David N. DeHart

Voltage-dependent Anion Channels Modulate Mitochondrial Metabolism in Cancer Cells REGULATION BY FREE TUBULIN AND ERASTIN

Background: Metabolites generating mitochondrial membrane potential (ΔΨ) enter through voltage-dependent anion channels (VDAC). Results: VDAC3 contributed to ΔΨ formation more than VDAC1/2. VDAC3 knockdown decreased ATP and NADH/NAD+. Tubulin decreased VDAC1/2 not VDAC3 conductance, an effect antagonized by erastin. Conclusion: Tubulin negatively modulates mitochondrial metabolism by closing VDAC1/2. Significance: Antagonism of tubulin-dependent VDAC closure reverses mitochondrial suppression in Warburg metabolism. Respiratory substrates and adenine nucleotides cross the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC), comprising three isoforms — VDAC1, 2, and 3. We characterized the role of individual isoforms in mitochondrial metabolism by HepG2 human hepatoma cells using siRNA. With VDAC3 to the greatest extent, all VDAC isoforms contributed to the maintenance of mitochondrial membrane potential, but only VDAC3 knockdown decreased ATP, ADP, NAD(P)H, and mitochondrial redox state. Cells expressing predominantly VDAC3 were least sensitive to depolarization induced by increased free tubulin. In planar lipid bilayers, free tubulin inhibited VDAC1 and VDAC2 but not VDAC3. Erastin, a compound that interacts with VDAC, blocked and reversed mitochondrial depolarization after microtubule destabilizers in intact cells and antagonized tubulin-induced VDAC blockage in planar bilayers. In conclusion, free tubulin inhibits VDAC1/2 and limits mitochondrial metabolism in HepG2 cells, contributing to the Warburg phenomenon. Reversal of tubulin-VDAC interaction by erastin antagonizes Warburg metabolism and restores oxidative mitochondrial metabolism.

ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells is Independent of the Adenine Nucleotide Translocator.

Non-proliferating cells oxidize respiratory substrates in mitochondria to generate a protonmotive force (Δp) that drives ATP synthesis. The mitochondrial membrane potential (ΔΨ), a component of Δp, drives release of mitochondrial ATP4− in exchange for cytosolic ADP3− via the electrogenic adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane, which leads to a high cytosolic ATP/ADP ratio up to >100-fold greater than matrix ATP/ADP. In rat hepatocytes, ANT inhibitors, bongkrekic acid (BA), and carboxyatractyloside (CAT), and the F1FO-ATP synthase inhibitor, oligomycin (OLIG), inhibited ureagenesis-induced respiration. However, in several cancer cell lines, OLIG but not BA and CAT inhibited respiration. In hepatocytes, respiratory inhibition did not collapse ΔΨ until OLIG, BA, or CAT was added. Similarly, in cancer cells OLIG and 2-deoxyglucose, a glycolytic inhibitor, depolarized mitochondria after respiratory inhibition, which showed that mitochondrial hydrolysis of glycolytic ATP maintained ΔΨ in the absence of respiration in all cell types studied. However in cancer cells, BA, CAT, and knockdown of the major ANT isoforms, ANT2 and ANT3, did not collapse ΔΨ after respiratory inhibition. These findings indicated that ANT did mediate mitochondrial ATP/ADP exchange in cancer cells. We propose that suppression of ANT contributes to low cytosolic ATP/ADP, activation of glycolysis, and a Warburg metabolic phenotype in proliferating cells.

Erastin-Like Anti-Warburg Agents Prevent Mitochondrial Depolarization Induced by Free Tubulin and Decrease Lactate Formation in Cancer Cells:

In Warburg metabolism, suppression of mitochondrial metabolism contributes to a low cytosolic ATP/ADP ratio favoring enhanced aerobic glycolysis. Flux of metabolites across the mitochondrial outer membrane occurs through voltage-dependent anion channels (VDAC). In cancer cells, free dimeric tubulin induces VDAC closure and dynamically regulates mitochondrial membrane potential (ΔΨ). Erastin, a small molecule that binds to VDAC, antagonizes the inhibitory effect of tubulin on VDAC and hyperpolarizes mitochondria in intact cells. Here, our aim was to identify novel compounds from the ChemBridge DIVERSet library that block the inhibitory effect of tubulin on ΔΨ using cell-based screening. HCC4006 cells were treated with nocodazole (NCZ) to increase free tubulin and decrease ΔΨ in the presence or absence of library compounds. Tetramethylrhodamine methylester (TMRM) fluorescence was assessed by high-content imaging to determine changes in ΔΨ. Compounds were considered positive if ΔΨ increased in the presence of NCZ. Using confocal microscopy, we identified and validated six lead molecules that antagonized the depolarizing effect of NCZ. Lead compounds and erastin did not promote microtubule stabilization, so changes in ΔΨ were independent of tubulin dynamics. The most potent lead compound also decreased lactate formation. These novel small molecules represent a potential new class of anti-Warburg drugs.

Opening of voltage dependent anion channels promotes reactive oxygen species generation, mitochondrial dysfunction and cell death in cancer cells

Graphical abstract Erastin and erastin‐like compounds revert tubulin‐dependent VDAC inhibition to increase VDAC conductance, leading to mitochondrial hyperpolarization (↑&Dgr;&PSgr;m), mitochondrial ROS generation (ROSm) blocked by NAC and MitoQ, mitochondrial dysfunction, and ferroptotic cell death. Figure. No caption available. ABSTRACT Enhancement of aerobic glycolysis and suppression of mitochondrial metabolism characterize the pro‐proliferative Warburg phenotype of cancer cells. High free tubulin in cancer cells closes voltage dependent anion channels (VDAC) to decrease mitochondrial membrane potential (&Dgr;&PSgr;), an effect antagonized by erastin, the canonical promotor of ferroptosis. Previously, we identified six compounds (X1–X6) that also block tubulin‐dependent mitochondrial depolarization. Here, we hypothesized that VDAC opening after erastin and X1–X6 increases mitochondrial metabolism and reactive oxygen species (ROS) formation, leading to ROS‐dependent mitochondrial dysfunction, bioenergetic failure and cell death. Accordingly, we characterized erastin and the two most potent structurally unrelated lead compounds, X1 and X4, on ROS formation, mitochondrial function and cell viability. Erastin, X1 and X4 increased &Dgr;&PSgr; followed closely by an increase in mitochondrial ROS generation within 30–60 min. Subsequently, mitochondria began to depolarize after an hour or longer indicative of mitochondrial dysfunction. N‐acetylcysteine (NAC, glutathione precursor and ROS scavenger) and MitoQ (mitochondrially targeted antioxidant) blocked increased ROS formation after X1 and prevented mitochondrial dysfunction. Erastin, X1 and X4 selectively promoted cell killing in HepG2 and Huh7 human hepatocarcinoma cells compared to primary rat hepatocytes. X1 and X4‐dependent cell death was blocked by NAC. These results suggest that ferroptosis induced by erastin and our erastin‐like lead compounds was caused by VDAC opening, leading to increased &Dgr;&PSgr;, mitochondrial ROS generation and oxidative stress‐induced cell death.

Voltage-Dependent Anion Channels and Tubulin: Bioenergetic Controllers in Cancer Cells

The Warburg phenotype is characterized by enhanced glycolysis and suppression of mitochondrial metabolism. Sustained glycolysis depends on a low cytosolic ATP/ADP ratio that, in turn, occurs when mitochondrial metabolism is decreased. We propose that the voltage-dependent anion channel (VDAC) is a global controller of cancer cell bioenergetics. VDAC located in the mitochondrial outer membrane allows the flux of respiratory substrates, ADP, and Pi into mitochondria and the release of mitochondrial ATP to the cytosol. The tricarboxylic acid cycle generates NADH from the oxidation of respiratory substrates. NADH enters the respiratory chain to generate a proton motive force that maintains mitochondrial membrane potential (ΔΨ) and is utilized to generate ATP. The respiratory chain is also the major cellular source of mitochondrial reactive oxygen species (ROS). α-β tubulin heterodimers decrease VDAC conductance in lipid bilayers. Free tubulin by closing VDAC limits the ingress of respiratory substrates and ATP, thereby decreasing mitochondrial ΔΨ in tumor cells. By this mechanism, fluctuations of free tubulin dynamically regulate VDAC conductance and globally control mitochondrial metabolism, the intracellular flow of energy, and ROS formation. Erastin, a VDAC-binding molecule lethal to certain types of cancer cells, and erastin-like compounds identified in a small molecule screening antagonize the inhibitory effect of tubulin on VDAC. Blockage of tubulin inhibition of VDAC opens the channel to increase mitochondrial metabolism and subsequently decrease glycolysis. Mitochondrial hyperpolarization after VDAC opening also leads to oxidative stress and mitochondrial dysfunction, bioenergetic failure, and cell death. In summary, antagonism of VDAC-tubulin interaction promotes cell death by a “double hit model” characterized by reversion of the pro-proliferative Warburg phenotype and promotion of oxidative stress.

Abstract A88: Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells

Background: Warburg metabolism is characterized by enhanced aerobic glycolysis and suppressed mitochondrial metabolism. Voltage dependent anion channels (VDAC) located in the mitochondrial outer membrane control flux of metabolites into mitochondria. Free α,β-tubulin closes VDAC in planar lipid bilayers (Rostovtseva et al., PNAS 105:18746), and high free tubulin in cancer cells decreases mitochondrial membrane potential (ΔΨ) by limiting ingress of respiratory substrates and ATP that supportΔΨ formation (Maldonado et al., Cancer Res. 70:10192). The small molecule erastin opens VDAC by antagonizing the inhibitory effect of tubulin on VDAC (Maldonado et al., JBC 288:11920). Here, we hypothesized that erastin and erastin-like antagonists of VDAC-tubulin interaction increase mitochondrial redox state, decrease glycolysis, increase mitochondrial formation of reactive oxygen species (ROS) and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of cancer cells. Our AIM was to evaluate the effects of erastin/erastin-like compounds on mitochondrialΔΨ, NAD(P)H+ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 hepatocarcinoma cells. Methods: Confocal/multiphoton fluorescence microscopy assessedΔΨ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF]; MitoSOX Red) and NAD(P)H (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry. Results: In both HepG2 and Huh7, erastin and small molecules X1 and X2 identified in a high-throughput screen increasedΔΨ and NAD(P)H. The three compounds also prevented mitochondrial depolarization by cytosolic high free tubulin induced by nocodazole (microtubule depolymerizing agent). Initial increases ofΔΨ were followed by mitochondrial depolarization occurring 1-2 h after X1 and X2 and 3-4 h after erastin. Lactate generation tested after X1 decreased by 60%. cmDCF and MitoSOX fluorescence increased 30 min after X1 and 60 min after X2 and erastin. The mitochondrially targeted antioxidant MitoQ blocked this increase of ROS. Additionally, erastin caused JNK activation with maximal phosphorylation within 1 h. Both X1 and X2 caused killing of cancer cells (~93% and ~76% respectively), which the antioxidant N-acetylcysteine (100µM) blocked. By contrast, X1 and X2 caused Conclusion: Antagonists of VDAC-tubulin interaction promote mitochondrial metabolism and inhibit glycolysis. These anti-Warburg drugs also cause mitochondrial generation of ROS, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants. Grants: T32DK083262 to DND; DK073336, DK037034 and 14.Z50.31.0028 to JJL; ACS 13-043-01 and COBRE Pilot Project GM103542 to ENM. Citation Format: David N. DeHart, Monika Gooz, John J. Lemasters, John J. Lemasters, Eduardo N. Maldonado. Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A88.