Src induces expression of carbonic anhydrase IX via hypoxia-inducible factor 1

Carbonic anhydrase IX (CA IX) belongs to the physiologically important enzymes which contribute to tumor physiology. Tumor-associated expression of CA IX is induced mainly due to its strong transcriptional activation via hypoxia-inducible factor 1 (HIF-1). Therefore, CA IX can serve as a surrogate marker of hypoxia and a prognostic indicator. HIF-1 is a master transcription factor that mediates essential homeostatic responses to cellular and systemic hypoxia by activating transcription of multiple genes including those encoding glycolytic enzymes and vascular endothelial growth factor. In addition to hypoxia, HIF-1alpha expression can be up-regulated by growth factors and oncogenic signals (e.g. Src oncogene). Consequently, induction of the HIF-1alpha transcription factor up-regulates target gene expression. The results from the present study suggest that Src oncogene induces CA IX expression under normoxic, as well as hypoxic conditions. Moreover, we demonstrate that Src-mediated induction of CA IX expression is critically dependent on HIF-1alpha activity. Transcriptional activity of the CA9 promoter was significantly increased by expression of v-Src or c-Src. The effect was more prominent in normoxia, most likely because of already high level of HIF-1alpha expression in hypoxia. By co-transfection with dominant-negative HIF-1alpha we confirmed that Src-induced stimulation of CA9 transcription is mediated via HIF-1alpha. Consistent with this, Src-expressing HeLa cells displayed higher levels of HIF-1alpha protein. Finally, these results indicate a novel regulatory pathway responsible for increased CA IX expression in tumor cells and define CA IX as a new down-stream target for Src oncogene.