Hypoxia-Inducible Factors Modulate the Stemness and Malignancy of Colon Cancer Cells by Playing Opposite Roles in Canonical Wnt Signaling

This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling. Under normoxia, we determined that both HIF-1 alpha and HIF-2 alpha are expressed in human colon cancer cells but not in their non-malignant counterparts. The stable knockdown of HIF-1 alpha or HIF-2 alpha expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1 alpha depletion exerting greater effects. Knockdown of these two HIF transcripts induced different and even opposite effects on beta-catenin transcriptional activity in colon cancer cells with different genetic Wnt signaling pathways. In SW480 cells, HIF-2 alpha knockdown did not affect beta-catenin levels, increasing the transcriptional activity of beta-catenin by inducing its nuclear accumulation, whereas HIF-1 alpha silencing negatively affected the stability and transcriptional activity of beta-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin. In addition, although HIF-1 alpha depletion induced a reversal of the epithelial-to-mesenchymal transition (EMT), HIF-2 alpha silencing altered the expression of the stem cell markers CD44, Oct4, and CD24 and of the differentiation marker CK20 in the opposite direction as HIF-1 alpha silencing. Remarkably, HIF-2 alpha knockdown also enhanced beta-catenin transcriptional activity under hypoxia in cells that displayed normal Wnt signaling, suggesting that the gene negatively modulates canonical Wnt signaling in colon cancer cells. Taken together, our results indicate that HIFs play opposing roles in canonical Wnt signaling and are essential for the stemness and malignancy maintenance of colon cancer cells.