C-terminal binding proteins link glycolytic metabolism with the adaptive response to tumor hypoxia and pH regulation

Abstract

Aberrant blood flow and avascular growth create areas of sub-physiological oxygen pressure within tumours (hypoxia), thereby negatively affecting therapy outcome. To maintain ATP production in hypoxia, cancer cells upregulate glycolysis. Both processes combined lead to the accumulation of acidic metabolites. Adaptive responses are mediated by the transcription factor Hypoxia-inducible factor-1 (HIF-1) which upregulates the expression of genes involved in glycolysis and pH regulation, including the transmembrane-spanning enzyme carbonic anhydrase 9 (CA9). CA9 mediates cancer cell survival by generating protons and bicarbonate from CO2 and water, thus maintaining an alkaline intracellular pH while further engraving extracellular acidosis. Cterminal binding proteins (CtBPs) couple glycolysis with gene transcription via their NADHdependent activation, thus functioning as co-regulators of transcription. It is unknown, whether CtBPs link glycolysis with the adaptive response to hypoxia to promote tumour cell survival. In hypoxic MCF7 breast cancer cells, or in MCF7 incubated in 1 mM DMOG, the role of glycolysis was investigated by inhibition with 2-deoxyglucose (2-DG) and in fructose-grown MCF7. The role of CtBPs was determined by transfection with CtBP-targeting siRNA, the inhibitor MTOB, and in MCF7 either expressing mutant CtBP monomers or overexpressing wildtype CtBP2. Reverse transcription PCR and Western blotting demonstrated, that the HIF-induced CA9 mRNA and protein expression, but not of other HIF-target genes, is potentially regulated via a functional interaction between CtBPs and HIF. Colony forming assays showed that the transient silencing of p53 and CtBP or of p53 and CA9 impairs the survival of MCF7 breast cancer cells in hypoxiainduced acidosis. In epithelial-like breast cancer cells, this study establishes a novel link between glycolysis and pH regulation via the activation of CtBPs. Additionally, this work further strengthens CtBPs as a therapeutic target and contributes to the growing body of evidence that CtBPs are not mere co-repressors, but are versatile co-regulators of gene transcription instead.

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Identifiers

ORCID for Jeremy Blaydes: orcid.org/0000-0001-8525-0209

ORCID for Ali Tavassoli: orcid.org/0000-0002-7420-5063

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