Background: Intratumoural hypoxia is associated with an aggressive and metastatic phenotype and poor prognosis. The transcription factor Hypoxia-inducible Factor-1α (HIF-1α) is a key regulator of hypoxia, inducing the expression of genes which promote tumour cell survival, such as carbonic-anhydrase IX (CA-IX) playing a role in cell adhesion, pH regulation and dedifferentiation of the tumours. Here we investigated whether there is cross talk between the HIF-1 and Wnt/β-catenin pathway. We stably transfected a thyroid carcinoma cell line and control cell lines with a dominant negative form of HIF-1α (DN- HIF) where the DN-HIF retains the N-terminal regions known to interact with β-catenin.
Aims: To determine the potential interaction of HIF with β-catenin on the regulation of CA-IX.
Results: Using a luciferase reporter assay that the transcriptional activity of HIF-1α was inhibited in stably DN-HIF transfected cells. Western blot analysis revealed that DN cells have reduced expression of hypoxia-regulated genes but display high levels of CA-IX protein in air. siRNA against DN-HIF showed reduced CA-IX expression confirming the increase was due to DN-HIF and was not a clonal artefact. Increased CA-IX expression was also displayed in a fibrosarcoma cell line with hyperactive β-catenin activity and was not observed in a breast carcinoma cell line with lower β-catenin activity. We also found using immuno-fluorescence, altered expression and localisation of β-catenin in DN-HIF transfected cells.
Conclusions: Increased CA-IX expression observed in DN cells suggests the increase is due to hyperactive β-catenin activity. The results display the possibility of a unique regulatory mechanism of CA-IX and highlights the potential cross talk between the HIF-1 and Wnt signalling pathways.