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 those 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. What are the factors regulating the HIF protein in stem or differentiated cells is 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 a bait and found nine proteins interacting with PHD2. We focused on two genes that are implicated embryonic development and cancer initiation, MAGEA11 and DVL1. MAGEA11 is a cancer-testis antigen that is expressed in embryonic but not in adult tissues. It is aberrantly expressed in tumors of different histology. The DVL gene family is a major intermediate in a Wnt signal transduction pathway shown to play a role in stem cell maintenance. We obtained several lines of evidence that MAGEA11 inhibits PHD2, a major regulator of HIF degradation. We found that MAGEA11 expression is downregulated during retinoic acid-induced differentiation of the human teratocarcinoma cell line NTERA2 in concordance with HIF-1 and HIF-2. This year, in collaboration with the Laboratory of Jacek Capala we studied MAGE11 function in tumor formation using breast cancer xenograft mouse model. MAGE11 protein was downregulated by ectopic shRNA overexpression in MDA-MB-435 breast cancer cell line that was injected into nude mice. Both tumor growth and lung metastasis were unchanged in shRNA and control cells. The study of putative crosstalk between hypoxia and Wnt pathways- originated 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. Future plans include: (1) the study of the role of MAGE11 in tumor progression in a mouse xenograft model by creating a breast cancer cell line stably expressing shRNA that inhibits expression of MAGE11; (2) analysis of the MAGE11 function in tumor cell growth and migration/invasion; (3) utilization of a retroviral vector with shMAGE11 to transduce pluripotent teratocarcinoma cells and study the effects of MAGE11 on differentiation; (4) investigation of the mechanism of PHD2 mediated inhibition of the Wnt pathway and its role in maintaining pluripotent state of teratocarcinoma cells.