maintain cellular homeostasis in both physiological and pathological conditions, but the role of ion channels in cancer cells has not been studied thoroughly. In this work we demonstrated that activity of the Kv11.1 potassium channel plays a vital role in controlling the migration of colon cancer cells by reversing the epithelial-to-mesenchymal transition (EMT) into the mesenchymal-to-epithelial transition (MET). We discovered that pharmacological stimulation of the Kv11.1 channel with the activator molecule NS1643 produces a strong inhibition of colon cancer cell motility. In agreement with the reversal of EMT, NS1643 treatment leads to a depletion of mesenchymal markers such as SNAIL1, SLUG, TWIST, ZEB, N-cadherin, and c-Myc, while the epithelial marker E-cadherin was strongly upregulated. Investigating the mechanism linking Kv11.1 activity to reversal of EMT into MET revealed that stimulation of Kv11.1 produced a strong and fast inhibition of the TGFβ signaling. Application of NS1643 resulted in de-phosphorylation of the TGFβ downstream effectors R-SMADs by activation of the serine/threonine phosphatase PP2B (calcineurin). Consistent with the role of TGFβ in controlling cancer stemness, NS1643 also produced a strong inhibition of NANOG, SOX2, and OCT4 while arresting the cell cycle in G0/G1. Our data demonstrate that activation of the Kv11.1 channel reprograms EMT into MET by inhibiting TGFβ signaling, which results in inhibition of motility in colon cancer cells.

Synthetic alkyl-ether-lipid promotes TRPV2 channel trafficking trough PI3K/Akt-girdin axis in cancer cells and increases mammary tumour volume

The Transient Receptor Potential Vanilloid type 2 (TRPV2) channel is highly selective for Ca2+ and can be activated by lipids, such as LysoPhosphatidylCholine (LPC). LPC analogues, such as the synthetic alkyl-ether-lipid edelfosine or the endogenous alkyl-ether-lipid Platelet Activating Factor (PAF), modulates ion channels in cancer cells. This opens the way to develop alkyl-ether-lipids for the modulation of TRPV2 in cancer. Here, we investigated the role of 2-Acetamido-2-Deoxy-l-O-Hexadecyl-rac-Glycero-3-PhosphatidylCholine (AD-HGPC), a new alkyl-ether-lipid (LPC analogue), on TRPV2 trafficking and its impact on Ca2+ -dependent cell migration. The effect of AD-HGPC on the TRPV2 channel and tumour process was further investigated using calcium imaging and an in vivo mouse model. Using molecular and pharmacological approaches, we dissected the mechanism implicated in alkyl-ether-lipids sensitive TRPV2 trafficking. We found that TRPV2 promotes constitutive Ca2+ entry, leading to migration of highly metastatic breast cancer cell lines through the PI3K/Akt-Girdin axis. AD-HGPC addresses the functional TRPV2 channel in the plasma membrane through Golgi stimulation and PI3K/Akt/Rac-dependent cytoskeletal reorganization, leading to constitutive Ca2+ entry and breast cancer cell migration (without affecting the development of metastasis), in a mouse model. We describe, for the first time, the biological role of a new alkyl-ether-lipid on TRPV2 channel trafficking in breast cancer cells and highlight the potential modulation of TRPV2 by alkyl-ether-lipids as a novel avenue for research in the treatment of metastatic cancer.

Statins as inhibitors of voltage-gated potassium channels Kv1.3 in cancer cells

Voltage-gated potassium channels are integral membrane proteins selectively permeable for potassium ions and activated upon change of membrane potential. Voltage-gated potassium channels of the Kv1.3 type were discovered both in plasma membrane and in inner mitochondrial membrane (mito Kv1.3 channels). For some time Kv1.3 channels located both in plasma membrane and in mitochondria are considered as a potentially new molecular target in several pathologies including some cancer disorders. Lipophilic nontoxic organic inhibitors of Kv1.3 channels may potentially find a clinical application to support therapy of some cancer diseases such as breast, pancreas and lung cancer, melanoma or chronic lymphocytic leukaemia (B-CLL). Inhibition of T lymphocyte Kv1.3 channels may be also important in treatment of chronic and acute respiratory diseases including severe pulmonary complication in corona virus disease Covid 19, however further studies are needed to confirm this supposition.Statins are small-molecule organic compounds, which are lipophilic and are widely used in treatment of hypercholesterolemia and atherosclerosis. Electrophysiological studies performed in our laboratory showed that statins: pravastatin, mevastatin and simvastatin are effective inhibitors of Kv1.3 channels in cancer cells of human T cell line Jurkat. We showed that application of the statins in the concentration range from 1.5 μM to 50 μM inhibited the channels in a concentration-dependent manner. The inhibitory effect was the most potent in case of simvastatin and the least potent in case of pravastatin. The inhibition was partially irreversible in case of simvastatin and fully reversible in case of pravastatin and mevastatin. It was accompanied by a significant acceleration of the current inactivation rate without any significant change of the activation rate. Mechanism of the inhibition is probably complex, including a direct interaction with the channel protein and perturbation of lipid bilayer structure, leading to stabilization of the inactivated state of the channels.

Ion Channel Regulation by Sex Steroid Hormones and Vitamin D in Cancer: A Potential Opportunity for Cancer Diagnosis and Therapy.

Many ion channels are involved in tumor development, promoting cancer cell proliferation, migration, invasion, and survival. Accordingly, some of them have been suggested as tumor markers and novel targets for cancer therapy. Some sex steroid hormones (SSH), including estrogens and androgens, favor cancer progression. Meanwhile, other steroid hormones like vitamin D may have anticancer properties. SSH and vitamin D modulate the expression of a number of ion channels in cancer cells from hormone-sensitive tissues, including breast, ovary, prostate, and cervix. Moreover, rapid effects of SSH may be mediated by their direct action on membrane ion channels. Here, we reviewed the SSH and vitamin D regulation of ion channels involved in cancer, and analyzed the potential molecular pathways implicated. In addition, we described the potential clinical use of ion channels in cancer diagnosis and therapy, taking advantage of their regulation by SSH and vitamin D. Since SSH are considered risk factors for different types of cancer, and ion channels play important roles in tumor progression, the regulation of ion channels by SSH and vitamin D may represent a potential opportunity for early cancer diagnosis and therapeutic approaches in SSH and vitamin D sensitive tumors.

TTYH1 and TTYH2 Serve as LRRC8A-Independent Volume-Regulated Anion Channels in Cancer Cells.

Volume-regulated anion channels (VRACs) are involved in cellular functions such as regulation of cell volume, proliferation, migration, and cell death. Although leucine-rich repeat–containing 8A (LRRC8A) has been characterized as a molecular component of VRACs, here we show that Drosophila melanogaster tweety homologue 1 and 2 (TTYH1 and TTYH2) are critical for VRAC currents in cancer cells. LRRC8A-independent VRAC currents were present in the gastric cancer cell line SNU-601, but almost completely absent in its cisplatin-resistant derivative SNU-601-R10 (R10). The VRAC current in R10 was partially restored by treatment with trichostatin A (TSA), a histone deacetylase inhibitor. Based on microarray expression profiling of these cells, we selected two chloride channels, TTYH1 and TTYH2, as VRAC candidates. VRAC currents were completely absent from TTYH1- and TTYH2-deficient SNU-601 cells, and were clearly restored by expression of TTYH1 or TTYH2. In addition, we examined the expression of TTYH1 or TTYH2 in several cancer cell lines and found that VRAC currents of these cells were abolished by gene silencing of TTYH1 or TTYH2. Taken together, our data clearly show that TTYH1 and TTYH2 can act as LRRC8A-independent VRACs, suggesting novel therapeutic approaches for VRACs in cancer cells.

Pore-forming spider venom peptides show cytotoxicity to hyperpolarized cancer cells expressing K+ channels: A lentiviral vector approach.

Recent studies demonstrated the upregulation of K+ channels in cancer cells. We have previously found that a pore-forming peptide LaFr26, purified from the venom of the Lachesana sp spider, was selectively incorporated into K+ channel expressing hyperpolarized cells. Therefore, it is expected that this peptide would have selective cytotoxicity to hyperpolarized cancer cells. Here we have tested whether LaFr26 and its related peptide, oxyopinin-2b, are selectively cytotoxic to K+ channel expressing cancer cells. These peptides were cytotoxic to the cells, of which resting membrane potential was hyperpolarized. The vulnerabilities of K+ channel-expressing cell lines correlated with their resting membrane potential. They were cytotoxic to lung cancer cell lines LX22 and BEN, which endogenously expressed K+ current. Contrastingly, these peptides were ineffective to glioblastoma cell lines, U87 and T98G, of which membrane potentials were depolarized. Peptides have a drawback, i.e. poor drug-delivery, that hinders their potential use as medicine. To overcome this drawback, we prepared lentiviral vectors that can express these pore-forming peptides and tested the cytotoxicity to K+ channel expressing cells. The transduction with these lentiviral vectors showed autotoxic activity to the channel expressing cells. Our study provides the basis for a new oncolytic viral therapy.

STIM-Orai Channels and Reactive Oxygen Species in the Tumor Microenvironment.

The tumor microenvironment (TME) is shaped by cancer and noncancerous cells, the extracellular matrix, soluble factors, and blood vessels. Interactions between the cells, matrix, soluble factors, and blood vessels generate this complex heterogeneous microenvironment. The TME may be metabolically beneficial or unbeneficial for tumor growth, it may favor or not favor a productive immune response against tumor cells, or it may even favor conditions suited to hijacking the immune system for benefitting tumor growth. Soluble factors relevant for TME include oxygen, reactive oxygen species (ROS), ATP, Ca2+, H+, growth factors, or cytokines. Ca2+ plays a prominent role in the TME because its concentration is directly linked to cancer cell proliferation, apoptosis, or migration but also to immune cell function. Stromal-interaction molecules (STIM)-activated Orai channels are major Ca2+ entry channels in cancer cells and immune cells, they are upregulated in many tumors, and they are strongly regulated by ROS. Thus, STIM and Orai are interesting candidates to regulate cancer cell fate in the TME. In this review, we summarize the current knowledge about the function of ROS and STIM/Orai in cancer cells; discuss their interdependencies; and propose new hypotheses how TME, ROS, and Orai channels influence each other.

Selenium Enhances the Apoptotic Efficacy of Docetaxel Through Activation of TRPM2 Channel in DBTRG Glioblastoma Cells

The rate of mitosis of cancer cells is significantly higher than normal primary cells with increased metabolic needs, which in turn enhances the generation of reactive oxygen species (ROS) production. Higher ROS production is known to increase cancer cell dependence on ROS scavenging systems to counteract the increased ROS. Therapeutic options which selectively modulate the levels of intracellular ROS in cancers are likely candidates for drug discovery. Docetaxel (DTX) has demonstrated antitumor activity in preclinical and clinical studies. It is thought that DTX induces cell death through excessive ROS production and increased Ca2+ entry. The Ca2+ permeable TRPM2 channel is activated by ROS. Selenium (Se) has been previously used to stimulate apoptosis for the treatment of glioblastoma cells resistant to DTX. However, the potential mechanism(s) of the additive effect of DTX on TRPM2 channels in cancer cells remains unclear. The aim of this study was to evaluate the effect of combination therapy of DTX and Se on activation of TRPM2 in DBTRG glioblastoma cells. DBTRG cells were divided into four treatment groups: control, DTX (10nM for 10h), Se (1μM for 10h), and DTX+Se. Our study showed that apoptosis (Annexin V and propidium iodide), mitochondrial membrane depolarization (JC1), and ROS production levels were increased in DBTRG cells following treatment with Se and DTX respectively. Cell number and viability, and the levels of apoptosis, JC1, ROS, and [Ca2+]i, induced by DTX, were further increased following addition of Se. We also observed an additive increase in the activation of the NAD-dependent DNA repair enzyme poly (ADP-ribose) polymerase-1 (PARP-1) activity, which was accompanied by a decline in its essential substrate NAD+. As well, the Se- and DTX-induced increases in intracellular Ca2+ florescence intensity were decreased following treatment with the TRPM2 antagonist N-(p-amylcinnamoyl) anthranilic acid (ACA). Therefore, combination therapy with Se and DTX may represent an effective strategy for the treatment of glioblastoma cells and may be associated with TRPM2-mediated increases in oxidative stress and [Ca2+]i.

PO-268 The interaction of hERG1 potassium channels with integrin receptors perturbs the force transduction machinery in pancreatic cancer

IntroductionIon channels regulate cell proliferation, differentiation, and migration through cell-cell and cell–extracellular matrix interactions mediated by integrins. K+ flux through the human ether-à-gogo–related gene 1 (hERG1) channel shapes action potential firing in excitable cells, e.g. cardiomyocytes. Its abundance is often aberrantly high in human tumours, including the pancreatic ductal adenocarcinoma (PDAC). We recently demonstrated that the direct interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signalling pathways that depended on the conformational state of hERG1 and affected different aspects of tumour progression.1 We hypothesised that hERG1 channels compromise the PDAC mechano-reciprocity, the ability to dynamically respond to externally applied forces by exerting forces, which enhances invasion and compromises treatment.Material and methodsUsing elastic micropillar arrays of varying stiffness,2 we quantified the β1-integrin mediated forces exerted by PANC-1 cells. Micropillars coating with collagen and/or fibronectin derived peptides allowed us to direct cell surface receptor binding specificity (i.e. α2β1 and α5β1 integrins).Results and discussionsIndependently of the substrate coating, PANC-1 cells exerted higher forces as a function of substrate stiffness, as already demonstrated for other cell types. Remarkably, the disruption of the β1/hERG1 interaction through E4031 inhibition of hERG1 channels resulted in a significant increase in the detected cellular forces. Our results suggest that in addition to alter distinct signalling pathways1 the direct interaction of β1 integrins and hERG1 channels perturbs the force transduction machinery.ConclusionThese findings encouraged us to develop bispecific antibodies (scDb, single-chain diabody) binding to the β1/hERG1 complex that are now being validated in PDAC cell lines. In particular, we have developed a bispecific antibody (scDb-β1/hERG1),3 which is composed by the variable domains (VH and VL chains) of monoclonal antibodies binding two different antigens, hERG1 and β1 integrin.ReferencesBecchetti, et al. Science Signaling2017;10, eaaf3236.van Hoorn, et al. Nano Lett2014;14:4257.A. Arcangeli, C. Duranti, S. Crescioli, L. Carraresi. Patent Ref: 102017000083637 (University of Florence).

Abstract 183: HERG1 potassium channels perturb the β1 integrins mediated force transduction machinery in pancreatic cancer

Abstract Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-gogo-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in human tumors, including the pancreatic ductal adenocarcinoma (PDAC). We recently demonstrated that the direct interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression [1]. We hypothesized that hERG1 channels compromise the PDAC mechano-reciprocity, the ability to dynamically respond to externally applied forces by exerting forces, which enhances invasion and compromises treatment. Using elastic micropillar arrays of varying stiffness (20-150 kPa) [2], we quantified the β1-integrin mediated forces exerted by PANC-1 cells. Micropillars coating with collagen and/or fibronectin derived peptides allowed us to direct cell surface receptor binding specificity (i.e. α2β1 and α5β1 integrins). Independently of the substrate coating, PANC-1 cells exerted higher forces as a function of substrate stiffness, as already demonstrated for other cell types. Remarkably, the disruption of the β1/hERG1 interaction through E4031 inhibition of hERG1 channels resulted in a significant increase in the detected cellular forces by ~20%, accompanied by an increase of focal adhesion areas as demonstrated by staining with vinculin. Our results suggest that, in addition to alter distinct signaling pathways and tumor progression [1], the direct interaction of β1 integrins and hERG1 channels perturbs the force transduction machinery. These findings encouraged us to develop bispecific antibodies (scDb, single-chain diabody) binding to the β1/hERG1 complex that are now being validated in PDAC cell lines. In particular, we have developed a bispecific antibody (scDb-β1/hERG1) [3], which is composed by the variable domains (VH and VL chains) of monoclonal antibodies binding two different antigens, hERG1 and β1 integrin. Data obtained from in vitro studies demonstrates that the scDb antibody impacts cell viability and migratory behavior in Mia PaCa-2 cell lines, holding promises for translational impact after a further validation in vivo as a novel candidate in cancer therapeutics. [1] Becchetti et al. Science Signaling (2017), 10 (473), eaaf3236. [2] van Hoorn et al. Nano Lett (2014), 14, 4257-4262. [3] Patent: “Novel antibodies”. Inventors: Annarosa Arcangeli, Claudia Duranti, Silvia Crescioli, Laura Carraresi. Patent Deposit: July 2017. Patent Ref: 102017000083637 (University of Florence). Citation Format: Stefano Coppola, Claudia Duranti, Annarosa Arcangeli, Thomas Schmidt. HERG1 potassium channels perturb the β1 integrins mediated force transduction machinery in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 183.

Real-Time Imaging of Lithium ‘Hot-Spots’: An Analysis of Ion Conductance in Aquaporin-1 using Novel Photo-Switchable Sensor

Aquaporin-1 (AQP1) belongs to the aquaporin (AQP) protein family that facilitate both the flux of water and other solutes across membranes. In each of the four channel subunits, AQP1 has pores for water conductance, while the central pore of the tetrameric channel contributes the monovalent cation conductance. Among all 13 AQPs, AQP1 is the one that plays a crucial role in cell migration. In various studies, cells with genetic knockdown of AQP1 expression have demonstrated significant impaired cell motility, whereas reintroduction of AQP1 restored cell migration. The mechanism of AQP1 action on cell migration remains unknown, it seems likely to involve ion fluxes. Our previous works have also reported impaired migration of HT29 cancer cells when treated with 2 different AQP1 ion channel antagonists (AqB011 and Bacopaside I). To understating the role ion channel function of AQP1 in rapid cell motility would advance our knowledge of the physiological relevance of aquaporin ion channels. Here we report a newly designed ion sensor named ‘SHL’ that is Li+-selective and photo-switchable. SHL has been used to monitor AQP1 ion channel activity in living colon cancer cells to at real time by the appearance of lithium hot spots imaged using confocal microscopy. In HT29 colon cancer cells, which has higher AQP1-expressing, the Li+ hot spots are clustered in the lamellipodial leading edges. The hot-spots are blocked by the AQP1-ion channel antagonist AqB011. Lithium hot spots are not observable in cell line SW620 that lacks comparable membrane expression of AQP1. The correlation between the subcellular distribution and the visualized ion channel activity of AQP1 channels in cancer cells supports the proposed role of the AQP1 ion channel in cell migration which makes it a novel candidate for therapeutics.

Destroying Deep Lung Tumor Tissue through Lung-Selective Accumulation and by Activation of Caveolin Uptake Channels Using a Specific Width of Carbon Nanodrug.

The main difficulty with current anticancer nanotherapeutics comes from the low efficiency of tumor targeting. Although many strategies have been investigated, including cancer-specific antibody conjugation, lung tumors remain one of the invulnerable types of cancer that must be overcome in the near future. Meanwhile, despite their advantageous physiochemical properties, carbon nanotube structures are not considered safe medical drug delivery agents, but are considered a hazardous source that may cause pulmonary toxicity. However, high-aspect-ratio (width vs. length) nanostructures can be used as very efficient drug delivery agents due to their lung tissue accumulation property. Furthermore, selection of a specific width of the carbon nanostructures can activate additional caveolin uptake channels in cancer cells, thereby maximizing internalization of the nanodrug. The present study aimed to evaluate the therapeutic potential of carbon nanotube-based nanodrugs having various widths (10-30 nm, 60-100 nm, and 125-150 nm) as a delivery agent to treat lung tumors. The results of the present study provided evidence that both lung tissue accumulation (passive targeting) and caveolin-assisted uptake (active targeting) can simultaneously contribute to the destruction of lung tumor tissues of carbon nanotube.

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression.

Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-go-go-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the β1 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the β1 integrin-hERG1 interaction. Although open hERG1 channels did not interact as strongly with β1 integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/β1 integrin interaction was disrupted. We conclude that the interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.

Abstract B07: Two-pore domain potassium (K+) channel genes and triple-negative (TN) subtype in The Cancer Genome Atlas (TCGA) breast cancer dataset

Abstract Introduction: The family of 2-pore domain K+ (K2P) channel genes has 15 members, are background channels which enable the leak of K+ ions from cells, and are considered to be important for baseline activity of cells at rest including membrane potential, calcium homeostasis and volume regulation. Prior studies support the hypothesis that alterations of expression or function of K2P channels in cancer cells may play a significant role in tumor development and progression. In the vast majority of tumors, the abnormally expressed channel is wild type. The role of K2P channel genes in breast cancer is currently emerging. A recent microarray database study suggests that all but 5 K2P gene family members showed altered expression in breast cancer. Other studies suggest potential clinical utility of K2P channel genes as biomarkers associated with TN subtype. A weighted gene co-expression network analysis study showed that upregulation of KCNK5 was associated with poor outcome for TN related tumors. TN tumors are also known to occur more frequently in women of non-Hispanic (nH) black race and association between black race and unique KCNK4 and KCNK9 breast tumor methylation patterns have been suggested; KCNK9 association with TN subtype has also been observed. However, there is a paucity of studies characterizing K2P genes in clinical breast cancer, and the goal of our study was the systematic evaluation of the relationship between K2P gene DNA methylation/expression and TN breast tumor subtype, in the large publically available TCGA dataset. Methods: TCGA invasive breast cancer data was available for 1040 women of which 767 had Illumina HM450 methylation beta-values and 959 had RSEM mRNA expression z-scores (single values from level 3 data). We evaluated the direction and association of all K2P gene expression/methylation loci and TN subtype using age and race adjusted glm models. Age and race adjusted Cox models were used for analysis of disease free (DFS) and overall survival (OS). CpG methylation loci within 25kb from either end of the genes of interest were included for examination (UCSC genome browser, hg37). A total of 724 CG loci were included using this approach, but after exclusion of probes with null reads, the number of loci available for study was reduced to 608. Methylation glm model results were sorted on p-values (smallest to largest) and the top 10 associated loci were selected for reporting and further analysis. Selected loci were then checked for functionality related to expression using Spearman's correlation. Bonferroni corrected p-values were used where appropriate. Results: Overexpression of KCNK5, KCNK9, KCNK10 and KCNK12, and underexpression of KCNK6 and KCNK15, were significantly associated with TN subtype (all p<0.0033). nH black race was also associated with overexpression of KCNK5 and KCNK12, and underexpression of KCNK6 and KCNK15. With regards to survival, only KCNK9 was significantly associated with DFS (hazard ratio 1.05; 95% confidence interval, 1.01-1.09) and marginally associated with OS. Monotonicity of increased survival hazards was also observed when KCNK9 gene expression was modeled as an indicator variable categorized as tertiles. For nH black race, the most significantly associated CG loci were: 4 hypermethylated loci in the promoter region of KCNK15, 3 loci (both hyper- and hypomethylated) near KCNK4 (both body and promoter), 2 hypomethylated loci in KCNK5 gene body, and 1 hypomethylated locus in KCNK2 gene body; we did not find any race associated loci on KCNK9. In age and race adjusted models, the top K2P methylation loci associated with TN subtype were found on KCNK2, KCNK4, KCNK5, KCNK9, KCNK10, KCNK12 and KCNK15 (all p<8.22*(10)-8); and loci on KCNK4, KCNK5, KCNK9, and KCNK12 were hypomethylated. In addition, specific loci on KCNK2 (cg04923840, gene body), KCNK5 (cg05255811, cg18705155, cg09130674, gene body) and KCNK9 (cg21415530, TSS1500) were significantly correlated with negative gene expression within TN subtype. Another locus (cg00981060, located between MSH2 and KCNK12) was correlated with positive gene expression. Only one CG locus among the top reported for association with TN subtype (cg01708924 near KCNK4/PRDX5) was significantly associated with DFS. Conclusions: TN subtype is associated with specific K2P channel gene over and underexpression patterns, and similar expression patterns observed in blacks is consistent with more frequent TN disease. Our findings also suggest a link between KCNK9 overexpression and worse survival in breast cancer. Both KCNK5 and KCNK9 showed hypomethylation patterns correlated with negative expression, and overall gene overexpression related to TN subtype, and these findings appear to be consistent with prior literature. Citation Format: Keith A. Dookeran, Maria Argos. Two-pore domain potassium (K+) channel genes and triple-negative (TN) subtype in The Cancer Genome Atlas (TCGA) breast cancer dataset. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B07.

Activation of Ca 2+ ‐activated K + channel (SK4) Rescues Squamous Cancer Cells from Iononmycin‐induced Cell Death

The ion channels in cancer cells are drawing attention regarding cell proliferation and migration. Intracellular Ca2+ ([Ca2+]i)-dependent signaling has crucial influence to the fate of cancer. Here we investigate the role of Ca2+-activated K+ channels in head and neck squamous carcinoma cells (HNSCC); SNU-1076, OSC-19 and HN5. Treatment with 1 μM ionomycin (24-48 h) induced cell death in all the three cell lines. Whole- cell patch clamp study revealed the functional expression of Ca2+-activated Cl- chan- nels (CaCC, Ano-1) and Ca2+-activated nonselective cation channels (CAN). Ca2+- activated K+ channel (SK4) activity was variable between cell lines; at a raised[Ca2+]i (0.6 μM) or with 1 μM ionomycin, only SNU-1076 showed prominent SK4 current (ISK4). However, an application of SK4 activator (1-EBIO) induced robust ISK4 with membrane hyperpolarization in OSC-19 as well as SNU-1076 while not in in HN5 cells. The EBIO-induced ISK4 was completely suppressed by TRAM-34, a selec-tive SK4 blocker. Interestingly, the ionomycin-induced cell death was effectively prevented by 1 - 30 μM 1-EBIO in SNU-1076 and OSC-19. Consistently, the rescue effect was annihilated by combined treatment with TRAM-34. The rescue by 1-EBIO was not effective in HN5. Above results newly demonstrate the role of SK4 and membrane hyperpolarization in HNSCCs' proliferation and death. Pharmacological modulation of SK4 might provide an intriguing novel tool for the anti-cancer strategy in HNSCC.