Bakiri L, Macho-Maschler S, Custic I, Niemiec J, Guío-Carrión A, Hasenfuss S C, Eger A, Müller M, Beug H, Wagner E F
Genes, Development and Disease Group, F-BBVA Cancer Cell Biology Programme, National Cancer Research Centre (CNIO), Madrid, Spain.
Institute for Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria.
Cell Death Differ. 2015 Feb;22(2):336-50. doi: 10.1038/cdd.2014.157. Epub 2014 Oct 10.
Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfβ1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFβ signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes a basis for future in vivo genetic manipulations and preclinical studies using mouse models.
上皮-间质转化（EMT）对于胚胎形态发生和伤口愈合至关重要，对肿瘤细胞的侵袭和扩散也起着关键作用。AP-1转录因子Fra-1与人类乳腺癌的肿瘤发生及肿瘤相关的EMT有关。我们在来自多个阵列数据集的一大群乳腺癌患者中观察到Fra-1 mRNA表达与无远处转移生存期之间存在显著的负相关。Fos基因之间的这种独特相关性促使我们评估Fra-1在人和小鼠细胞EMT中的功能之间的进化保守性。在完全极化的、非致瘤性的小鼠乳腺上皮EpH4细胞中异位表达Fra-1会诱导间充质表型，其特征是上皮标志物丢失和间充质标志物增加。由此产生的EpFra1细胞的增殖、运动性和侵袭性也增加，并且这些细胞具有致瘤性，移植后能有效地在肺部定植。分子分析显示Tgfβ1以及诱导EMT的转录因子Zeb1、Zeb2和Slug的表达增加。从机制上讲，Fra-1与tgfb1和zeb2启动子以及zeb1第一个内含子中的一个进化保守区域结合。此外，在EpFra1细胞中检测到zeb2启动子报告基因的活性增加，并且显示其依赖于AP-1结合位点。抑制EpFra1细胞中的TGFβ信号传导适度增加了上皮标志物的表达，而沉默zeb1或zeb2则恢复了上皮表型并降低了体外迁移和体内致瘤性。因此，Fra-1诱导编码EMT相关转录因子的基因表达变化，导致上皮细胞获得间充质、侵袭和致瘤能力。本研究定义了Fra-1/AP-1通过直接结合基因组调控区域来调节tgfb1、zeb1和zeb2表达的新功能，这为未来使用小鼠模型进行体内基因操作和临床前研究奠定了基础。
