Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.

Background Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved. Results Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes. Conclusions CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells. Electronic supplementary material The online version of this article (10.1186/s40170-019-0200-4) contains supplementary material, which is available to authorized users.

BACKGROUND: Tumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) is critical for maintaining intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers, correlates with increased tumour progression and poor patient survival.RESULTS: Here, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I-V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS). CONCLUSIONS:Our study highlights an important role for CHCHD4 in regulating tumour cell metabolism, and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology. BACKGROUNDMetabolic reprogramming and altered mitochondrial metabolism is a feature of cancer, and thus has become an attractive area for therapeutic exploitation [1,2]. Given the importance of mitochondria in controlling normal physiological processes, understanding how mitochondrial metabolism underlies tumorigenesis is important for ascertaining its therapeutic potential in cancer.Previously, we discovered the essential redox-sensitive mitochondrial intermembrane space (IMS) protein CHCHD4, is a critical for regulating intracellular oxygen consumption rate and metabolic responses to low oxygen (hypoxia) in tumour cells [3,4]. Overexpression of Thomas et al. 3CHCHD4 in human cancers significantly correlates with the hypoxia gene signature, tumour progression, disease recurrence and poor patient survival [3].CHCHD4 provides an import and oxidoreductase-mediated protein folding function along with the sulfhydryl oxidase GFER (ALR/Erv1) as a key part of the disulphide relay system (DRS) within the mitochondrial IMS [5][6][7]. As such, CHCHD4 controls the import of a number of mitochondrial proteins that contain a twin-CX 9 C or twin-CX 3 C motif [8][9][10].Additio...

Background Tumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus, mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) is critical for regulating intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers correlates with increased tumour progression and poor patient survival. Results Here, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I–V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS). Conclusions Our study highlights an important role for CHCHD4 in regulating tumour cell metabolism and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology. Electronic supplementary material The online version of this article (10.1186/s40170-019-0194-y) contains supplementary material, which is available to authorized users.

BACKGROUND: Mitochondrial metabolism involves oxidative phosphorylation (OXPHOS) via the respiratory chain and is required for the maintenance of tumour cell proliferation and regulation of epithelial/mesenchymal transition (EMT) related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study we interrogate the role of CHCHD4 regulated respiratory chain activity and metabolism in tumour cell proliferation and EMT-related phenotypes. RESULTS: We show that CHCHD4 is essential for the proliferation of tumour cells irrespective of their aetiology. In human tumours, elevated CHCHD4 expression is correlated with a mitochondrial OXPHOS gene signature and with a proliferative gene signature associated with the mTORC1 signalling pathway. Elevated CHCHD4 increases tumour cell proliferation, in a manner that is dependent on complex I (CI) activity, glutamine consumption and mTORC1 activation. CHCHD4 expression is inversely correlated with EMT gene expression both in vitro and in vivo. Finally, we show CHCHD4 regulates the intracellular distribution of the EMT marker vimentin, in a CI mediated manner. CONCLUSIONS: CHCHD4 regulates tumour cell proliferation and metastatic (EMT related) phenotypes through its control of CI-mediated mitochondrial metabolism.

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