Abstract
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-1α and HIF-2α are expressed in human colon cancer cells but not in their non-malignant counterparts. The stable knockdown of HIF-1α or HIF-2α 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-1α depletion exerting greater effects. Knockdown of these two HIF transcripts induced different and even opposite effects on β-catenin transcriptional activity in colon cancer cells with different genetic Wnt signaling pathways. In SW480 cells, HIF-2α knockdown did not affect β-catenin levels, increasing the transcriptional activity of β-catenin by inducing its nuclear accumulation, whereas HIF-1α silencing negatively affected the stability and transcriptional activity of β-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin. In addition, although HIF-1α depletion induced a reversal of the epithelial-to-mesenchymal transition (EMT), HIF-2α 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-1α silencing. Remarkably, HIF-2α knockdown also enhanced β-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.
Highlights
Wnt signaling has been well-characterized as one of the most important contributors to tumorigenesis in many types of solid tumors
Because high hypoxia-inducible factors (HIFs) expression has been detected in tumor cells in the absence of hypoxia, we compared the expression of these factors in cultured colon cancer cells under normoxic and hypoxic conditions with cultured nonmalignant 112CoN cells that were incubated under the same conditions by western blot
We showed that only colon carcinoma cells co-express HIF-1a and HIF-2a under normoxic conditions, in contrast to non-malignant colon cells, which do not express these factors under these conditions
Summary
Wnt signaling has been well-characterized as one of the most important contributors to tumorigenesis in many types of solid tumors. The Wnt pathway has been demonstrated to play an important role in the development and regulation of adult stem cell systems, and canonical Wnt signaling supports the formation and maintenance of both stem and cancer stem cells (CSC) [3]. Without stimulation by Wnt, b-catenin is assembled into the so-called destruction complex, in which APC plays a central role, and this complex includes axin, GSK-3b and Casein kinase 1. This complex directs a series of phosphorylation events in b-catenin that make it a target for ubiquitination and subsequent proteolysis via the proteasome [4]. Stimulation by Wnt leads to the inhibition of b-catenin breakdown, allowing b-catenin to accumulate, enter the nucleus, and activate Wnt target genes such as CYCLIN D1 and C-MYC proto-oncogenes, which promote the entry of the cell into the S phase of the cell cycle [5]
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