Characterizing CHCHD2 subcellular localization in response to hypoxia and redox perturbations in glioblastoma cells

BACKGROUND: Glioblastoma (GBM) is the most common and malignant form of primary brain tumor in adults and remains incurable. Inter-organelle communication between mitochondria and nuclei is a potential compensatory signaling pathway that may contribute to therapeutic resistance and cellular plasticity in the face of a hypoxic tumor microenvironment. Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) is a protein that colocalizes to the mitochondrial inter-membrane space and the nucleus, where it exhibits hypoxia-sensitive transcription factor capabilities. We demonstrated previously that CHCHD2 knockout in hypoxic U87 GBM cells harboring epidermal growth factor receptor (EGFR) variant III (U87vIII) abrogated the increase in proliferation and invasion exhibited by U87vIII WT cells expressing CHCHD2. The C-terminus of CHCHD2 contains redox-sensitive cysteines amenable to thiol/disulfide formation, which determine protein folding. The objective of this study was to characterize the effect of hypoxia and cellular redox status on CHCHD2 subcellular localization. We hypothesized that reducing the C-terminal CX9C motif would induce CHCHD2 mitochondrial export and nuclear accumulation. METHODS: U87 and U87vIII cells were incubated in standard culture oxygen conditions (~21% O2), 7% O2, 4% O2, and pathophysiological hypoxia (1% O2). Confocal immunofluorescence microscopy was used to observe CHCHD2 subcellular localization, and nuclear fluorescence intensity was quantified using Axiovision software. Western blot of whole cell lysates was used to quantify CHCHD2 protein levels in response to decreasing oxygen tensions. Chemical reduction of cells was achieved using dithiothreitol (DTT) at designated concentrations and time courses. RESULTS: The amount of nuclear CHCHD2 did not differ between 21% and 7% O2. Incubation in 1% O2 increased the amount of nuclear CHCHD2 in both U87 and U87vIII cells, with the latter consistently exhibiting greater amounts of nuclear CHCHD2 at all oxygen tensions. Total CHCHD2 protein levels in U87 cells were maximal at 4% O2, but reduced at 1% O2, a trend also observed in U87vIII cells. Chemical reduction with 1 mM DTT for either 15 min or 24 h induced significantly greater CHCHD2 nuclear accumulation in U87vIII cells compared to U87. Increasing DTT dose 10-fold resulted in rapid accumulation of CHCHD2 in nuclei after 15 min, and complete disappearance from mitochondria after 30 min. CONCLUSIONS: Hypoxia and chemical reduction are capable of driving CHCHD2 translocation from mitochondria to the nucleus in U87 GBM cells, which is enhanced in the presence of the EGFRvIII mutant. Ongoing work to expand our understanding of how CHCHD2 protein folding governs protein localization and function may inspire novel therapeutic strategies to undermine mitonuclear signaling in GBM. Citation Format: Jan C. Lumibao, H. Rex Gaskins. Characterizing CHCHD2 subcellular localization in response to hypoxia and redox perturbations in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2650.