Genrich Geeske, Kruppa Marcus, Lenk Lennart, Helm Ole, Broich Anna, Freitag-Wolf Sandra, Röcken Christoph, Sipos Bence, Schäfer Heiner, Sebens Susanne
Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17, 24105, Kiel, Germany.
Institute of Medical Informatics and Statistics, UKSH Campus Kiel, Brunswiker Str. 10, 24105, Kiel, Germany.
BMC Cancer. 2016 Feb 25;16:155. doi: 10.1186/s12885-016-2191-7.
Nuclear factor E2 related factor-2 (Nrf2) is an oxidative stress inducible transcription factor being essential in regulating cell homeostasis. Thus, acute induction of Nrf2 in epithelial cells exposed to inflammation confers protection from oxidative cell damage and mutagenesis supporting an anti-tumorigenic role for Nrf2. However, pancreatic ductal adenocarcinoma (PDAC) is characterized by persistent Nrf2 activity conferring therapy resistance which points to a pro-tumorigenic role of Nrf2. A similar dichotomous role in tumorigenesis is described for the Transforming Growth Factor-beta 1 (TGF-β1). The present study therefore aimed at elucidating whether the switch of Nrf2 function towards a tumor promoting one relates to the modulation of TGF-β1 induced cell responses and whether this might occur early in PDAC development.
In situ analysis comprised immunohistochemical stainings of activated (phosphorylated) Nrf2 and Ki67 in pancreatic tissues containing normal ducts and pancreatic intraepithelial neoplasia (PanINs). In vitro, Nrf2 levels in benign (H6c7-pBp), premalignant (H6c7-kras) and malignant (Colo357) pancreatic ductal epithelial cells were modulated by Nrf2 specific siRNA or Nrf2 overexpression. Then, the effect of Nrf2 alone and in combination with TGF-β1 on cell growth and survival was investigated by cell counting, Ki67 staining and apoptosis assays. The underlying cell signaling was investigated by western blotting. Statistical analysis was performed by Shapiro-Wilk test for normal distribution. Parametric data were analyzed by one-way ANOVA, while non-parametric data were analyzed by Kruskal-Wallis one-way ANOVA on ranks.
Significantly elevated expression of activated Nrf2 and Ki67 could be detected in PanINs but not in normal pancreatic ductal epithelium. While the effect of Nrf2 on basal cell growth of H6c7-pBp, H6c7-kras and Colo357 cells was minor, it clearly attenuated the growth inhibiting effects of TGF-β1 in all cell lines. This enhanced Nrf2-mediated cell survival was predominantly based on an enhanced proliferative activity. Accordingly, expression of p21 expression along with expression of phospho-p38 and phospho-Smad3 was diminished whereas Erk-phosphorylation was enhanced under these conditions.
Overall, our data demonstrate that Nrf2 being elevated in early precursor lesions counteracts the growth inhibiting function of TGF-β1 already in benign and premalignant pancreatic ductal epithelial cells. This could represent one fundamental mechanism underlying the functional switch of both- TGF-β1 and Nrf2 - which may manifest already in early stages of PDAC development.
核因子E2相关因子2（Nrf2）是一种氧化应激诱导转录因子，对调节细胞稳态至关重要。因此，在暴露于炎症的上皮细胞中急性诱导Nrf2可保护细胞免受氧化损伤和诱变，支持Nrf2的抗肿瘤作用。然而，胰腺导管腺癌（PDAC）的特征是Nrf2持续激活，赋予治疗抗性，这表明Nrf2具有促肿瘤作用。转化生长因子β1（TGF-β1）在肿瘤发生中也有类似的双重作用。因此，本研究旨在阐明Nrf2功能向促肿瘤方向的转变是否与TGF-β1诱导的细胞反应调节有关，以及这是否可能在PDAC发展的早期发生。
原位分析包括对含有正常导管和胰腺上皮内瘤变（PanINs）的胰腺组织中活化（磷酸化）Nrf2和Ki67进行免疫组化染色。在体外，通过Nrf2特异性siRNA或Nrf2过表达来调节良性（H6c7-pBp）、癌前（H6c7-kras）和恶性（Colo357）胰腺导管上皮细胞中的Nrf2水平。然后，通过细胞计数、Ki67染色和凋亡检测研究Nrf2单独以及与TGF-β1联合对细胞生长和存活的影响。通过蛋白质印迹法研究潜在的细胞信号传导。采用Shapiro-Wilk检验进行正态分布统计分析。参数数据采用单因素方差分析，非参数数据采用Kruskal-Wallis秩和单向方差分析。
在PanINs中可检测到活化Nrf2和Ki67的表达显著升高，但在正常胰腺导管上皮中未检测到。虽然Nrf2对H6c7-pBp、H6c7-kras和Colo357细胞的基础细胞生长影响较小，但它明显减弱了TGF-β1在所有细胞系中的生长抑制作用。这种增强的Nrf2介导的细胞存活主要基于增殖活性的增强。因此，在这些条件下，p21的表达以及磷酸化p38和磷酸化Smad3的表达降低，而Erk磷酸化增强。
总体而言，我们的数据表明，在早期前体病变中升高的Nrf2已经在良性和癌前胰腺导管上皮细胞中抵消了TGF-β1的生长抑制功能。这可能代表了TGF-β1和Nrf2功能转换的一个基本机制，这可能在PDAC发展的早期阶段就已表现出来。
