Abstract 3211: Inhibition of Hsp90 impairs expression of VDAC in plasma and mitochondrial membrane influencing cancer cell metabolism

Abstract

Abstract Voltage dependent anion channels (VDACs) play an important role in transport through the outer mitochondrial membrane. Importantly, mitochondrial VDACs (mVDACs) are crucial for glycolysis, the predominant metabolic pathway in cancer cells (Warburg effect), where they bind hexokinase II (HK II) and supply ATP necessary for metabolic activity of this enzyme. In addition, overexpression of the molecular chaperone heat-shock protein 90 (Hsp90) in cancer cells has been described. Interestingly, TOM complex, responsible for import of proteins (e.g. VDACs) into mitochondria, is an Hsp90 client protein. Therefore, we hypothesised that targeting Hsp90 would affect HK II binding to VDAC and in consequence decrease glycolysis. Interestingly, in cancer cells, VDACs have been shown also to localise to plasma membrane and iron uptake was suggested to be one of the probable plasma membrane (pm) activity of VDACs. Here, we have also evaluated the possible role for Hsp90 in cell trafficking of pmVDACs. To determine the effects of Hsp90 inhibitor 17-DMAG (17-(dimethylaminoethylamino)-17-demethoxygeldanamycin) on cancer cell metabolism and VDAC localisation we have adopted human gastric and pancreatic cancer cell lines (TMK1, MiaPaCa2). In vitro, localisation of proteins was determined using Western blotting and immunocytochemistry. Spectrophotometric technique was employed to evaluate HK enzyme activity and intracellular iron level whilst co-immunoprecipitation was used for protein complex formation. In vivo, murine Panc02 pancreatic adenocarcinoma cells were used in subcutaneous tumor model. Staining of HK II, VDAC and mitochondria in the cells showed large areas of co-localisation upon control conditions which was significantly reduced when cells were treated with 17-DMAG. Overall HK activity in the mitochondrial fraction was decreased by 25 ± 8,1% and 57 ± 9,7% in cells and tissues treated with 17-DMAG compared to control conditions. Furthermore, expression of VDACs in plasma membrane fraction was significantly decreased after 4 h 17-DMAG treatment. Complex formation of VDACs with Hsp90 was confirmed by co-immunoprecipitation. 4 min incubation of cells in the presence of different concentrations of extracellular iron resulted in a dose dependent iron uptake. Moreover, in the presence of extracellular iron, dose-dependent iron uptake was decreased when cells were incubated with VDACs blocker - phosphorothioate randomer (∼20 ± 3,5 %) or preincubated with 17-DMAG (∼40 % ± 2,5 %) for both cell lines. Overall, the results have shown that (1) Targeting Hsp90 affects glucose metabolism in cancer cells via inhibition of VDAC incorporation into the mitochondrial membrane resulting in HK II - VDAC binding impairment. (2) VDACs are also expressed in the plasma membrane of cancer cells and their trafficking depends on Hsp90. (3) One of the potential roles of pmVDAC is iron transport. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3211. doi:1538-7445.AM2012-3211