VDAC Regulation of Warburg Metabolism in Hepatocarcinoma

 描述(由申请人提供)：增强糖酵解和抑制线粒体代谢是肝癌和其他癌症的Warburg现象的特征。代谢产物通过外膜上的一个通道进入和离开线粒体：电压依赖性阴离子通道(VDAC)。这一建议的中心假设是，癌细胞中的高游离微管蛋白阻断了VDAC并抑制了VDAC代谢中的氧化磷酸化，逆转了VDAC的微管蛋白抑制具有抗WARBURG效应，增强了氧化磷酸化，减少了糖酵解。基于令人兴奋的新的初步数据，我们进一步假设，VDAC-微管蛋白拮抗剂，包括erastin，逆转了依赖微管蛋白的VDAC抑制，从而导致线粒体超极化，增加了ROS的产生和细胞死亡。因此，在特定的目标1中，我们将表征erastin和其他VDAC-微管蛋白拮抗剂对人肝癌细胞(HepG2、Huh7和Focus)细胞生物能量学(ATP、ADP、AMP、PI、NADH氧化还原状态、磷酸化AMP激酶以及呼吸和糖酵解速率)的影响。我们还将在Huh7小鼠异种移植模型中评估erastin/VDAC-微管蛋白拮抗剂对线粒体膜电位(ΔΨ)和糖酵解表型的影响。高通量筛选后出现的Erastin类化合物将被电生理学确认为VDAC-微管蛋白拮抗剂，进一步评估对细胞生物能量学的影响，并用于创建药效团。在特定的目标2中，我们将确定蛋白激酶A(PKA)激动剂和PKA抑制剂是否在存在和不存在Erastin/VDAC-微管蛋白拮抗剂的情况下以及在VDAC亚型双敲除细胞中改变Huh7、Focus和HepG2细胞的生物能量学。我们还将评估PKA过度表达/沉默对细胞生物能量学的影响。此外，蛋白质组学分析将确定VDAC亚型磷酸化的特定位点。在特定的目标3中，我们将确定Erastin诱导的ROS形成导致细胞死亡的机制。我们预计erastin和其他VDAC-微管蛋白拮抗剂将增加线粒体代谢，ΔΨ和ROS的形成，导致线粒体通透性转变和线粒体生物能量衰竭，最终导致细胞死亡。最终，我们预计拮抗VDAC-微管蛋白相互作用将抑制体内肿瘤的生长。总体而言，该项目将在抑制肝癌细胞线粒体代谢的潜在机制方面产生基本的新知识，并寻找新的药物来阻断VDAC-微管蛋白相互作用，以逆转促增殖的Warburg代谢表型，并选择性地促进细胞毒性氧化应激。

 DESCRIPTION (provided by applicant): 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 (ΔΨ) 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, ΔΨ 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.