Application of two-dimensional gel-based mass spectrometry to functionally dissect resistance to targeted cancer therapy

Authors

  • Oliver Klein,

    1. Berlin-Brandenburg Center for Regenerative Therapies, Charité – Universitätsmedizin Berlin, Berlin, Germany
    2. Core Unit Proteomics, Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany
    3. Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Berlin, Germany
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    • These authors contributed equally to this work.

  • Nadine Rohwer,

    1. Department of Hepatology and Gastroenterology, Charité – Universitätsmedizin Berlin, Berlin, Germany
    2. Molekulares Krebsforschungszentrum (MKFZ), Charité – Universitätsmedizin Berlin, Berlin, Germany
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    • These authors contributed equally to this work.

  • Katja Freitag de Molina,

    1. German Consortium for Translational Cancer Research (DKTK), Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany
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  • Stefan Mergler,

    1. Department of Ophthalmology, Charité – Universitätsmedizin Berlin, Berlin, Germany
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  • Petra Wessendorf,

    1. Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Berlin, Germany
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  • Marion Herrmann,

    1. Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Berlin, Germany
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  • Joachim Klose,

    Corresponding author
    1. Berlin-Brandenburg Center for Regenerative Therapies, Charité – Universitätsmedizin Berlin, Berlin, Germany
    2. Core Unit Proteomics, Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany
    3. Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Berlin, Germany
    • Correspondence: Professor Joachim Klose, Berlin-Brandenburg Center for Regenerative Therapies, Core Unit Proteomics and Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany

      E-mail: joachim.klose@charite.de

      Fax: +49-30-450-566-904

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    • These authors jointly directed this work.

  • Thorsten Cramer

    1. Department of Hepatology and Gastroenterology, Charité – Universitätsmedizin Berlin, Berlin, Germany
    2. Molekulares Krebsforschungszentrum (MKFZ), Charité – Universitätsmedizin Berlin, Berlin, Germany
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    • These authors jointly directed this work.


  • Colour Online: See the article online to view Figs. 1, 4 and 6 in colour.

Abstract

Purpose

The majority of gastric cancers are diagnosed at advanced stages, characterized by robust therapy resistance. The oncoprotein hypoxia-inducible factor 1 (HIF-1) is associated with therapy resistance, partly via activation of the DNA damage response. We have noted a robust ability of gastric cancer cells to functionally compensate the loss of HIF-1 in vitro. The purpose of this study was to identify molecular pathways that underlie this compensation.

Experimental design

We performed 2DE to compare the nuclear proteome of wild-type and HIF-1-deficient gastric cancer cells. Differently expressed protein spots were identified via MS). After bioinformatic evaluation, functional validation of selected identified pathways was performed.

Results

2DE displayed a total of 2523 protein spots, from which 87 were identified as regulated by HIF-1. Seventy of the identified spots were different proteins and 17 were isoforms. Bioinformatic analyses revealed that a significant amount of the identified proteins were related to cellular survival pathways. Specifically, members of the proteasome pathway were found upregulated upon loss of HIF-1. Combined inhibition of HIF-1 and the proteasome inflicted significant DNA damage, supporting the hypothesis that the proteasome is of functional importance to compensate the loss of HIF-1.

Conclusions and clinical relevance

Our data show robust and functional changes of the nuclear proteome upon inactivation of the HIF-1 oncoprotein in gastric cancer cells. We propose that 2DE-MS represents a useful tool to functionally dissect resistance mechanisms to targeted therapy and to identify novel targets for antiproliferative combination therapy.

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