title: 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.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17, 24105, Kiel, Germany. stu94401@mail.uni-kiel.de.
authors.forename: Geeske
authors.initials: G
authors.lastname: Genrich
authors.affiliation: Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17, 24105, Kiel, Germany. marcus.kruppa@gmail.com.
authors.forename: Marcus
authors.initials: M
authors.lastname: Kruppa
authors.affiliation: Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17, 24105, Kiel, Germany. lennartlenk@email.uni-kiel.de.
authors.forename: Lennart
authors.initials: L
authors.lastname: Lenk
authors.affiliation: Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17, 24105, Kiel, Germany. olehelm@email.uni-kiel.de.
authors.forename: Ole
authors.initials: O
authors.lastname: Helm
authors.affiliation: Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17, 24105, Kiel, Germany. anna-k-broich@web.de.
authors.forename: Anna
authors.initials: A
authors.lastname: Broich
authors.affiliation: Institute of Medical Informatics and Statistics, UKSH Campus Kiel, Brunswiker Str. 10, 24105, Kiel, Germany. freitag@medinfo.uni-kiel.de.
authors.forename: Sandra
authors.initials: S
authors.lastname: Freitag-Wolf
authors.affiliation: Department of Pathology, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 14, 24105, Kiel, Germany. Christoph.Roecken@uk-sh.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Röcken
authors.affiliation: Department of Pathology and Neuropathology, University Hospital Tübingen, Liebermeisterstr. 8, 72076, Tübingen, Germany. Bence.Sipos@med.uni-tuebingen.de.
authors.forename: Bence
authors.initials: B
authors.lastname: Sipos
authors.affiliation: Laboratory of Molecular Gastroenterology & Hepatology, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 6, 24105, Kiel, Germany. hschaef@1med.uni-kiel.de.
authors.forename: Heiner
authors.initials: H
authors.lastname: Schäfer
authors.affiliation: Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17, 24105, Kiel, Germany. susanne.sebens@email.uni-kiel.de.
authors.forename: Susanne
authors.initials: S
authors.lastname: Sebens
mesh_terms: D017209:Apoptosis / Q000235:genetics; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D010183:Pancreatic Ducts / Q000378:metabolism* / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011230:Precancerous Conditions; D011485:Protein Binding; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D019394:Ki-67 Antigen; D051267:NF-E2-Related Factor 2; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; D020928:Mitogen-Activated Protein Kinases