Enhanced glycolysis and suppression of mitochondrial metabolism are features of the Warburg phenomenon in hepatocarcinoma and other cancers. Metabolites enter and exit mitochondria through one channel in the outer membrane: the voltage dependent anion channel (VDAC). The central hypothesis of this proposal is that high free tubulin in cancer cells blocks VDAC and suppresses oxidative phosphorylation in Warburg metabolism and that reversal of tubulin inhibition of VDAC has an anti-Warburg effect that enhances oxidative phosphorylation and decreases glycolysis. Based on exciting new preliminary data, we further hypothesize that VDAC-tubulin antagonists, including erastin, reverse tubulin- dependent VDAC inhibition with consequent mitochondrial hyperpolarization, increased ROS generation and cell death. Accordingly in Specific Aim 1, we will characterize the effects of erastin and other VDAC-tubulin antagonists on cellular bioenergetics (ATP, ADP, AMP, Pi, NADH redox state, phospho-AMP kinase, and rates of respiration and glycolysis) in human hepatocarcinoma cells (HepG2, Huh7 and FOCUS). We will also assess in a Huh7 mouse xenograft model the effect of erastin/VDAC-tubulin antagonists on mitochondrial membrane potential (??) and the glycolytic phenotype. Erastin-like compounds that emerged from high throughput screening will be confirmed by electrophysiology as VDAC-tubulin antagonists, further evaluated for effects on cellular bioenergetics and used to create a pharmacophore.
In Specific Aim 2, we will determine if protein kinase A (PKA) agonists and PKA inhibitors alter the bioenergetics of Huh7, FOCUS and HepG2 cells in the presence and absence of erastin/VDAC- tubulin antagonists and in VDAC isoform double knockdown cells. We will also assess the effects of PKA overexpression/silencing on cellular bioenergetics. Additionally, proteomic analysis will determine specific sites of VDAC isoform phosphorylation.
In Specific Aim 3 we will determine mechanisms of erastin-induced ROS formation that leads to cell death. We expect that erastin and other VDAC-tubulin antagonists will increase mitochondrial metabolism, ?? and ROS formation, leading to the mitochondrial permeability transition and mitochondrial bioenergetic failure, culminating in cell death. Ultimately, we expect that antagonizing VDAC- tubulin interaction will suppress tumor growth in vivo. Overall, the project will generate fundamental new knowledge on mechanisms underlying suppression of mitochondrial metabolism in hepatocarcinoma cells and identify new agents that block VDAC-tubulin interaction to revert the pro-proliferative Warburg metabolic phenotype and selectively promote cytotoxic oxidative stress.

Public Health Relevance

Enhanced glycolysis and suppression of mitochondrial metabolism are features of the Warburg phenomenon in cancer cells. Metabolites enter and exit mitochondria through one channel in the outer membrane: the voltage dependent anion channel (VDAC). This project will evaluate the hypothesis that suppression of mitochondrial metabolism in cancer cells is caused by free tubulin-mediated VDAC closure (pro-Warburg effect) and that antagonists of the inhibitory effect of free tubulin on VDAC enhance mitochondrial metabolism (anti-Warburg effect) leading to enhanced formation of reactive oxygen species (ROS), decreased tumor growth and eventually selective death of cancer cells.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Hepatobiliary Pathophysiology Study Section (HBPP)
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Willis, Kristine Amalee
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Medical University of South Carolina
Schools of Pharmacy
United States
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Ramshesh, Venkat K; Lemasters, John J (2018) Imaging of Mitochondrial pH Using SNARF-1. Methods Mol Biol 1782:351-356
DeHart, David N; Lemasters, John J; Maldonado, Eduardo N (2018) Erastin-Like Anti-Warburg Agents Prevent Mitochondrial Depolarization Induced by Free Tubulin and Decrease Lactate Formation in Cancer Cells. SLAS Discov 23:23-33
DeHart, David N; Fang, Diana; Heslop, Kareem et al. (2018) Opening of voltage dependent anion channels promotes reactive oxygen species generation, mitochondrial dysfunction and cell death in cancer cells. Biochem Pharmacol 148:155-162
Fang, Diana; Maldonado, Eduardo N (2018) VDAC Regulation: A Mitochondrial Target to Stop Cell Proliferation. Adv Cancer Res 138:41-69
Lemasters, John J (2017) Evolution of Voltage-Dependent Anion Channel Function: From Molecular Sieve to Governator to Actuator of Ferroptosis. Front Oncol 7:303
Maldonado, Eduardo N (2017) VDAC-Tubulin, an Anti-Warburg Pro-Oxidant Switch. Front Oncol 7:4
Maldonado, Eduardo N; DeHart, David N; Patnaik, Jyoti et al. (2016) ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells Is Independent of the Adenine Nucleotide Translocator. J Biol Chem 291:19642-50