Regulation of Hypoxia-Inducible Factors in Pluripotent Cancer Cells.

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The direct role of Hypoxia-inducible factors (HIF) in the self-renewal and maintenance of stem and pluripotent cells is supported by several observations. First, HIF is an important partner of the Notch signaling pathway involved in transcriptional activation of Notch target genes necessary to maintain neural and myogenic stem cells in an undifferentiated state. Second, HIF-2 transcriptionally activates Oct-4 gene expression, thereby contributing to defective hematopoietic stem cell differentiation, and large embryonic stem cell-derived tumors characterized by an increased number of undifferentiated cells. The mechanism of HIF stabilization in these cells may partially be explained by low oxygen availability in tissues harboring stem cells; however, HIF is stabilized in circulating hematopoietic stem cells under normoxic conditions compared to differentiated cells from the same microenvironment. The factors regulating HIF protein in stem or differentiated cells are mainly unclear. To test the hypothesis that stem cells may stabilize HIFs by inhibiting PHDs we performed a yeast two-hybrid screen using PHD2 as bait and found 9 proteins interacting with PHD2. We focused on two genes that are implicated embryonal development and cancer initiation, MAGEA11 and DVL1. MAGEA11 is a cancer-testis antigen that is expressed in embryonic but not in adult tissues and is aberrantly expressed in tumors of different histology. The DVL gene family is a major intermediate in the Wnt signal transduction pathway, shown to play a role in stem cells maintenance. We obtained several lines of evidence that MAGEA11 inhibits PHD2, a major regulator of HIF degradation through increased stabilization and transcriptional activation of HIF-1a . We found that MAGEA11 expression is downregulated during retinoic acid-induced differentiation of the human teratocarcinoma cell line NTERA2 in concordance with HIF-1a and HIF-2a. The manuscript on MAGEA11 inhibition of PHD2 and activation of HIF is currently in preparation. The study on putative crosstalk between hypoxia and Wnt pathways originating from PHD2-DVL1 interaction confirmed that these two proteins bind and that an increase in abundance of either of them negatively regulates the endpoint of the corresponding pathway. For example, overexpression of PHD2 slows down the Wnt signal transduction and an overexpression of DVL inhibits PHD2 activity. Work is underway to identify the biological system where this interaction effectively functions to regulate stem/differentiation potential of the cell.