Tveitarås Maria K, Reigstad Inga, Leiss Lina, Reed Rolf K, Stuhr Linda
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway; Matrix Biology group, Haukeland University Hospital, Norway; Center for Cancer Biomarkers (CCBIO), University of Bergen, Norway.
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway; Matrix Biology group, Haukeland University Hospital, Norway.
Exp Cell Res. 2017 Oct 1;359(1):257-265. doi: 10.1016/j.yexcr.2017.07.021. Epub 2017 Jul 20.
Epithelial to mesenchymal transition (EMT) is considered to be important for cancer invasion and metastasis. Tumour hypoxia, in addition to Transforming Growth Factor-β (TGF-β) and Notch, amongst others, have been suggested to be involved in EMT. We therefore investigated if hypoxia, TGF-β1 and the Notch ligand Jagged-1 alone induced morphological changes with corresponding EMT signatures in different epithelial breast cancer cell lines in vitro. Furthermore, we also studied whether or not TGF-β1, or Jagged-1 in combination with hypoxia added any effect on EMT.
The cells were exposed to normoxia or hypoxia alone or in combination with TGF-β1 or Jagged-1. Morphological responses to treatment were investigated by light microscopy, and changes in markers for EMT and hypoxia were evaluated by western blot analysis and immunofluorescence studies.
One of the four cell lines (MCF7) became elongated and highly multipolar, indicative of EMT, following hypoxia, TGF-β1 and Jagged-1 treatment per se with the most distinct morphological shift seen with Jagged-1 treatment in combination with hypoxia. Also, when regarding hypoxia, MCF7 cells showed the greatest change in EMT-markers of the four cell lines tested, but these changes were not consistent with a typical EMT pattern. The morphology of BT474 cells was not altered following Jagged-1 treatment, however, Jagged-1 increased E-cadherin levels. Morphology was changed following TGF-β1 treatment of BT474 cells, but it did not affect E-cadherin levels. Neither Jagged-1 nor TGF-β1 altered the levels of Vimentin in the BT474 cell line. The E-cadherin responses to hypoxia varied with end-point in both MCF7 and BT474 cells, and in most cases were not consistent with EMT.
Our results using four different breast cancer cell lines in vitro do not provide evidence that EMT is induced by hypoxia alone or in combination with TGF-β1 or the Notch ligand Jagged-1. The inconsistency in morphological appearance and EMT-markers, as well as the time dependent variation in E-cadherin responses could not support EMT. Importantly, there was not one single common response pattern to the stimuli used, suggesting that cell lines with different hormone statuses display individual traits that respond differently to the stimuli applied. Thus, based on the present results, common statements that single factors by themselves can induce EMT seem questionable.
上皮-间质转化（EMT）被认为对癌症侵袭和转移至关重要。除转化生长因子-β（TGF-β）和Notch等外，肿瘤缺氧也被认为与EMT有关。因此，我们研究了缺氧、TGF-β1和Notch配体Jagged-1单独作用时，是否会在体外不同的上皮性乳腺癌细胞系中诱导形态学变化及相应的EMT特征。此外，我们还研究了TGF-β1或Jagged-1与缺氧联合作用是否会对EMT产生额外影响。
将细胞单独暴露于常氧或缺氧环境，或与TGF-β1或Jagged-1联合处理。通过光学显微镜观察处理后的形态学反应，并通过蛋白质印迹分析和免疫荧光研究评估EMT和缺氧标志物的变化。
在四种细胞系之一（MCF7）中，缺氧、TGF-β1和Jagged-1单独处理后细胞变长且高度多极，提示发生EMT，其中Jagged-1与缺氧联合处理时形态学变化最为明显。此外，就缺氧而言，MCF7细胞在测试的四种细胞系中EMT标志物变化最大，但这些变化与典型的EMT模式不一致。Jagged-1处理后BT474细胞的形态未改变，然而，Jagged-1增加了E-钙黏蛋白水平。TGF-β1处理BT474细胞后形态发生改变，但未影响E-钙黏蛋白水平。Jagged-1和TGF-β1均未改变BT474细胞系中波形蛋白的水平。在MCF7和BT474细胞中，E-钙黏蛋白对缺氧的反应随终点不同而变化，且在大多数情况下与EMT不一致。
我们使用四种不同乳腺癌细胞系进行的体外研究结果并未提供证据表明缺氧单独或与TGF-β1或Notch配体Jagged-1联合可诱导EMT。形态学表现和EMT标志物的不一致，以及E-钙黏蛋白反应的时间依赖性变化均不支持EMT。重要的是，对所用刺激没有单一的共同反应模式，这表明具有不同激素状态的细胞系表现出对施加刺激的不同个体特征。因此，基于目前的结果，认为单一因素本身可诱导EMT的普遍说法似乎值得怀疑。
