The anti-oxidative transcription factor Nuclear factor E2 related factor-2 (Nrf2) counteracts TGF-β1 mediated growth inhibition of pancreatic ductal epithelial cells -Nrf2 as determinant of pro-tumorigenic functions of TGF-β1.

Geeske Genrich,Marcus Kruppa,Christoph Röcken,Lennart Lenk,Heiner Schäfer,Sandra Freitag-Wolf,Ole Helm,Anna Broich,Susanne Sebens,Bence Sipos

doi:10.1186/s12885-016-2191-7

Abstract

BackgroundNuclear 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.MethodsIn 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.ResultsSignificantly 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.ConclusionsOverall, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2191-7) contains supplementary material, which is available to authorized users.

Highlights

Nuclear factor E2 related factor-2 (Nrf2) is an oxidative stress inducible transcription factor being essential in regulating cell homeostasis
This elevated proliferative activity of pancreatic intraepithelial neoplasia (PanIN) was significantly associated with increased p-Nrf2 expression (Table 3) supporting the view that Nrf2 activity accounts for higher proliferation of pancreatic ductal epithelial cells already in early PanINs
Nrf2 antagonizes the growth inhibiting effect of Transforming Growth Factor beta-1 (TGF-β1) on benign, premalignant and malignant pancreatic ductal epithelial cells Animal based studies revealed that oncogene activation can lead to enhanced Nrf2 mediated gene transcription accounting for increased proliferation and tumorigenesis [25]

Summary

Introduction

Nuclear factor E2 related factor-2 (Nrf2) is an oxidative stress inducible transcription factor being essential in regulating cell homeostasis. Pancreatic ductal adenocarcinoma (PDAC) is characterized by persistent Nrf activity conferring therapy resistance which points to a pro-tumorigenic role of Nrf. The present study aimed at elucidating whether the switch of Nrf 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. Pancreatic ductal adenocarcinoma (PDAC) is still a leading cause of cancer related deaths in western countries with a poor 5-year survival rate of 6 % [1]. This can be mainly explained by the late diagnosis when the disease has been already progressed to an advanced stage and a profound therapy resistance. One hallmark of PDAC is its pronounced stromal microenvironment comprising stellate cells, myofibroblasts and diverse immune cells together with extracellular matrix [5,6,7,8] which start to accumulate in earliest PanINs [9]

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