HIGD2A silencing impairs hepatocellular carcinoma growth via inhibiting mitochondrial function and the MAPK/ERK pathway.

BACKGROUND: The Hypoxia inducible gene domain family member 2A (HIGD2A) protein is indispensable for the assembly of the mitochondrial respiratory supercomplex, which has been implicated in cell proliferation and cell survival under hypoxic conditions. Because the liver has a naturally low oxygen microenvironment, the role of HIGD2A in the development of hepatocellular carcinoma (HCC) remains largely unknown. METHODS: Gene expression data and clinical information were obtained from multiple public databases. A lentivirus-mediated gene knockdown approach was conducted to explore the function and mechanism of HIGD2A activity in HCC cells. In vivo and in vitro assays were performed to investigate the biological roles of HIGD2A. RESULTS: HIGD2A was overexpressed in HCC tissues and cell lines and was associated with a worse prognosis. Silencing HIGD2A expression significantly attenuated cell proliferation and migration, caused S-phase cell cycle arrest, and decreased tumor formation in nude mice. Mechanistically, HIGD2A depletion greatly decreased cellular ATP levels by disrupting mitochondrial ATP production. Moreover, HIGD2A knockdown cells displayed impaired mitochondrial function, such as mitochondrial fusion, increased expression of the mitochondrial stress response protein, and decreased oxygen consumption. Furthermore, knockdown of HIGD2A markedly attenuated the activation of the MAPK/ERK pathway. CONCLUSIONS: HIGD2A promoted liver cancer cell growth by fueling mitochondrial ATP synthesis and activating the MAPK/ERK pathway, suggested that targeting HIGD2A may represent a new strategy for HCC therapy.

Dynamic Distribution of HIG2A between the Mitochondria and the Nucleus in Response to Hypoxia and Oxidative Stress.

Mitochondrial respiratory supercomplex formation requires HIG2A protein, which also has been associated with cell proliferation and cell survival under hypoxia. HIG2A protein localizes in mitochondria and nucleus. DNA methylation and mRNA expression of the HIGD2A gene show significant alterations in several cancers, suggesting a role for HIG2A in cancer biology. The present work aims to understand the dynamics of the HIG2A subcellular localization under cellular stress. We found that HIG2A protein levels increase under oxidative stress. H2O2 shifts HIG2A localization to the mitochondria, while rotenone shifts it to the nucleus. HIG2A protein colocalized at a higher level in the nucleus concerning the mitochondrial network under normoxia and hypoxia (2% O2). Hypoxia (2% O2) significantly increases HIG2A nuclear colocalization in C2C12 cells. In HEK293 cells, chemical hypoxia with CoCl2 (>1% O2) and FCCP mitochondrial uncoupling, the HIG2A protein decreased its nuclear localization and shifted to the mitochondria. This suggests that the HIG2A distribution pattern between the mitochondria and the nucleus depends on stress and cell type. HIG2A protein expression levels increase under cellular stresses such as hypoxia and oxidative stress. Its dynamic distribution between mitochondria and the nucleus in response to stress factors suggests a new communication system between the mitochondria and the nucleus.

HIGD-Driven Regulation of Cytochrome c Oxidase Biogenesis and Function.

The biogenesis and function of eukaryotic cytochrome c oxidase or mitochondrial respiratory chain complex IV (CIV) undergo several levels of regulation to adapt to changing environmental conditions. Adaptation to hypoxia and oxidative stress involves CIV subunit isoform switch, changes in phosphorylation status, and modulation of CIV assembly and enzymatic activity by interacting factors. The latter include the Hypoxia Inducible Gene Domain (HIGD) family yeast respiratory supercomplex factors 1 and 2 (Rcf1 and Rcf2) and two mammalian homologs of Rcf1, the proteins HIGD1A and HIGD2A. Whereas Rcf1 and Rcf2 are expressed constitutively, expression of HIGD1A and HIGD2A is induced under stress conditions, such as hypoxia and/or low glucose levels. In both systems, the HIGD proteins localize in the mitochondrial inner membrane and play a role in the biogenesis of CIV as a free unit or as part as respiratory supercomplexes. Notably, they remain bound to assembled CIV and, by modulating its activity, regulate cellular respiration. Here, we will describe the current knowledge regarding the specific and overlapping roles of the several HIGD proteins in physiological and stress conditions.

Distinct Roles of Mitochondrial HIGD1A and HIGD2A in Respiratory Complex and Supercomplex Biogenesis.

The mitochondrial respiratory chain enzymes are organized as individual complexes and supercomplexes, whose biogenesis remains to be fully understood. To disclose the role of the human Hypoxia Inducible Gene Domain family proteins HIGD1A and HIGD2A in these processes, we generate and characterize HIGD-knockout (KO) cell lines. We show that HIGD2A controls and coordinates the modular assembly of isolated and supercomplexed complex IV (CIV) by acting on the COX3 assembly module. In contrast, HIGD1A regulates CIII and CIII-containing supercomplex biogenesis by supporting the incorporation of UQCRFS1. HIGD1A also clusters with COX4-1 and COX5A CIV subunits and, when overexpressed, suppresses the CIV biogenesis defect of HIGD2A-KO cells. We conclude that HIGD1A and HIGD2A have both independent and overlapping functions in the biogenesis of respiratory complexes and supercomplexes. Our data illuminate the existence of multiple pathways to assemble these structures by dynamic HIGD-mediated CIV biogenesis, potentially to adapt to changing environmental and nutritional conditions.

Biosystem Analysis of the Hypoxia Inducible Domain Family Member 2A: Implications in Cancer Biology.

The expression of HIGD2A is dependent on oxygen levels, glucose concentration, and cell cycle progression. This gene encodes for protein HIG2A, found in mitochondria and the nucleus, promoting cell survival in hypoxic conditions. The genomic location of HIGD2A is in chromosome 5q35.2, where several chromosomal abnormalities are related to numerous cancers. The analysis of high definition expression profiles of HIGD2A suggests a role for HIG2A in cancer biology. Accordingly, the research objective was to perform a molecular biosystem analysis of HIGD2A aiming to discover HIG2A implications in cancer biology. For this purpose, public databases such as SWISS-MODEL protein structure homology-modelling server, Catalogue of Somatic Mutations in Cancer (COSMIC), Gene Expression Omnibus (GEO), MethHC: a database of DNA methylation and gene expression in human cancer, and microRNA-target interactions database (miRTarBase) were accessed. We also evaluated, by using Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), the expression of Higd2a gene in healthy bone marrow-liver-spleen tissues of mice after quercetin (50 mg/kg) treatment. Thus, among the structural features of HIG2A protein that may participate in HIG2A translocation to the nucleus are an importin -dependent nuclear localization signal (NLS), a motif of DNA binding residues and a probable SUMOylating residue. HIGD2A gene is not implicated in cancer via mutation. In addition, DNA methylation and mRNA expression of HIGD2A gene present significant alterations in several cancers; HIGD2A gene showed significant higher expression in Diffuse Large B-cell Lymphoma (DLBCL). Hypoxic tissues characterize the "bone marrow-liver-spleen" DLBCL type. The relative quantification, by using RT-qPCR, showed that Higd2a expression is higher in bone marrow than in the liver or spleen. In addition, it was observed that quercetin modulated the expression of Higd2a gene in mice. As an assembly factor of mitochondrial respirasomes, HIG2A might be unexpectedly involved in the change of cellular energetics happening in cancer. As a result, it is worth continuing to explore the role of HIGD2A in cancer biology.