Ethylisopropylamiloride Research Articles

Abstract 375: Lipogenic phenotype is an adaptation to nutrient stress in the tumor microenvironment

Abstract The nutrient and oxygen poor microenvironment combined with acidosis arising from fermentative metabolism imparts strong selection pressure on cancer cells that promotes evolution of more aggressive cancer types. Poor nutrient availability in the tumor microenvironment forces cancer cells to adapt to alternate routes of metabolism, nutrient scavenging and autophagy leading to a shift in their metabolic phenotype. These adaptations promote evolution of invasive and aggressive phenotypes. To test this hypothesis and to characterize the evolution of cancer cells under nutrient stress, we generated nutrient stress adapted cells from MCF7-GFP cells by subjecting them to multiple cycles of nutrient starvation and repletion. Distinct clones that outgrew were selected as nutrient stress adapted cells (NSA). Here we show that MCF7-NSA cells that are adapted to grow in low nutrient conditions exhibited robust accumulation of lipid droplets compared to parental MCF7-GFP cells as shown by confocal microscopy after neutral lipid staining as well as immunofluorescence staining for PLIN2 (Perilipin 2), a lipid droplet coat protein. In addition, MCF7-NSA cells showed elevated expression of the nuclear receptor PPARγ and the adipokine FABP4 indicating an adipogenic/lipogenic signature. Strikingly, these nutrient stress adapted cells were capable of nutrient scavenging by macropinocytosis compared to control cells even under nutrient replete conditions. Macropinocytosis was assessed by DQ-BSA processing and TMR-Dextran uptake. NHE inhibitor EIPA (ethyl isopropyl amiloride), that blocks macropinocytosis, but not receptor mediated endocytosis confirmed dextran uptake through macropinocytosis. Similarly, macropinocytosis inhibitor KH7 blocked dextran uptake in these adapted cells. Furthermore, MCF7-NSA cells showed high expression of TFEB and TFE3, master regulators of autophagy and lysosomogenesis suggesting that the necrotic cell debris scavenged through macropinocytosis and processed through autolysosome machinery might be contributing to de novo lipogenesis. Although control and adapted cells exhibited similar growth kinetics in vitro, when implanted in mammary fat pads (MFP) of NSG mice, NSA cells formed significantly smaller and slow growing primary tumors. Interestingly, 80% of these mice showed metastasis to other organs (lung, liver, kidney) whereas control mice did not develop metastasis as parental MCF7 is not an aggressive cell line in xenograft models. MCF7-NSA cells were more migratory in vitro in wound healing assays and were capable of developing lung metastases in a mouse model where primary tumor from mammary fat pad was resected to allow for metastases to develop. Taken together, our data suggest that lipogenic phenotype confers survival advantage in the harsh tumor microenvironment and selects for more invasive and aggressive phenotypes. Citation Format: Crystal Griffith, Dominique Abrahams, Antonio Ortiz, Alexandra Tassielli, Joseph Johnson, Robert Gatenby, Smitha Pillai. Lipogenic phenotype is an adaptation to nutrient stress in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 375.

Loss of Dja2 accompanies pH deviation in lysosomes and lysosome-related organelles.

The Dja2 knockout (Dja2-/- ) mice had respiratory distress, and >60% died within 2 days after birth. The surviving adult Dja2-/- mice were infertile and the lungs of Dja2-/- mice showed several abnormalities, including the processing defect of prosurfactant protein C in the alveolar epithelial type II cells and the accumulation of glycolipids in enlarged alveolar macrophages. The luminal pH of acidic organelles in Dja2-/- cells was shifted to pH 5.37-5.45. This deviated pH was immediately restored to control levels (pH 4.56-4.65) by the addition of a diuretic, ethyl isopropyl amiloride (EIPA). Although the role of DJA2 in maintaining the pH homeostasis of lysosome-related organelles is currently obscure, this rapid and remarkable pH resilience is best explained by an EIPA-sensitive proton efflux machinery that is disorganized and overactivated due to the loss of Dja2.

NHE1 is Involved in the Cytoskeletal Remodeling and ECM Deposition of Lung Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a progressive lung disease characterized by the deregulation of the wound healing process leaving an accumulation of fibroblasts and scarring tissue. Fibroblasts, under normal conditions, support growth and health of connective tissues and maintain the extracellular matrix (ECM). However, as the disease progresses, increases in profibrotic agonists lead to activation of fibroblasts causing stiffening of the cells, increasing in secretion of ECM proteins in the bronchial. The chronic nature of the disease leads fibroblast differentiating into myofibroblast cells making the tissue less compliant. The affected patient’s alveolar architecture becomes damaged which then leads to lung inflexibility, disruption of gas exchange, respiratory failure, and death. Sodium Hydrogen Exchanger Isoform 1 (NHE1) is an ion transporter protein found in the membrane that mediates cellular pH, cell migration, and cytoskeletal anchoring. NHE1 has also been found to impact the pathophysiological development of cancer and other diseases. Interestingly, a number of signaling pathways which contribute to the activation of tumor cells are also involved in profibrotic behavior in fibroblasts and myofibroblast, the purpose of this study was to investigate if NHE1 worsened IPF and if inhibiting NHE1 would decrease the profibrotic activity of fibrosis. We measured two of the indicators of progressing fibrosis: the remodeling of skeletal proteins through formation of actin stress fibers, and the production and secretion of ECM proteins such as collagen and fibronectin. Actin stress fibers have an important role in cell migration and contractility. To stimulate this formation, cells were treated with three agonists that have all been found to increase fibrotic activity: Transforming Growth Factor‐β (TGF‐β), Serotonin (5HT), and Lysophosphatidic Acid (LPA) in the presence and absence of Ethyl‐Isopropyl Amiloride (EIPA), a NHE1 inhibitor. LPA induced stress fibers 43.94 +/‐ 4.4 % over the control, and both TGF‐β and 5HT also stimulated the formation of stress fibers by 32.08 +/‐ 2.1 and 39.1 +/‐ 4.2 %, respectively, over the control non‐treated cells. In each case, the addition of EIPA blocked stress fibers indicating that all three profibrotic factors required NHE1 activity for cytoskeletal remodeling. Additionally, when any combination of agonists were added in combination or a mixture of all three, it was observed that these combinations seemed to block stress fiber formation in comparison to the control. An initial interpretation hints at a competitive or feed‐back loop to controlling fibrosis. The role of NHE1 in TGF‐ β induced fibroblast‐myofibroblast differentiation is also determined and is identified with expression of alpha smooth muscle actin (α‐SMA) with and without EIPA. Finally, the role of NHE1 in production and secretion of ECM proteins, such as collagen and fibronectin, were investigated to detect changes in these proteins in the presence and absence of agonists and EIPA. This work highlights that NHE1 has a role in supporting profibrotic behavior and may be a novel target to fight IPF.

Amyloid Beta Is Internalized via Macropinocytosis, an HSPG- and Lipid Raft-Dependent and Rac1-Mediated Process.

Intracellular amyloid β peptide (Aβ) accumulation has drawn attention in relation to the pathophysiology of Alzheimer’s disease in addition to its extracellular deposition as senile plaque. Cellular uptake of extracellular Aβ is one of the possible mechanisms by which intracellular Aβ deposits form. Given the relevance of Aβ inside cells, it is important to understand the mechanism by which it is taken up by them. In this study, we elucidated that Neuro2A and SH-SY5Y cells internalize specifically oligomerized Aβ in a time- and dose-dependent manner. The depletion of plasma membrane cholesterol with methyl-β-cyclodextrin or treatment with trypsin diminished the internalization of oAβ, suggesting that the oAβ uptake might be both a lipid raft-dependent and heparan sulfate proteoglycan-mediated process. Treatment with a macropinocytosis inhibitor (ethylisopropyl amiloride and wortmannin) also drastically reduced the uptake of oligomer-Aβ (oAβ). oAβ-treated cells exhibited an increase in Rac1 activity, indicating that macropinocytosis induced by oAβ is regulated by these small GTPases. These findings suggest that macropinocytosis is a major endocytic route through which oAβ42 enters cells.

The proton ATPase inhibitor bafilomycin A1 reduces the release of rhinovirus C and cytokines from primary cultures of human nasal epithelial cells

Rhinovirus species C (RV-C) causes more severe asthma attacks than other rhinovirus species. However, the modulation of RV-C replication by drugs has not been well studied. Primary human nasal epithelial (HNE) cells cultured on filter membranes with air-liquid interface methods were infected with RV-C03, and the levels of RV-C03 RNA collected from the airway surface liquid (ASL) of HNE cells were measured with a SYBR Green assay. Pretreatment of HNE cells with the specific vacuolar H+-ATPase inhibitor bafilomycin A1 reduced the RV-C03 RNA levels in the ASL; inflammatory cytokines, including interleukin (IL)-1β, IL-6 and IL-8, in the supernatant; the mRNA expression of the RV-C receptor cadherin-related family member 3 (CDHR3) in the cells; and the number of acidic endosomes where RV-B RNA enters the cytoplasm. The levels of RV-C03 RNA in the ASL obtained from HNE cells with the CDHR3 rs6967,330 G/A genotype tended to be higher than those obtained from HNE cells with the G/G genotype. Pretreatment with the Na+/H+ exchanger inhibitor ethyl-isopropyl amiloride or either of the macrolides clarithromycin or EM900 also reduced RV-C03 RNA levels in the ASL and the number of acidic endosomes in HNE cells. In addition, significant levels of RV-A16, RV-B14 and RV-C25 RNA were detected in the ASL, and bafilomycin A1 also decreased the RV-C25 RNA levels. These findings suggest that bafilomycin A1 may reduce the release of RV-Cs and inflammatory cytokines from human airway epithelial cells. RV-Cs may be sensitive to drugs, including bafilomycin A1, that increase endosomal pH, and CDHR3 may mediate virus entry through receptor-mediated endocytosis in human airway epithelial cells.

Vacuolar H+-ATPase and Na+/K+-ATPase energize Na+ uptake mechanisms in the nuchal organ of the hyperregulating freshwater crustacean Daphnia magna.

The nuchal organ of the embryos and neonates of the cladoceran, Daphnia magna, has been shown to be a site of Na+ influx and H+, NH4+ and Cl- efflux. This study combines the scanning ion-selective electrode technique with application of inhibitors of specific transporters to assess the mechanisms of Na+ transport across the nuchal organ. Na+ influx across the nuchal organ was inhibited both by inhibitors of the Na+/K+-ATPase (ouabain, bufalin) and by inhibitors of the vacuolar H+-ATPase (bafilomycin, N-ethylmaleimde, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, KM91104, S-nitrosoglutathione). Na+ influx was unaffected by the epithelial Na+ channel blocker benzamil, but was sensitive to ethylisopropyl amiloride and elevated external ammonium concentrations, consistent with roles for Na+/H+ and Na+/NH4+ exchangers in the apical membrane but not Na+ channels. Transport across the basolateral membrane into the haemolymph is proposed to involve the Na+/K+-ATPase and a thiazide-sensitive Na+/Cl- cotransporter.

Ethyl isopropyl amiloride decreases oxidative phosphorylation and increases mitochondrial fusion in clonal untransformed and cancer cells.

Many cancer cells, regardless of their tissue origin or genetic landscape, have increased expression or activity of the plasma membrane Na-H exchanger NHE1 and a higher intracellular pH (pHi) compared with untransformed cells. A current perspective that remains to be validated is that increased NHE1 activity and pHi enable a Warburg-like metabolic reprogramming of increased glycolysis and decreased mitochondrial oxidative phosphorylation. We tested this perspective and find it is not accurate for clonal pancreatic and breast cancer cells. Using the pharmacological reagent ethyl isopropyl amiloride (EIPA) to inhibit NHE1 activity and decrease pHi, we observe no change in glycolysis, as indicated by secreted lactate and intracellular pyruvate, despite confirming increased activity of the glycolytic enzyme phosphofructokinase-1 at higher pH. Also, in contrast to predictions, we find a significant decrease in oxidative phosphorylation with EIPA, as indicated by oxygen consumption rate (OCR). Decreased OCR with EIPA is not associated with changes in pathways that fuel oxidative phosphorylation or with mitochondrial membrane potential but occurs with a change in mitochondrial dynamics that includes a significant increase in elongated mitochondrial networks, suggesting increased fusion. These findings conflict with current paradigms on increased pHi inhibiting oxidative phosphorylation and increased oxidative phosphorylation being associated with mitochondrial fusion. Moreover, these findings raise questions on the suggested use of EIPA-like compounds to limit metabolic reprogramming in cancer cells.

NHE1 exacerbates profibrotic activity in idiopathic pulmonary fibrosis and post‐COVID‐19 infection

Idiopathic pulmonary fibrosis (IPF) is a destructive, aging-related pulmonary disease characterized by formation of lung scar tissue. Like other coronaviruses, SARS-COV-2 leads to long term lung damage in some patients in the form of lung fibrosis. In either disease, aggregation of fibroblasts, cell differentiation and excessive tissue remodeling causes the lung tissue to lose lung compliance and oxygen exchange.Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound transport protein that functions to support cell movement, regulate cell activity, and balance intracellular pH. A multitude of signaling pathways involved in the regulating of NHE1 function are also involved in the profibrotic activity of fibroblasts and myofibroblasts; therefore we investigated if NHE1 is involved in profibrotic activity and tissue damage associated with IPF and COVID-19 infection. Because NHE1 is key in the remodeling of actin stress fibers and is involved in stiffness of both disorders, we first investigated the impact of NHE1 on stress fiber formation using agonists involved with both fibrosis and COVID response. Lung fibroblasts were treated with three agonists: transforming growth factor-β (TGF-β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of an NHE1 inhibitor, ethyl-isopropyl amiloride (EIPA). LPA induced stress fiber formation 65.40+/-3.3 percent over the control, and both 5HT and TGF-β also stimulated the formation of stress fibers by 50.27+/−5.7 and 50.91+/−1.4 percent over the untreated cells. In each case, inhibiting NHE1 with EIPA blocked agonist-activated stress fiber formation, delineating that all three profibrotic factors need NHE1 activity for cytoskeletal remodeling. Another indicator of fibrosis is the fibroblast to myofibroblast transdifferentiation. Myofibroblasts are apoptosis-resistant cells that excessively deposit extracellular matrix proteins; the build up of these cells further compromises the structural integrity of the alveoli and contributes to lung dysfunction. TGF- β, a proinflammatory cytokine, significantly contributes to profibrotic activity by stimulating both fibroblast-myofibroblast transition and excess collagen production. Cells treated with TGF-β to induce cell differentiation showed delayed or significantly reduced myofibroblast transformation in the presence of pharmacological inhibitors of NHE1. Specifically, expression of α-smooth muscle actin (α-SMA), the marker for myofibroblast differentiation, was diminished in each CCL39, LL29, and WI38 cell lines by at least 30 percent and a several day delay of the marker's expression. This study demonstrates the potential for NHE1 as a novel target to fight IPF and lung fibrosis in post-COVID-19 infection.

Investigating the Potential for NHE1 Inhibitors to Function as Adjuvant Therapies in the Treatment of Ovarian Cancer

Each year in the United States over 22,000 women are diagnosed with ovarian cancer leading to more than 14,000 deaths. Differential expression and activity of the Na+-H+ Exchanger Isoform 1 (NHE1) has been demonstrated to regulate cell proliferation in ovarian cancer cells from patients. NHE1 is an 815 amino acid transmembrane transport protein that regulates intracellular pH. NHE1 is hyperactivated in various types of cancer cells leading to a pH inversion that supports both cell proliferation and invasion. A significant issue in ovarian cancer therapy is the development of drug resistance and disease recurrence. In an effort to combat these outcomes, it is hypothesized that combining common ovarian cancer therapeutic agents along with compounds that directly inhibit NHE1 activation will enhance the ability of the therapeutic agents to slow ovarian cancer progression. Two ovarian adenocarcinoma cell lines; CAOV-3 and SKOV-3 are used in this research. Utilizing a proliferation assay, the impact of the ovarian cancer therapeutics, Rucaparib, Olaparib, Carboplatin and Cisplatin, were assessed in the presence and absence of the NHE1 inhibitors Cariporide and Ethylisopropyl Amiloride (EIPA). Dose response curves have been generated for each therapeutic (mean ± SE n=36) to evaluate the impact of combination therapies and supplement IC50 and percent reduction values for each pair. One example includes a 46% reduction in IC50 value for carboplatin alone (47.09 mM) as compared to carboplatin plus Cariporide (25.28 mM) in CAOV-3 cells. Reductions in IC50 values for each of the therapeutics in combination with NHE1 inhibitors ranging from 24% to 92% were seen in both cell lines. This work has been expanded to a three-dimensional environment, investigating the impact of combination therapies in a spheroid assay. To begin to identify a mechanism for NHE1 involvement in ovarian cancer progression and treatment, a steady-state intracellular pH was performed. It has been shown that when chemotherapeutics are paired with NHE1 inhibitors, a synergistic effect is exhibited in which the combination is more proficient at reducing cancer growth than either compound can alone. To confirm this relationship, NHE1 knockout cell lines are being created via CRISPR-Cas9 gene editing. If this relationship is validated, it could allow a reduction in the required drug dosage while maintaining efficacy and hopefully reducing harmful side effects in patients. Our research indicates that the inhibition of NHE1 in combination with standard chemotherapeutics may serve as a potential therapeutic target in ovarian cancer treatment.

Investigating the Role of NHE1 in the Regulation of Changes in Cellular Functions Related to the Development of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by the thickening, stiffening, and scaring of tissue within the lungs. Affected individuals develop shortness of breath and the progressive reduction of respiratory function. Fibrosis ultimately results in low blood flow and hypoxia in the tissue, a microenvironment which is closely associated with the upregulation of the sodium-hydrogen exchanger isoform 1 (NHE1). NHE1 is a ubiquitously expressed protein that exchanges one extracellular Na+ for one intracellular H+. This regulates intracellular pH and cell progression through the cell cycle. When NHE1 is upregulated it supports increased cell proliferation and migration, indicating that it may play a vital role in the behavior of fibroblasts in the development of idiopathic pulmonary fibrosis. We hypothesized that inhibition of NHE1 activation would decrease cell progression towards a fibrotic phenotype in lung fibroblasts. For this assessment, we compared differences in cellular behavior in three cell lines to evaluate the role of NHE1 in these processes. LL29 cells are human fibroblasts derived from a patient diagnosed with idiopathic pulmonary fibrosis; WI-38 cells which are derived from healthy lung tissue; and PSN cells, which are derived from Chinese hamster lung fibroblasts and express only human NHE1.Transforming growth factor beta 1 (TGF-b1), Lysophosphatidic Acid (LPA) and Serotonin (5-hydroxytryptamine) were evaluated for their ability to alter cell proliferation, stress fiber formation, and myofibroblast differentiation in all three cell lines. TGF-b1 is a cytokine that is a growth factor implicated in the pathogenesis of pulmonary fibrosis and is involved in differentiation, proliferation, and matrix production of fibroblasts. Serotonin (5-HT) is a signaling molecule that binds to G-protein coupled receptors supporting the increase in TGF-b1 and its effects in cells. This hormone is involved in wound healing and stimulating inflammation. LPA is a lipid signaling molecule that plays a role in mediating inflammation and activating TGF-b1 signaling. All experiments were performed in the presence and absence of Ethylisopropylamiloride (EIPA), a potent inhibitor of NHE1, to evaluate the role of NHE1 in fibrotic transformation. In PSN cells, stimulation with 10 µM LPA increased stress fiber formation over 2-fold and this stimulation was absent in cells treated with 10 µM EIPA indicating a role for NHE1 in the regulation of stress fiber formation and related cellular processes.

Tissue distribution of polystyrene nanoplastics in mice and their entry, transport, and cytotoxicity to GES-1cells.

With the widespread use of plastics and nanotechnology products, nanoplastics (NPs) have become a potential threat to human health. It is of great practical significance to study and evaluate the distribution of NPs in mice as mammal models and their entry, transport, and cytotoxicity in human cell lines. In this study, we detected the tissue distribution of fluorescent polystyrene nanoplastics (PS-NPs) in mice and assessed their endocytosis, transport pathways, and cytotoxic effects in GES-1cells. We found that PS-NPs were clearly visible in gastric, intestine, and liver tissues of mice and in GES-1cells treated with PS-NPs. Entry of PS-NPs into GES-1cells decreased with the inhibition of caveolae-mediated endocytosis (nystatin), clathrin-mediated endocytosis (chlorpromazine HCl), micropinocytosis (ethyl-isopropyl amiloride), RhoA (CCG-1423), and F-actin polymerization (lantrunculin A). Rac1 inhibitors (NSC 23766) had no significant effect on PS-NPs entering GES-1cells. F-actin levels significantly decreased in CCG-1423-pretreated GES-1cells exposed to PS-NPs. GES-1cell ultrastructural features indicated that internalized PS-NPs can be encapsulated in vesicles, autophagosomes, lysosomes, and lysosomal residues. RhoA, F-actin, RAB7, and LAMP1 levels in PS-NPs-treated GES-1cells were remarkably up-regulated and the Rab5 level was significantly down-regulated compared to levels in untreated cells. PS-NPs treatment decreased cell proliferation rates and increased cell apoptosis. The formation of autophagosomes and autolysosomes and levels of LC3II increased with the length of PS-NPs treatment. The results indicated that cells regulated endocytosis in response to PS-NPs through the RhoA/F-actin signaling pathway and internalized PS-NPs in the cytoplasm, autophagosomes, or lysosomes produced cytotoxicity. These results illustrate the potential threat of NPs pollution to human health.

Abstract A01: Vps34 promotes macropinocytosis in Tsc2-deficient cells

Abstract Purpose: The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is constitutively active in many human cancers and in tuberous sclerosis complex (TSC). mTORC1 hyperactivation drives extensive metabolic reprogramming via several interconnected mechanisms, including glucose and glutamine utilization, nucleic acid synthesis, and lipid synthesis. We recently found that treatment with chloroquine (CQ), a lysosomal/autophagy inhibitor, upregulates the uptake and endosomal processing of cholesterol selectively in TSC2-deficient cells, creating a TSC2-specific dependence on exogenous cholesterol. This dependence on exogenous nutrients and the mechanisms of nutrient uptake may be therapeutically targetable. Our central hypothesis is that Tsc2-deficient cells depend on the uptake of exogenous nutrients via macropinocytosis to maintain cellular metabolic homeostasis and proliferation. Methods and Results: To investigate the role of macropinocytosis-mediated nutrient uptake in Tsc2-deficient cells, we measured the uptake of dextran, a polysaccharide taken up via macropinocytosis. Tsc2-deficient cells showed a striking increase in both dextran (3.6-fold, p<0.001) and exogenous protein uptake (~2-fold, p<0.001) relative to Tsc2-expressing cells. Interestingly, mTOR inhibition using Torin1 decreased dextran uptake (3-fold, p<0.001). Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of dextran in Tsc2-deficient cells (~50%, p<0.0001). Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms (25%, p<0.001), suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Additionally, exogenous protein activates mTORC1 signaling of Tsc2-deficient cells grown in amino acid-free media. Treatment with ethylisopropylamiloride (EIPA) resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p<0.0001). Genetic inhibition of autophagy (Atg5-/- MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p<0.0001). Finally, pharmacologic inhibition of lysosomal function with CQ and macropinocytosis with SAR405 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Conclusions: These data suggest that macropinocytosis is upregulated in Tsc2-deficient cells via a Vps34-dependent mechanism to support tumorigenesis. Macropinocytosis-mediated nutrient uptake represents an unexplored opportunity for therapeutic intervention in TSC and may also be relevant to other diseases characterized by mTORC1 hyperactivation. Citation Format: Charilaos Filippakis, Amine Belaid, Brian Siroky, Constance Wu, Nicola Alesi, Thomas Alesi, Julie Nijmeh, Hilaire Colletta Lam, Ellizabeth Henske. Vps34 promotes macropinocytosis in Tsc2-deficient cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A01.

Native expression of ASIC1a and ASIC1b human homologues in the HEK 293 cell line allows pharmacological evaluation of analgesics targeting acid sensation in humans.

Nociceptors arising from the dorsal root ganglia (DRG) express acid-sensing ion channel-1 (ASIC1) subtypes to mediate the perception of inflammatory and neuropathic pain, and as such, these receptors are attractive targets for the development of analgesics for these painful conditions. Nevertheless, given that the human and rodent DRG differ considerably in subtype proportions of ASIC1 and that the pharmacological properties of rodent ASIC1 subtypes and their human homologues are distinct, ASIC1 inhibitors that demonstrate analgesic properties in rodents may not necessarily be effective in preventing pain in humans. In this study, we show that human embryonic kidney (HEK) 293 cells, which are routinely used as a cellular vehicle for the heterologous expression and pharmacological characterization of receptors and ion channels, natively transcribe the human homologues of ASIC1a and ASIC1b at similar proportions to those found in the human DRG. Importantly, HEK 293 ASIC1 is sensitive to inhibition by amiloride, ethylisopropyl amiloride, and the snake toxin mambalgin-1, but insensitive to inhibition by the ASIC1a inhibitor psalmotoxin-1 when applied at a physiological conditioning pH. Given that the human DRG transcribes the same set of ASIC1 subtypes as HEK 293 cells, our data support the notion that mambalgin-1 may be effective against acid pain sensation in humans. Moreover, our data suggest that the HEK 293 cell line may be a suitable tool for pharmacological screening and characterization of heteromeric human ASIC1.

NHE1 activity is required for profibrotic agonist activation of cytoskeleton remodeling in lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is an untreatable chronic lung disorder that is characterized by an accumulation of fibroblasts and scarring tissue due to aberrant tissue remodeling. The lung tissue loses flexibility as the disease upsurges and the affected patient’s alveolar architecture is destroyed. This leads to a disruption of gas exchange, eventual respiratory failure, and death. Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound protein that moderates cellular pH, supports cell migration, and regulates cell behavior. NHE1 has also been found to impact the pathophysiology of cancer. Considering that a myriad of signaling pathways that activate tumor cells are also involved in profibrotic activity of fibroblasts and myofibroblasts, the purpose of this study was to investigate if NHE1 exacerbates IPF and if inhibiting NHE1 would decrease the advancing fibrosis. We measured one of the indicators of progressing fibrosis, the remodeling of skeletal proteins, through the formation of actin stress fibers. Actin stress fibers have an important role in cell migration and contractility in fibroblasts. To stimulate this formation, cells were treated with three agonists that increase fibrotic activity: transforming growth factor ‐ β(TGF‐β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of ethyl‐isopropyl amiloride (EIPA). LPA induced stress fibers 43.94+/−4.4 percent over the control, and both TGF‐βand 5HT also stimulated the formation of stress fibers by 32.08+/−2.1 and 39.1+/−4.2 percent over the control, non‐treated cells. In each case, addition of EIPA blocked agonist‐induced stress fiber formation, indicating that all three profibrotic factors require NHE1 activity for cytoskeletal remodeling. Interestingly, we also observed that stimulation in any combination of agonists had seemed to block stress fiber formation in comparison to the control. An initial interpretation indicates that the agonists negatively regulate the others in stimulating stress fiber formation. The role of each agonist in fibroblasts isolated from fibrotic patients and control, non‐diseased tissue are presented. Myofibroblasts are a specialized cell type that contribute to fibrosis. The role of NHE1 in TGF‐βinduced fibroblast‐myofibroblast differentiation is also determined. The marker for myofibroblast differentiation, α‐smooth muscle actin (α‐SMA) was measured with and without EIPA to determine the role of NHE1 in advancing disease. Finally, the role of NHE1 in myofibroblasts was investigated with EIPA and stress fiber changes with agonists. This work highlights for the first time that NHE1 has a role supporting profibrotic factors and may be a novel target or adjuvant to fight IPF.

Regulation of Cellular Proliferation and Migration by Palmitoylation of the Na+‐H+ Exchanger Isoform 1 (NHE1)

The Na+‐H+ Exchanger Isoform 1 (NHE1) is an important factor in cellular proliferation and migration of cells in both healthy and diseased tissues. NHE1 has been extensively studied in cancer biology, as activation of NHE1 contributes to the inversion of the pH gradient that is characteristic of the transformation from healthy function to a tumorigenic state. NHE1 is a 12‐pass transmembrane protein consisting of 815 amino acids. The N‐terminal transport domain resides in the plasma membrane and exchanges a single extracellular sodium ion for one intracellular hydrogen ion. NHE1 also has an extended C‐terminal cytoplasmic tail that function as the transporters regulatory domain. The cytoplasmic regulatory domain includes an extensive series of binding motifs for proteins and lipids as well as numerous phosphorylation sites. Recently, it was recognized that NHE1 also undergoes reversible palmitoylation. We hypothesize that this palmitoylation may be utilized to regulate NHE1 activation, thereby regulating cell proliferation and migration. In order to evaluate the role of palmitoylation, 2‐bromopalmitate (2BP), an inhibitor for palmitoylation, along with ethyl‐isopropyl‐amiloride (EIPA), which acts as an inhibitor for NHE1, will be employed in migration and proliferation assays in Human Embryonic Kidney cells (HEK293). CRISPR‐Cas9 technology will also be used to create a knock‐out version of this cell line (HEK293NHE1KO). Further analysis with then be performed to assess effects of palmitoylation in the absence of NHE1. We hypothesize that inhibition of endogenous NHE1 activity and lack of cellular palmitoylation will decrease the potential for growth and migration.

Slc4a11 disruption causes duct cell loss and impairs NaCl reabsorption in female mouse submandibular glands.

Slc4a11, a member of the Slc4 HCO3 − transporter family, has a wide tissue distribution. In mouse salivary glands, the expression of Slc4a11 mRNA was more than eightfold greater than the other nine members of the Slc4 gene family. The Slc4a11 protein displayed a diffuse subcellular distribution in both the acinar and duct cells of mouse submandibular glands (SMG). Slc4a11 disruption induced a significant increase in the Na+ and Cl− concentrations of stimulated SMG saliva, whereas it did not affect the fluid secretion rate in response to either β‐adrenergic or cholinergic receptor stimulation. Heterologous expressed mouse Slc4a11 acted as a H+/OH− transporter that was uncoupled of Na+ or Cl− movement, and this activity was blocked by ethyl‐isopropyl amiloride (EIPA) but not 4,4′‐Diisothiocyanato‐2,2′‐stilbenedisulfonic acid (DIDS). Slc4a11 disruption revealed that Slc4a11 does not play a major role in intracellular pH regulation in mouse salivary gland cells. In contrast, NaCl reabsorption was impaired in the SMG saliva of female compared to male Slc4a11 null mice, which correlated with the loss of duct cells and a decrease in expression of the duct‐cell‐specific transcription factor Ascl3. Together, our results suggest that Slc4a11 expression regulates the number of ducts cells in the mouse SMG and consequently NaCl reabsorption.

Wnt canonical pathway activates macropinocytosis and lysosomal degradation of extracellular proteins

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Wnt treatment markedly increased the endocytosis and degradation in lysosomes of BSA. In this study, we report that in addition to receptor-mediated endocytosis, Wnt also triggers the intake of large amounts of extracellular fluid by macropinocytosis, a nonreceptor-mediated actin-driven process. Macropinocytosis induction is rapid and independent of protein synthesis. In the presence of Wnt, large amounts of nutrient-rich packages such as proteins and glycoproteins were channeled into lysosomes after fusing with smaller receptor-mediated vesicles containing glycogen synthase kinase 3 (GSK3) and protein arginine ethyltransferase 1 (PRMT1), an enzyme required for canonical Wnt signaling. Addition of Wnt3a, as well as overexpression of Disheveled (Dvl), Frizzled (Fz8), or dominant-negative Axin induced endocytosis. Depletion of the tumor suppressors adenomatous polyposis coli (APC) or Axin dramatically increased macropinocytosis, defined by incorporation of the high molecular weight marker tetramethylrhodamine (TMR)-dextran and its blockage by the Na+/H+ exchanger ethylisopropyl amiloride (EIPA). Macropinocytosis was blocked by dominant-negative vacuolar protein sorting 4 (Vps4), indicating that the Wnt pathway is dependent on multivesicular body formation, a process called microautophagy. SW480 colorectal cancer cells displayed constitutive macropinocytosis and increased extracellular protein degradation in lysosomes, which were suppressed by restoring full-length APC. Accumulation of the transcriptional activator β-catenin in the nucleus of SW480 cells was inhibited by methyltransferase inhibition, EIPA, or the diuretic amiloride. The results indicate that Wnt signaling switches metabolism toward nutrient acquisition by engulfment of extracellular fluids and suggest possible treatments for Wnt-driven cancer progression.

Vps34-mediated macropinocytosis in Tuberous Sclerosis Complex 2-deficient cells supports tumorigenesis

Tuberous Sclerosis Complex (TSC), a rare genetic disorder with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivation, is characterized by multi-organ hamartomatous benign tumors including brain, skin, kidney, and lung (Lymphangioleiomyomatosis). mTORC1 hyperactivation drives metabolic reprogramming including glucose and glutamine utilization, protein, nucleic acid and lipid synthesis. To investigate the mechanisms of exogenous nutrients uptake in Tsc2-deficient cells, we measured dextran uptake, a polysaccharide internalized via macropinocytosis. Tsc2-deficient cells showed a striking increase in dextran uptake (3-fold, p < 0.0001) relative to Tsc2-expressing cells, which was decreased (3-fold, p < 0.0001) with mTOR inhibitor, Torin1. Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of Dextran in Tsc2-deficient cells. Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms, suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Treatment with a macropinocytosis inhibitor, ethylisopropylamiloride (EIPA), resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p < 0.0001). Genetic inhibition of autophagy (Atg5−/− MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p < 0.0001). Finally, genetic downregulation of Vps34 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Our findings show that macropinocytosis is upregulated with Tsc2-deficiency via a Vps34-dependent mechanism to support their anabolic state. The dependence of Tsc2-deficient cells on exogenous nutrients may provide novel approaches for the treatment of TSC.

Soft-shelled turtle iridovirus enters cells via cholesterol-dependent, clathrin-mediated endocytosis as well as macropinocytosis.

Ranaviruses are nucleoplasmic large DNA viruses that can cause major economic losses in the aquaculture industry and pose a severe threat to global ecological diversity. The available literature demonstrates that classifiable members of the genus Ranavirus enter cells via multiple and complicated routes. Here, we demonstrated the underlying cellular entry mechanism of soft-shelled turtle iridovirus (STIV) using green fluorescence tagged recombinant virus. Treatment with chlorpromazine, sucrose, ethyl-isopropyl amiloride, chloroquine or bafilomycin A1 all significantly decreased STIV infection, suggesting that STIV uses clathrin-mediated endocytosis and macropinocytosis to enter cells via a pH-dependent pathway. Depletion of cellular cholesterol with methyl-β-cyclodextrin significantly inhibited STIV entry, but neither filipin III nor nystatin did, suggesting that STIV entry was cholesterol dependent but caveola independent. Treatment with dynasore, genistein, ML-7 or cytochalasin D all significantly inhibited STIV infection, indicating that Rac GTPase and myosin II activity were required for the macropinocytosis-like pathway as well as actin polymerization. Our findings suggest that the molecular events involved in STIV entry are not identical to those of other ranavirus isolates. Our results also extend our understanding of the molecular mechanism of iridovirus entry and pathogenesis.

Evaluating the Role of the Na+‐H+ Exchanger Isoform 1 (NHE1) in Non‐small Cell Lung Cancer

Lung cancer is a debilitating disease with 222,000 new cases diagnosed each year and causing 156,000 deaths each year in the United States. Approximately 80% of lung cancers are categorized as non‐small cell lung cancer (NSCLC). Of these NSCLCs, 50% are comprised of adenocarcinomas, or tumors that originated within glandular epithelium, while 30% are due to squamous cell carcinomas, or a cancer that began at in a squamous epithelial lining. The Na+‐H+ Exchanger Isoform 1 (NHE1) acts as a key regulator in cellular proliferation, migration, and metastasis in a variety of solid tumors. NHE1 exchanges 1 extracellular Na+ ion for 1 intracellular H+ ion driving changes in both intracellular and extracellular pH in these developing tumors. To evaluate the role of NHE1 in NSCLC, we employed three cells types: 1) NCI‐H1299 a carcinoma cell line, 2) NCI‐H520 a squamous cell carcinoma cell line, and 3) NCI‐H23 and adenocarcinoma cell line. For each cell type, changing the media conditions from 0.5% serum to 10% serum increased the rate of cell proliferation 2 to 3 fold. This increased proliferation was blocked when the NHE1 inhibitors EIPA (ethylisopropylamiloride) or cariporide were present. Similarly, the change in serum concentration increase cell migrations rates in an NHE1‐dependent manner. To further evaluate the role of NHE1 in these cells, western blot analysis was completed to evaluate NHE1 expression in both low and normal serum conditions for each cell type. We also employed CRISPR‐Cas9 technology to develop an NHE1‐knockout (NHE1‐KO) cell line for each cell type. The rates of cell proliferation and migration where then evaluated in the NHE1‐KO cells as compared to NHE1 expressing cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.