Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression
Jun Yang, … , Adrian L. Harris, Margaret Ashcroft
Jun Yang, … , Adrian L. Harris, Margaret Ashcroft
Published January 3, 2012
Citation Information: J Clin Invest. 2012;122(2):600-611. https://doi.org/10.1172/JCI58780.
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Research Article Oncology

Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression

Abstract

Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.

Authors

Jun Yang, Oliver Staples, Luke W. Thomas, Thomas Briston, Mathew Robson, Evon Poon, Maria L. Simões, Ethaar El-Emir, Francesca M. Buffa, Afshan Ahmed, Nicholas P. Annear, Deepa Shukla, Barbara R. Pedley, Patrick H. Maxwell, Adrian L. Harris, Margaret Ashcroft

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Figure 4

CHCHD4 proteins are critical for HIF-1α induction and function in response to hypoxia.

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CHCHD4 proteins are critical for HIF-1α induction and function in respon...
(A) Two independent CHCHD4 siRNAs or a NSC siRNA were transfected into p53–/– HCT116 cells. Cells were incubated in normoxia (Norm) or hypoxia (1% O2) (Hyp). Western blots showing HIF-1α and CHCHD4 proteins. Tubulin was used as a loading control. (B–D) HCT116 cells were transiently transfected with CHCHD4 siRNA or a NSC siRNA, and cells were incubated in normoxia or hypoxia (1% O2) for 16 hours. (B) Western blot showing GLUT-1 protein, (C) graph showing VEGF (pg/ml) protein, and (D) Western blot showing LOX protein (hypoxia samples are shown in duplicate). (E–G) HCT116 cells transiently transfected with CHCHD4 siRNA or NSC siRNA were labeled with the fluorescence tracker CMFDA and trypsinized. Cells were (E) serum starved for 4 hours and assessed for their ability to migrate toward FCS (5%) as a chemoattractant using an HTS FluoroBlok insert (BD Biosciences) (original magnification, ×20), (F) replated and assessed for haptomigration in a scratch assay, or (G) replated and assessed for invasion using the BD Biosciences invasion assay system. Cells in E–G were incubated in hypoxia (1% O2) for 16 hours then fixed and examined by microscopy. Graphs represent (E) the average number of migrated cells, (F) the percentage of haptomigrated cells, and (G) the average number of invaded cells. Cells were counted from at least 3 independent fields of view. *P < 0.05.