Regulation of Hypoxia-Inducible Factors in Pluripotent Cancer Cells.

低氧诱导因子(HIF)在干细胞和多能细胞自我更新和维持中的直接作用得到了多项观察的支持。首先，HIF是Notch信号通路的重要伙伴，Notch信号通路参与了Notch靶基因的转录激活，这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型胚胎干细胞来源的肿瘤。HIF在这些细胞中稳定的机制可能部分是由于含有干细胞的组织中的低氧可获得性；然而，与来自相同微环境的分化细胞相比，在常氧条件下，HIF在循环中的造血干细胞中稳定。干细胞或分化细胞中HIF蛋白的调节因素主要是什么，目前尚不清楚。为了验证干细胞可能通过抑制PhD来稳定HIFs的假设，我们以PHD2为诱饵进行了酵母双杂交筛选，发现了9种与PHD2相互作用的蛋白质。我们重点研究了两个与胚胎发育和癌症启动有关的基因，MAGEA11和DVL1。MAGEA11是一种癌症-睾丸抗原，在胚胎组织中表达，但在成人组织中不表达。它在不同组织学类型的肿瘤中异常表达。DVL基因家族是Wnt信号转导途径中的主要中间体，被证明在干细胞维持中发挥作用。我们获得了MAGEA11抑制PHD2的几条证据，PHD2是HIF降解的主要调节因子。我们发现，在维甲酸诱导的人畸胎癌细胞株NTERA2分化过程中，MAGEA11的表达下调，与HIF-1和HIF-2的表达一致。今年，我们与Jacek Capala实验室合作，利用乳腺癌异种移植小鼠模型研究了MAGE11在肿瘤形成中的作用。异位shRNA过表达可下调裸鼠体内MAGE11蛋白的表达。ShRNA和对照细胞的肿瘤生长和肺转移均无变化。对低氧和Wnt通路之间可能存在的串扰的研究--源于PHD2-DVL1相互作用--证实了这两种蛋白的结合，并且它们中任何一种丰度的增加都对相应途径的终点进行了负面调节。例如，PHD2的过表达减缓了Wnt信号转导的速度，而DVL的过表达抑制了PHD2的活性。未来计划包括：(1)通过建立稳定表达抑制MAGE11基因表达的shRNA的乳腺癌细胞系，研究MAGE11在肿瘤进展中的作用；(2)分析MAGE11在肿瘤细胞生长和迁移/侵袭中的功能；(3)利用含有shMAGE11的逆转录病毒载体转导多能畸胎癌细胞，并研究其对分化的影响；(4)研究PHD2介导抑制Wnt通路的机制及其在维持畸形癌细胞多能状态中的作用。

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.