Abstract
The transcription factor hypoxia-inducible factor 1 (HIF1) is the crucial regulator of genes that are involved in metabolism under hypoxic conditions, but information regarding the transcriptional activity of HIF1 in normoxic metabolism is limited. Different tumor cells were treated under normoxic and hypoxic conditions with various drugs that affect cellular metabolism. HIF1α was silenced by siRNA in normoxic/hypoxic tumor cells, before RNA sequencing and bioinformatics analyses were performed while using the breast cancer cell line MDA-MB-231 as a model. Differentially expressed genes were further analyzed and validated by qPCR, while the activity of the metabolites was determined by enzyme assays. Under normoxic conditions, HIF1 activity was significantly increased by (i) glutamine metabolism, which was associated with the release of ammonium, and it was decreased by (ii) acetylation via acetyl CoA synthetase (ACSS2) or ATP citrate lyase (ACLY), respectively, and (iii) the presence of L-ascorbic acid, citrate, or acetyl-CoA. Interestingly, acetylsalicylic acid, ibuprofen, L-ascorbic acid, and citrate each significantly destabilized HIF1α only under normoxia. The results from the deep sequence analyses indicated that, in HIF1-siRNA silenced MDA-MB-231 cells, 231 genes under normoxia and 1384 genes under hypoxia were transcriptionally significant deregulated in a HIF1-dependent manner. Focusing on glycolysis genes, it was confirmed that HIF1 significantly regulated six normoxic and 16 hypoxic glycolysis-associated gene transcripts. However, the results from the targeted metabolome analyses revealed that HIF1 activity affected neither the consumption of glucose nor the release of ammonium or lactate; however, it significantly inhibited the release of the amino acid alanine. This study comprehensively investigated, for the first time, how normoxic HIF1 is stabilized, and it analyzed the possible function of normoxic HIF1 in the transcriptome and metabolic processes of tumor cells in a breast cancer cell model. Furthermore, these data imply that HIF1 compensates for the metabolic outcomes of glutaminolysis and, subsequently, the Warburg effect might be a direct consequence of the altered amino acid metabolism in tumor cells.
Highlights
Aerobic glycolysis enables tumor cells to consume high levels of glucose for proliferation, producing lactate as a waste production, even in the presence of oxygen [1,2]
We demonstrated that ammonia/ammonium are responsible for the normoxic stabilization and activation of hypoxia-inducible factor 1 (HIF1) [13] and that HIF1 might have a function in the detoxification of ammonia/ammonia
The normoxic stabilization of HIF1α at the protein level was found in various tumor cell lines after the application of several growth factors (e.g., EGF or insulin) or fetal bovine serum only in the presence of the amino acid glutamine and as a result of glutaminolysis [13]
Summary
Aerobic glycolysis ( known as the Warburg effect) enables tumor cells to consume high levels of glucose for proliferation, producing lactate as a waste production, even in the presence of oxygen [1,2]. Parks and coauthors suggested a possible function of HIF1 in the metabolic adaptation of aerobic glycolysis [27], a finding that is in line with the association of HIF1-mediated transcriptional upregulation of glycolytic genes in a tumor model of Drosophila [25]; in this tumor model, the glycolytic genes were upregulated, by the HIF1 associated with hypoxia, and due to the activity of HIF under normoxia, which seems to be responsible for the development of glycolytic tumors [25] This assumption is further strengthened by a recent review summarizing the role of HIF1 in creating the Warburg effect [28], which is a normoxic phenomenon
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