Regulation of Hypoxia-Inducible Factors in Pluripotent Cancer Cells.

多能癌细胞中缺氧诱导因子的调节。

• 批准号: 8349179
• 负责人: John Niederhuber
• 金额: $ 4.97万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349179
• 关键词: Adult; Breast Cancer Cell; Cancer cell line; Cell Line; Cell Maintenance; Cells; Collaborations; Data; Down-Regulation; Embryo; Embryonic Development; Equilibrium; Future; Gene Expression; Gene Family; Gene Mutation; Gene Targeting; Genes; Genetic; Goals; Growth Factor; Hematopoietic; Hematopoietic stem cells; Histology; Human; Hypoxia; Hypoxia Inducible Factor; Investigation; Laboratories; Lung; Maintenance; Mediating; Methylation; Mus; Neoplasm Metastasis; Neurons; Notch Signaling Pathway; Nude Mice; Oxygen; Pathway interactions; Phenotype; Play; Pluripotent Stem Cells; Procollagen-Proline Dioxygenase; Protein Binding; Proteins; Regulation; Retroviral Vector; Role; Signal Transduction; Signal Transduction Pathway; Stem cells; Teratocarcinoma; Testing; Tissues; Transcriptional Activation; Translations; Tretinoin; Tumor Suppressor Genes; Tumor-Derived; Ubiquitination; Undifferentiated; Xenograft Model; Xenograft procedure; Yeasts; bHLH-PAS factor HLF; cancer cell; cancer initiation; cancer stem cell; cancer testis antigen; cell growth; cell motility; embryonic stem cell; hypoxia inducible factor 1; malignant breast neoplasm; mouse model; neoplastic cell; notch protein; overexpression; programs; protein protein interaction; relating to nervous system; self-renewal; small hairpin RNA; stem; stem cell differentiation; stem cell population; transcription factor; trophoblast; tumor; tumor growth; tumor progression; tumorigenic; yeast two hybrid system

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.

缺氧诱导因子（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，所述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稳定化的机制可以部分地通过含有干细胞的组织中的低氧可用性来解释;然而，与来自相同微环境的分化细胞相比，在常氧条件下，HIF在循环造血干细胞中是稳定的。在干细胞或分化的细胞中调节HIF蛋白的因素主要是不清楚的。为了验证干细胞可以通过抑制PHD来稳定HIF的假设，我们使用PHD 2作为诱饵进行了酵母双杂交筛选，发现了9种与PHD 2相互作用的蛋白质。我们集中在两个基因，涉及胚胎发育和癌症的启动，MAGEA 11和DVL 1。MAGEA 11是一种癌-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，显示在干细胞维持中起作用。我们获得了MAGEA 11抑制PHD 2的几条证据，PHD 2是HIF降解的主要调节因子。我们发现，MAGEA 11表达下调视黄酸诱导分化的人畸胎瘤细胞系NTERA 2与HIF-1和HIF-2一致。今年，我们与Jacek Capala实验室合作，使用乳腺癌异种移植小鼠模型研究了MAGE 11在肿瘤形成中的功能。在注射到裸鼠体内的MDA-MB-435乳腺癌细胞系中，MAGE 11蛋白通过异位shRNA过表达而下调。肿瘤生长和肺转移在shRNA和对照细胞中均无变化。 对缺氧和Wnt途径之间的假定串扰的研究-起源于PHD 2-DVL 1相互作用-证实了这两种蛋白质结合，并且它们中任一种的丰度的增加负调节相应途径的终点。例如，PHD 2的过表达减缓Wnt信号转导，而DVL的过表达抑制PHD 2活性。 今后的计划包括：（1）通过建立稳定表达抑制MAGE 11表达的shRNA的乳腺癌细胞系，研究MAGE 11在小鼠异种移植模型中的肿瘤进展中的作用;（2）分析MAGE 11在肿瘤细胞生长和迁移/侵袭中的功能;（3）利用含shMAGE 11的逆转录病毒载体转染多能性畸胎瘤细胞，研究MAGE 11对畸胎瘤细胞分化的影响;（4）研究PHD 2抑制Wnt通路的机制及其在维持畸胎瘤细胞多能性中的作用。