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In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer

Nature Medicine volume 20, pages 1138–1146 (2014)Cite this article

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Abstract

In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.

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Figure 1: A transposon-based mouse model of liver cancer shows therapy resistance resembling that of human HCC.
Figure 2: Outline and results of an in vivo RNAi screen to identify sorafenib treatment response modifiers.
Figure 3: Functional genetic validation of Mapk14 as a sorafenib sensitizer gene.
Figure 4: Pharmacological inhibition of Mapk14 sensitizes to sorafenib therapy.
Figure 5: Second-generation Mapk14 inhibitors (skepinone-L and PH-797804) are effective for HCC treatment in combination with sorafenib and exert no adverse effects.
Figure 6: Cross-talk of Mapk14 and Mek-Erk signaling in HCC and its role in sorafenib resistance.

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References

  1. Poulikakos, P.I. & Rosen, N. Mutant BRAF melanomas—dependence and resistance. Cancer Cell 19, 11–15 (2011).

    Article  CAS  Google Scholar 

  2. Hartsough, E., Shao, Y. & Aplin, A.E. Resistance to RAF inhibitors revisited. J. Invest. Dermatol. 134, 319–325 (2014).

    Article  CAS  Google Scholar 

  3. Berasain, C. Hepatocellular carcinoma and sorafenib: too many resistance mechanisms? Gut 62, 1674–1675 (2013).

    Article  CAS  Google Scholar 

  4. Bruix, J., Gores, G.J. & Mazzaferro, V. Hepatocellular carcinoma: clinical frontiers and perspectives. Gut 63, 844–855 (2014).

    Article  CAS  Google Scholar 

  5. Yau, T., Chan, P., Epstein, R. & Poon, R.T. Evolution of systemic therapy of advanced hepatocellular carcinoma. World J. Gastroenterol. 14, 6437–6441 (2008).

    Article  Google Scholar 

  6. Lord, R., Suddle, A. & Ross, P.J. Emerging strategies in the treatment of advanced hepatocellular carcinoma: the role of targeted therapies. Int. J. Clin. Pract. 65, 182–188 (2011).

    Article  CAS  Google Scholar 

  7. Llovet, J.M. et al. Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359, 378–390 (2008).

    Article  CAS  Google Scholar 

  8. Wilhelm, S. et al. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat. Rev. Drug Discov. 5, 835–844 (2006).

    Article  CAS  Google Scholar 

  9. Wuestefeld, T. et al. A direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration. Cell 153, 389–401 (2013).

    Article  CAS  Google Scholar 

  10. Kang, T.W. et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 479, 547–551 (2011).

    Article  CAS  Google Scholar 

  11. Carlson, C.M., Frandsen, J.L., Kirchhof, N., McIvor, R.S. & Largaespada, D.A. Somatic integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor development in the mouse. Proc. Natl. Acad. Sci. USA 102, 17059–17064 (2005).

    Article  CAS  Google Scholar 

  12. Braumüller, H. et al. T-helper-1-cell cytokines drive cancer into senescence. Nature 494, 361–365 (2013).

    Article  Google Scholar 

  13. Holohan, C., Van, S.S., Longley, D.B. & Johnston, P.G. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer 13, 714–726 (2013).

    Article  CAS  Google Scholar 

  14. Margutti, S. & Laufer, S.A. Are MAP kinases drug targets? Yes, but difficult ones. ChemMedChem 2, 1116–1140 (2007).

    Article  CAS  Google Scholar 

  15. Sawey, E.T. et al. Identification of a therapeutic strategy targeting amplified FGF19 in liver cancer by oncogenomic screening. Cancer Cell 19, 347–358 (2011).

    Article  CAS  Google Scholar 

  16. Wagner, E.F. & Nebreda, A.R. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat. Rev. Cancer 9, 537–549 (2009).

    Article  CAS  Google Scholar 

  17. Nebreda, A.R. & Porras, A. p38 MAP kinases: beyond the stress response. Trends Biochem. Sci. 25, 257–260 (2000).

    Article  CAS  Google Scholar 

  18. Zhang, J., Shen, B. & Lin, A. Novel strategies for inhibition of the p38 MAPK pathway. Trends Pharmacol. Sci. 28, 286–295 (2007).

    Article  Google Scholar 

  19. Goldstein, D.M., Kuglstatter, A., Lou, Y. & Soth, M.J. Selective p38alpha inhibitors clinically evaluated for the treatment of chronic inflammatory disorders. J. Med. Chem. 53, 2345–2353 (2010).

    Article  CAS  Google Scholar 

  20. Dominguez, C., Powers, D.A. & Tamayo, N. p38 MAP kinase inhibitors: many are made, but few are chosen. Curr. Opin. Drug Discov. Devel. 8, 421–430 (2005).

    CAS  PubMed  Google Scholar 

  21. Koeberle, S.C. et al. Skepinone-L is a selective p38 mitogen-activated protein kinase inhibitor. Nat. Chem. Biol. 8, 141–143 (2012).

    Article  CAS  Google Scholar 

  22. Hope, H.R. et al. Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogen-activated protein kinase: preclinical-to-clinical translation. J. Pharmacol. Exp. Ther. 331, 882–895 (2009).

    Article  CAS  Google Scholar 

  23. Fischer, S. et al. Dibenzosuberones as p38 mitogen-activated protein kinase inhibitors with low ATP competitiveness and outstanding whole blood activity. J. Med. Chem. 56, 241–253 (2013).

    Article  CAS  Google Scholar 

  24. MacNee, W., Allan, R.J., Jones, I., De Salvo, M.C. & Tan, L.F. Efficacy and safety of the oral p38 inhibitor PH-797804 in chronic obstructive pulmonary disease: a randomised clinical trial. Thorax 68, 738–745 (2013).

    Article  Google Scholar 

  25. Calvisi, D.F. et al. Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer. J. Hepatol. 54, 311–319 (2011).

    Article  CAS  Google Scholar 

  26. Calvisi, D.F. et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 130, 1117–1128 (2006).

    Article  CAS  Google Scholar 

  27. Guichard, C. et al. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma. Nat. Genet. 44, 694–698 (2012).

    Article  CAS  Google Scholar 

  28. Steelman, L.S. et al. JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. Leukemia 18, 189–218 (2004).

    Article  CAS  Google Scholar 

  29. Hindley, A. & Kolch, W. Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases. J. Cell Sci. 115, 1575–1581 (2002).

    CAS  PubMed  Google Scholar 

  30. Turner, N.C. et al. A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor. EMBO J. 27, 1368–1377 (2008).

    Article  CAS  Google Scholar 

  31. Swanton, C. et al. Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs. Cancer Cell 11, 498–512 (2007).

    Article  CAS  Google Scholar 

  32. Giroux, V., Iovanna, J. & Dagorn, J.C. Probing the human kinome for kinases involved in pancreatic cancer cell survival and gemcitabine resistance. FASEB J. 20, 1982–1991 (2006).

    Article  CAS  Google Scholar 

  33. Huang, S. et al. MED12 controls the response to multiple cancer drugs through regulation of TGF-beta receptor signaling. Cell 151, 937–950 (2012).

    Article  CAS  Google Scholar 

  34. Burgess, D.J. et al. Topoisomerase levels determine chemotherapy response in vitro and in vivo. Proc. Natl. Acad. Sci. USA 105, 9053–9058 (2008).

    Article  Google Scholar 

  35. Huesken, D. et al. Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995–1001 (2005).

    Article  CAS  Google Scholar 

  36. Mitchell, C. & Willenbring, H. A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nat. Protoc. 3, 1167–1170 (2008).

    Article  CAS  Google Scholar 

  37. Schlaeger, C. et al. Etiology-dependent molecular mechanisms in human hepatocarcinogenesis. Hepatology 47, 511–520 (2008).

    Article  CAS  Google Scholar 

  38. Dow, L.E. et al. A pipeline for the generation of shRNA transgenic mice. Nat. Protoc. 7, 374–393 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Rinkel, N. Struever, C. Hermann, V. Geissler, U. Koppenhoefer, H. Riedesel, M. Jarek, M. Scharfe and the HZI Genome Analytics Group team for technical assistance. We thank the tissue bank of the National Center for Tumor Diseases Heidelberg for providing human HCC tissues. This work was supported by the German Research Foundation, DFG (Emmy Noether Programme ZE 545/2-1 to L.Z., the “Rebirth” Cluster of Excellence, project “Liver regeneration”, SFB/TRR77 and SFB685), the Helmholtz Association of German Research Centers (VH-NG-424 to L.Z.), the European Commission (project 'Heptromic') and the Wilhelm Sander Stiftung.

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Author notes

  1. Ramona Rudalska and Daniel Dauch: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany

    Ramona Rudalska, Daniel Dauch, Torsten Wuestefeld, Tae-Won Kang, Anja Hohmeyer, Marina Pesic, Josef Leibold & Lars Zender

  2. Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany

    Thomas Longerich & Peter Schirmacher

  3. The Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Katherine McJunkin

  4. Translational Gastrointestinal Oncology Group within the German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany

    Tae-Won Kang, Anja Hohmeyer & Lars Zender

  5. Comprehensive Cancer Center Mainfranken, University of Wuerzburg, Wuerzburg, Germany

    Anne von Thun & Martin Eilers

  6. Theodor Boveri Institute, Biocenter, University of Wuerzburg, Wuerzburg, Germany

    Anne von Thun & Martin Eilers

  7. Research Institute of Molecular Pathology, Vienna, Austria

    Johannes Zuber

  8. Department of Gastroenterology, University Hospital Heidelberg, Heidelberg, Germany

    Karl-Heinz Weiss

  9. Cancer Genome Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Scott Powers

  10. Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany

    Nisar P Malek

  11. Institute of Pathology, University of Tuebingen, Tuebingen, Germany

    Bence Sipos

  12. Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Scott W Lowe

  13. Howard Hughes Medical Institute, New York, New York, USA

    Scott W Lowe

  14. Helmholtz Centre for Infection Research, Braunschweig, Germany

    Robert Geffers

  15. Department of Pharmaceutical and Medicinal Chemistry, University of Tuebingen, Tuebingen, Germany

    Stefan Laufer

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  1. Ramona Rudalska
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Contributions

The study was designed by L.Z. Research was conducted by R.R., D.D., T.L., K.M., T.W., T.-W.K., A.H., M.P., J.L., A.v.T., P.S., J.Z., K.-H.W., S.P., N.P.M., M.E., B.S., S.W.L., R.G., S.L. and L.Z. The manuscript was written by L.Z., R.R. and D.D.

Corresponding author

Correspondence to Lars Zender.

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The authors declare no competing financial interests.

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Results from in vivo RNAi screen. (XLS 169 kb)

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Rudalska, R., Dauch, D., Longerich, T. et al. In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer. Nat Med 20, 1138–1146 (2014). https://doi.org/10.1038/nm.3679

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