Identification of hypoxia-induced metabolism-associated genes in canine tumors.

The energy metabolism of tumors is biased toward glycolysis called as the Warburg effect. The tumor microenvironment is known to be hypoxic, which leads to activating hypoxia-inducible factor 1a (HIF1a) in tumor cells. The high expression of HIF1a plays a functional role in the enhancement of glycolysis and is correlated to poor outcomes in human cancers. However, in dogs, the molecular mechanisms involved in hypoxic tumor cells remain to be elucidated. In the present study, we investigated upregulated genes in hypoxia by RNA-seq analysis, the expression of proteins related to glycolysis in hypoxia by western blotting (WB), and the expression of the monocarboxylate transporter4 (MCT4) in a total of 96 canine tumor tissue by immunohistochemical (IHC) staining. The glycolysis and HIF-1 signaling pathways were upregulated in hypoxic melanoma cells by RNA-seq and WB. In addition, the experiment using HIF1a knockout melanoma cells showed a marker of glycolysis MCT4 was regulated by HIF1a activation. Hypoxia induced high lactate secretion due to the enhancement of glycolysis in canine melanoma cells. IHC analysis revealed membrane-localization of MCT4 protein was observed in urothelial carcinoma and lung adenocarcinoma tissues. We concluded canine MCT4 protein has a role in lactic acid efflux from glycolytic cells and might be a novel marker of hypoxia and glycolysis in canine tumors. This data may also be important in finding a new therapeutic target for MCT4 in the future. Overall design: RNAseq analysis of canine malignant melanoma cells incubated under conditions of normoxia or hypoxia.