Regulation of Hypoxia-Inducible Factors in Pluripotent Cancer Cells.

缺氧诱导因子（HIF）在干细胞和多能细胞的自我更新和维持中的直接作用得到了一些观察结果的支持。首先，HIF是Notch信号通路的重要伙伴，参与Notch靶基因的转录激活，而Notch靶基因是维持神经和肌源性干细胞处于未分化状态所必需的。其次，HIF-2转录激活Oct-4基因表达，从而导致造血干细胞分化缺陷，以及以未分化细胞数量增加为特征的大胚胎干细胞衍生肿瘤。这些细胞中HIF稳定的机制可以部分解释为干细胞所在组织中的低氧可用性；然而，与来自相同微环境的分化细胞相比，HIF在常氧条件下在循环造血干细胞中是稳定的。在干细胞或分化细胞中调控HIF蛋白的因素主要不清楚。为了验证干细胞可能通过抑制博士来稳定hif的假设，我们以PHD2为诱饵进行了酵母双杂交筛选，发现了9个与PHD2相互作用的蛋白。我们重点研究了两个与胚胎发育和癌症发生有关的基因MAGEA11和DVL1。MAGEA11是一种癌睾丸抗原，在胚胎组织中表达，而在成人组织中不表达，并且在不同组织学的肿瘤中异常表达。DVL基因家族是Wnt信号转导通路的主要中间体，在干细胞维持中发挥作用。我们获得了几条证据，表明MAGEA11通过增加HIF-1a的稳定性和转录激活来抑制PHD2， PHD2是HIF降解的主要调节因子。我们发现，在视黄酸诱导的人畸胎瘤细胞系NTERA2分化过程中，MAGEA11的表达下调，与HIF-1a和HIF-2a一致。MAGEA11抑制PHD2和激活HIF的稿件目前正在准备中。对缺氧与源于PHD2-DVL1相互作用的Wnt通路之间的串扰的研究证实，这两种蛋白结合，其中任何一种蛋白丰度的增加都对相应通路的终点产生负性调节。例如，PHD2过表达会减缓Wnt信号转导，而DVL过表达会抑制PHD2活性。工作正在进行中，以确定这种相互作用有效地调节细胞的干细胞/分化潜能的生物系统。

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.