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The antioxidant transcription factor Nrf2 modulates the stress response and phenotype of malignant as well as premalignant pancreatic ductal epithelial cells by inducing expression of the ATF3 splicing variant ΔZip2

Oncogenevolume 38pages14611476 (2019) | Download Citation

Abstract

Pancreatic ductal adenocarcinoma (PDAC) exhibits one of the worst survival rates of all cancers. While death rates show declining trends in the majority of cancers, PDAC registers rising rates. Based on the recently described crosstalk between TGF-β1 and Nrf2 in the PDAC development, the involvement of ATF3 and its splice variant ΔZip2 in TGF-β1- and Nrf2-driven pancreatic tumorigenesis was investigated. As demonstrated here, PDAC (Panc1, T3M4) cells or premalignant H6c7 pancreatic ductal epithelial cells differentially express ΔZip2- and ATF3, relating to stronger Nrf2 activity seen in Panc1 cells and TGF-ß1 activity in T3M4 or H6c7 cells, respectively. Treatment with the electrophile/oxidative stress inducer tBHQ or the cytostatic drug gemcitabine strongly elevated ΔZip2 expression in a Nrf2-dependent fashion. The differential expression of ATF3 and ΔZip2 in response to Nrf2 and TGF-ß1 relates to differential ATF3-gene promoter usage, giving rise of distinct splice variants. Nrf2-dependent ΔZip2 expression confers resistance against gemcitabine-induced apoptosis, only partially relating to interference with ATF3 and its proapoptotic activity, e.g., through CHOP-expression. In fact, ΔZip2 autonomously activates expression of cIAP anti-apoptotic proteins. Moreover, ΔZip2 favors and ATF3 suppresses growth and clonal expansion of PDAC cells, again partially independent of each other. Using a Panc1 tumor xenograft model in SCID-beige mice, the opposite activities of ATF3 and ΔZip2 on tumor-growth and chemoresistance were verified in vivo. Immunohistochemical analyses confirmed ΔZip2 and Nrf2 coexpression in cancerous and PanIN structures of human PDAC and chronic pancreatitis tissues, respectively, which to some extent was reciprocal to ATF3 expression. It is concluded that depending on selective ATF3-gene promoter usage by Nrf2, the ΔZip2 expression is induced in response to electrophile/oxidative (here through tBHQ) and xenobiotic (here through gemcitabine) stress, providing apoptosis protection and growth advantages to pancreatic ductal epithelial cells. This condition may substantially add to pancreatic carcinogenesis driven by chronic inflammation.

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Acknowledgements

The authors thank Maike Witt-Ramdohr, Iris Kosmol, and Dagmar Leisner (from the Institute of Experimental Cancer Research) as well as Sandra Krüger and Deniza Hajzeri (both from the Institute of Pathology) for excellent technical assistance. Special thanks also to Dr. Christian Röder (Biomaterial Bank of the Comprehensive Cancer Center Kiel) for supporting gene expression analysis on PDAC patient tissues and Dr. Claudia Geismann (Department of Internal Medicine) for assistance with mice experiments.

Funding

Financial support by the Medical Faculty of the CAU Kiel (to HS and OH) and the Cluster of Excellence “Inflammation at Interfaces” (to HS, AA, and SS) is greatly acknowledged.

Author contributions

MLK, LH, and FD conducted the experiments. CR and BS contributed to histochemistry experiments and data analysis. SS, AA, OH, and HS designed the study. OH and HS wrote the manuscript. All authors read and approved the contents of the manuscript and its publication.

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Author notes

  1. Part of a doctoral thesis (MLK).

Affiliations

  1. Laboratory of Molecular Gastroenterology & Tumor Biology, Institute for Experimental Cancer Research, Christian-Albrechts-University & UKSH Campus Kiel, Bldg. 17, Arnold-Heller-Straße 3, 24105, Kiel, Germany

    • My-Lan Kha
    • , Lisa Hesse
    • , Florian Deisinger
    •  & Heiner Schäfer
  2. Department of Pathology and Neuropathology, University Hospital Tübingen, Liebermeisterstr. 8, 72076, Tübingen, Germany

    • Bence Sipos
  3. Institute of Pathology, Christian-Albrechts-University & UKSH Campus Kiel, Bldg. 14, Arnold-Heller-Straße 3, 24105, Kiel, Germany

    • Christoph Röcken
  4. Biomaterial Bank of the Comprehensive Cancer Center Kiel, UKSH Campus Kiel, Bldg. 17, Arnold-Heller-Straße 3, 24105, Kiel, Germany

    • Christoph Röcken
    •  & Susanne Sebens
  5. Laboratory of Gastrointestinal Signal Transduction, Department of Internal Medicine I, UKSH Campus Kiel, Bldg. 6, Arnold-Heller-Straße 3, 24105 Kiel, Germany

    • Alexander Arlt
  6. Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University & UKSH Campus Kiel, Bldg. 17, Arnold-Heller-Straße 3, 24105, Kiel, Germany

    • Susanne Sebens
    •  & Ole Helm

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethics statement

Ethics committees (University Hospital Tübingen, reference: 470/210BO1; Medical Faculty of Kiel University, reference: D400/14; P2N_2018-035) approved the study. Written informed consent was obtained from all patients.

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Correspondence to Heiner Schäfer.

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https://doi.org/10.1038/s41388-018-0518-3