• An Erratum to this article was published on 07 April 2015

This article has been updated

Abstract

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP–based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell–autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.

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Change history

  • 31 October 2014

     In the version of this article initially published online, affiliation 12 was missing for Roland Rad, Roland M. Schmid and Dieter Saur. The list should have read: “Roland Rad1,5,12,13, Roland M Schmid1,12,13 & Dieter Saur1,12,13.” The error has been corrected for the print, PDF and HTML versions of this article.

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References

  1. 1.

    , & Cancer statistics, 2013. CA Cancer J. Clin. 63, 11–30 (2013).

  2. 2.

    et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4, 437–450 (2003).

  3. 3.

    et al. Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Cancer Cell 11, 291–302 (2007).

  4. 4.

    et al. A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors. Proc. Natl. Acad. Sci. USA 105, 10137–10142 (2008).

  5. 5.

    , & RAS oncogenes: weaving a tumorigenic web. Nat. Rev. Cancer 11, 761–774 (2011).

  6. 6.

    , , , & The Nestin progenitor lineage is the compartment of origin for pancreatic intraepithelial neoplasia. Proc. Natl. Acad. Sci. USA 104, 4437–4442 (2007).

  7. 7.

    et al. Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice. Proc. Natl. Acad. Sci. USA 105, 18913–18918 (2008).

  8. 8.

    et al. Context-dependent transformation of adult pancreatic cells by oncogenic K-Ras. Cancer Cell 16, 379–389 (2009).

  9. 9.

    , & KRAS, Hedgehog, Wnt and the twisted developmental biology of pancreatic ductal adenocarcinoma. Nat. Rev. Cancer 10, 683–695 (2010).

  10. 10.

    et al. In vivo diagnosis of murine pancreatic intraepithelial neoplasia and early-stage pancreatic cancer by molecular imaging. Proc. Natl. Acad. Sci. USA 108, 9945–9950 (2011).

  11. 11.

    & Genetically engineered mouse models of pancreatic cancer: unravelling tumour biology and progressing translational oncology. Gut 61, 1488–1500 (2012).

  12. 12.

    et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324, 1457–1461 (2009).

  13. 13.

    , , , & Oncogenic Kras-induced GM-CSF production promotes the development of pancreatic neoplasia. Cancer Cell 21, 836–847 (2012).

  14. 14.

    et al. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell 21, 418–429 (2012).

  15. 15.

    , & Mosaic Cre-mediated recombination in pancreas using the pdx-1 enhancer/promoter. Genesis 26, 143–144 (2000).

  16. 16.

    et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7, 469–483 (2005).

  17. 17.

    et al. Selective requirement of PI3K/PDK1 signaling for Kras oncogene-driven pancreatic cell plasticity and cancer. Cancer Cell 23, 406–420 (2013).

  18. 18.

    & Vagaries of conditional gene targeting. Nat. Immunol. 8, 665–668 (2007).

  19. 19.

    & Mast cells: the Jekyll and Hyde of tumor growth. Trends Immunol. 25, 235–241 (2004).

  20. 20.

    Mast cells and pancreatic cancer. N. Engl. J. Med. 358, 1860–1861 (2008).

  21. 21.

    et al. Independent prognostic value of eosinophil and mast cell infiltration in colorectal cancer tissue. J. Pathol. 189, 487–495 (1999).

  22. 22.

    et al. Stromal mast cells in invasive breast cancer are a marker of favourable prognosis: a study of 4,444 cases. Breast Cancer Res. Treat. 107, 249–257 (2008).

  23. 23.

    et al. Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity. Immunity 35, 832–844 (2011).

  24. 24.

    et al. Mast cells are required for angiogenesis and macroscopic expansion of Myc-induced pancreatic islet tumors. Nat. Med. 13, 1211–1218 (2007).

  25. 25.

    et al. Mast cells in tumor microenvironment promotes the in vivo growth of pancreatic ductal adenocarcinoma. Clin. Cancer Res. 17, 7015–7023 (2011).

  26. 26.

    et al. Interstitial cells of Cajal integrate excitatory and inhibitory neurotransmission with intestinal slow-wave activity. Nat. Commun. 4, 1630 (2013).

  27. 27.

    , & Uncoupling cancer mutations reveals critical timing of p53 loss in sarcomagenesis. Cancer Res. 71, 4040–4047 (2011).

  28. 28.

    et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137, 835–848 (2009).

  29. 29.

    et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 491, 399–405 (2012).

  30. 30.

    et al. The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma. Nature 486, 266–270 (2012).

  31. 31.

    et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011).

  32. 32.

    et al. Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism. Cell 149, 656–670 (2012).

  33. 33.

    et al. Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice. J. Clin. Invest. 122, 639–653 (2012).

  34. 34.

    et al. A modular and flexible ESC-based mouse model of pancreatic cancer. Genes Dev. 28, 85–97 (2014).

  35. 35.

    et al. Generation of primary tumors with Flp recombinase in FRT-flanked p53 mice. Dis. Model. Mech. 5, 397–402 (2012).

  36. 36.

    et al. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat. Genet. 29, 418–425 (2001).

  37. 37.

    et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell 119, 847–860 (2004).

  38. 38.

    , , , & A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

  39. 39.

    , , & An Flp indicator mouse expressing alkaline phosphatase from the ROSA26 locus. Nat. Genet. 29, 257–259 (2001).

  40. 40.

    et al. Essential role of PDK1 in regulating cell size and development in mice. EMBO J. 21, 3728–3738 (2002).

  41. 41.

    & High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS ONE 2, e162 (2007).

  42. 42.

    et al. In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A. Genesis 43, 129–135 (2005).

  43. 43.

    et al. E-cadherin regulates metastasis of pancreatic cancer in vivo and is suppressed by a SNAIL/HDAC1/HDAC2 repressor complex. Gastroenterology 137 361–371, 371 e361–e365 (2009).

  44. 44.

    et al. PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice. Science 330, 1104–1107 (2010).

  45. 45.

    et al. CXCR4 expression increases liver and lung metastasis in a mouse model of pancreatic cancer. Gastroenterology 129, 1237–1250 (2005).

  46. 46.

    et al. A porcine model of familial adenomatous polyposis. Gastroenterology 143, 1173–1175. e1171–1177 (2012).

  47. 47.

    et al. Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations. Cancer Res. 66, 95–106 (2006).

  48. 48.

    et al. Efemp1 and p27(Kip1) modulate responsiveness of pancreatic cancer cells towards a dual PI3K/mTOR inhibitor in preclinical models. Oncotarget 4, 277–288 (2013).

  49. 49.

    & Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc., B 57, 289–300 (1995).

  50. 50.

    et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

  51. 51.

    et al. Single-nucleotide promoter polymorphism alters transcription of neuronal nitric oxide synthase exon 1c in infantile hypertrophic pyloric stenosis. Proc. Natl. Acad. Sci. USA 101, 1662–1667 (2004).

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Acknowledgements

The authors thank A. Berns (Netherlands Cancer Institute), S. Dymecki (Harvard Medical School), T. Jacks (Massachusetts Institute of Technology), L. Luo (Stanford University), J. Martinez-Barbera (University College London) and D. Tuveson (Cold Spring Harbor Laboratory) for providing transgenic animals, C. Wright (Vanderbilt University) for the mouse Pdx1 promoter construct, P. Soriano (Mount Sinai School of Medicine) for the Flp-o expression vector and the R26 targeting vector, T. Schmidt and M. Bewerunge-Hudler (DKFZ Microarray Core Facility) for mRNA analyses, and J. Götzfried, U. Götz and S. Jaeckel for technical assistance. This work was supported by funding from Deutsche Forschungsgemeinschaft (DFG SA 1374/4-1 to D.S. and SFB824, TP C9 to G.S. and D.S.), the Helmholtz Alliance Preclinical Comprehensive Cancer Center (to H.-R.R., R.R., R.M.S. and D.S.), the German Cancer Consortium (DKTK) (to R.R., R.M.S. and D.S.), the Wilhelm-Sander Foundation (2012.084.1 to G.S.), the Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal (I.V.), the European Union (ERC Advanced Grant No.233074 to H.-R.R.), and the National Cancer Institute USA (R01 CA138265 to D.G.K. and CA155620 to A.M.L.).

Author information

Author notes

    • Nina Schönhuber
    • , Barbara Seidler
    • , Kathleen Schuck
    •  & Christian Veltkamp

    These authors contributed equally to this work.

Affiliations

  1. Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, München, Germany.

    • Nina Schönhuber
    • , Barbara Seidler
    • , Kathleen Schuck
    • , Christian Veltkamp
    • , Christina Schachtler
    • , Magdalena Zukowska
    • , Stefan Eser
    • , Mariel C Paul
    • , Sabine Klein
    • , Roland Rad
    • , Roland M Schmid
    • , Günter Schneider
    •  & Dieter Saur
  2. Division for Cellular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

    • Thorsten B Feyerabend
    •  & Hans-Reimer Rodewald
  3. Gene Center and Department of Biochemistry, Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-Universität München, München, Germany.

    • Philipp Eser
  4. Moores Cancer Center, Division of Surgical Oncology, University of California San Diego, La Jolla, California, USA.

    • Andrew M Lowy
  5. Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, UK.

    • Ruby Banerjee
    • , Fangtang Yang
    • , Allan Bradley
    •  & Roland Rad
  6. Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina, USA.

    • Chang-Lung Lee
    •  & David G Kirsch
  7. Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.

    • Everett J Moding
    •  & David G Kirsch
  8. Helmholtz Zentrum München, Research Unit Comparative Medicine, Neuherberg, Germany.

    • Angelika Scheideler
  9. MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK.

    • Dario R Alessi
  10. Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-Sodercan), Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain.

    • Ignacio Varela
  11. Livestock Biotechnology, Technische Universität München, Freising, Germany.

    • Alexander Kind
    •  & Angelika E Schnieke
  12. German Cancer Consortium (DKTK), Heidelberg, Germany.

  13. German Cancer Research Center (DKFZ), Heidelberg, Germany.

    • Roland Rad
    • , Roland M Schmid
    •  & Dieter Saur

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Contributions

B.S. and D.S. designed research; N.S., B.S., K.S., C.V., C.S., M.Z., S.E., M.C.P., P.E., S.K., R.B., F.Y., A.S., I.V., R.R., G.S. and D.S., performed research; T.B.F., A.M.L., C.-L.L., E.J.M., D.G.K., A.S., D.R.A., I.V., A.B., A.K., A.E.S., H.-R.R., R.R. and R.M.S. contributed new reagents/analytic tools; N.S., B.S., K.S., C.V., C.S., M.Z., S.E., M.C.P., P.E., S.K., R.B., F.Y., I.V., R.R., G.S. and D.S. analyzed data; and B.S. and D.S. wrote the paper. N.S., B.S., K.S. and C.V. contributed equally to this manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dieter Saur.

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    Supplementary Text and Figures

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https://doi.org/10.1038/nm.3646

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