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MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer

Nature Genetics volume 39, pages 759–769 (2007)Cite this article

Abstract

We performed a high-throughput retroviral insertional mutagenesis screen in mouse mammary tumor virus (MMTV)-induced mammary tumors and identified 33 common insertion sites, of which 17 genes were previously not known to be associated with mammary cancer and 13 had not previously been linked to cancer in general. Although members of the Wnt and fibroblast growth factors (Fgf) families were frequently tagged, our exhaustive screening for MMTV insertion sites uncovered a new repertoire of candidate breast cancer oncogenes. We validated one of these genes, Rspo3, as an oncogene by overexpression in a p53-deficient mammary epithelial cell line. The human orthologs of the candidate oncogenes were frequently deregulated in human breast cancers and associated with several tumor parameters. Computational analysis of all MMTV-tagged genes uncovered specific gene families not previously associated with cancer and showed a significant overrepresentation of protein domains and signaling pathways mainly associated with development and growth factor signaling. Comparison of all tagged genes in MMTV and Moloney murine leukemia virus–induced malignancies showed that both viruses target mostly different genes that act predominantly in distinct pathways.

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Figure 1: Rspo3 promotes tumorigenicity of HC11 cells, a p53-deficient mammary epithelial cell line.
Figure 2: Mining data sets generated by insertional mutagenesis screens in mice to identify genes associated with cancer in humans.

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References

  1. Nusse, R. & Varmus, H.E. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 31, 99–109 (1982).

    Article  CAS  Google Scholar 

  2. Johansson, F.K. et al. Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging. Proc. Natl. Acad. Sci. USA 101, 11334–11337 (2004).

    Article  CAS  Google Scholar 

  3. Mikkers, H. et al. High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat. Genet. 32, 153–159 (2002).

    Article  CAS  Google Scholar 

  4. Suzuki, T. et al. New genes involved in cancer identified by retroviral tagging. Nat. Genet. 32, 166–174 (2002).

    Article  CAS  Google Scholar 

  5. Chatterjee, G. et al. Acceleration of mouse mammary tumor virus-induced murine mammary tumorigenesis by a p53 172H transgene: influence of FVB background on tumor latency and identification of novel sites of proviral insertion. Am. J. Pathol. 161, 2241–2253 (2002).

    Article  CAS  Google Scholar 

  6. Callahan, R. & Smith, G.H. MMTV-induced mammary tumorigenesis: gene discovery, progression to malignancy and cellular pathways. Oncogene 19, 992–1001 (2000).

    Article  CAS  Google Scholar 

  7. Lazo, P.A., Lee, J.S. & Tsichlis, P.N. Long-distance activation of the Myc protooncogene by provirus insertion in Mlvi-1 or Mlvi-4 in rat T-cell lymphomas. Proc. Natl. Acad. Sci. USA 87, 170–173 (1990).

    Article  CAS  Google Scholar 

  8. Lammie, G.A. et al. Proviral insertions near cyclin D1 in mouse lymphomas: a parallel for BCL1 translocations in human B-cell neoplasms. Oncogene 7, 2381–2387 (1992).

    CAS  PubMed  Google Scholar 

  9. Sakatani, T. et al. Loss of imprinting of Igf2 alters intestinal maturation and tumorigenesis in mice. Science 307, 1976–1978 (2005).

    Article  CAS  Google Scholar 

  10. van Roozendaal, C.E. et al. Loss of imprinting of IGF2 and not H19 in breast cancer, adjacent normal tissue and derived fibroblast cultures. FEBS Lett. 437, 107–111 (1998).

    Article  CAS  Google Scholar 

  11. Shackleford, G.M., MacArthur, C.A., Kwan, H.C. & Varmus, H.E. Mouse mammary tumor virus infection accelerates mammary carcinogenesis in Wnt-1 transgenic mice by insertional activation of int-2/Fgf-3 and hst/Fgf-4. Proc. Natl. Acad. Sci. USA 90, 740–744 (1993).

    Article  CAS  Google Scholar 

  12. Gallahan, D. & Callahan, R. Mammary tumorigenesis in feral mice: identification of a new int locus in mouse mammary tumor virus (Czech II)-induced mammary tumors. J. Virol. 61, 66–74 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Gallahan, D. & Callahan, R. The mouse mammary tumor associated gene INT3 is a unique member of the Notch gene family (Notch4). Oncogene 14, 1883–1890 (1997).

    Article  CAS  Google Scholar 

  14. Takahashi, K., Mitsui, K. & Yamanaka, S. Role of ERas in promoting tumour-like properties in mouse embryonic stem cells. Nature 423, 541–545 (2003).

    Article  CAS  Google Scholar 

  15. Zhang, S. et al. p16 INK4a gene promoter variation and differential binding of a repressor, the ras-responsive zinc-finger transcription factor, RREB. Oncogene 22, 2285–2295 (2003).

    Article  CAS  Google Scholar 

  16. Erny, K.M., Peli, J., Lambert, J.F., Muller, V. & Diggelmann, H. Involvement of the Tpl-2/cot oncogene in MMTV tumorigenesis. Oncogene 13, 2015–2020 (1996).

    CAS  PubMed  Google Scholar 

  17. Ceci, J.D. et al. Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation. Genes Dev. 11, 688–700 (1997).

    Article  CAS  Google Scholar 

  18. Sourvinos, G., Tsatsanis, C. & Spandidos, D.A. Overexpression of the Tpl-2/Cot oncogene in human breast cancer. Oncogene 18, 4968–4973 (1999).

    Article  CAS  Google Scholar 

  19. Chen, J.Z. et al. Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR. Mol. Biol. Rep. 29, 287–292 (2002).

    Article  CAS  Google Scholar 

  20. Lowther, W., Wiley, K., Smith, G.H. & Callahan, R. A new common integration site, Int7, for the mouse mammary tumor virus in mouse mammary tumors identifies a gene whose product has furin-like and thrombospondin-like sequences. J. Virol. 79, 10093–10096 (2005).

    Article  CAS  Google Scholar 

  21. Adams, N.C., Tomoda, T., Cooper, M., Dietz, G. & Hatten, M.E. Mice that lack astrotactin have slowed neuronal migration. Development 129, 965–972 (2002).

    CAS  PubMed  Google Scholar 

  22. Zagha, E. et al. DPP10 modulates Kv4-mediated A-type potassium channels. J. Biol. Chem. 280, 18853–18861 (2005).

    Article  CAS  Google Scholar 

  23. Theodorou, V. et al. Fgf10 is an oncogene activated by MMTV insertional mutagenesis in mouse mammary tumors and overexpressed in a subset of human breast carcinomas. Oncogene 23, 6047–6055 (2004).

    Article  CAS  Google Scholar 

  24. Dennis, G. Jr. et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, P3 (2003).

    Article  Google Scholar 

  25. Vogelstein, B. & Kinzler, K.W. Cancer genes and the pathways they control. Nat. Med. 10, 789–799 (2004).

    Article  CAS  Google Scholar 

  26. van de Vijver, M.J. et al. A gene-expression signature as a predictor of survival in breast cancer. N. Engl. J. Med. 347, 1999–2009 (2002).

    Article  CAS  Google Scholar 

  27. Payton, M., Scully, S., Chung, G. & Coats, S. Deregulation of cyclin E2 expression and associated kinase activity in primary breast tumors. Oncogene 21, 8529–8534 (2002).

    Article  CAS  Google Scholar 

  28. Kwan, H. et al. Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice. Mol. Cell. Biol. 12, 147–154 (1992).

    Article  CAS  Google Scholar 

  29. Peters, G., Lee, A.E. & Dickson, C. Concerted activation of two potential proto-oncogenes in carcinomas induced by mouse mammary tumour virus. Nature 320, 628–631 (1986).

    Article  CAS  Google Scholar 

  30. Dontu, G. et al. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res. 6, R605–R615 (2004).

    Article  CAS  Google Scholar 

  31. Kazanskaya, O. et al. R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev. Cell 7, 525–534 (2004).

    Article  CAS  Google Scholar 

  32. Kim, K.A. et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259 (2005).

    Article  CAS  Google Scholar 

  33. Liu, Y.J., Xu, Y. & Yu, Q. Full-length ADAMTS-1 and the ADAMTS-1 fragments display pro- and antimetastatic activity, respectively. Oncogene 25, 2452–2467 (2006).

    Article  CAS  Google Scholar 

  34. Takeuchi, J.K. et al. Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade. Development 130, 2729–2739 (2003).

    Article  CAS  Google Scholar 

  35. Prasad, V. et al. Haploinsufficiency of Atp2a2, encoding the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump, predisposes mice to squamous cell tumors via a novel mode of cancer susceptibility. Cancer Res. 65, 8655–8661 (2005).

    Article  CAS  Google Scholar 

  36. Bissell, M.J. & Radisky, D. Putting tumours in context. Nat. Rev. Cancer 1, 46–54 (2001).

    Article  CAS  Google Scholar 

  37. Tlsty, T.D. Stromal cells can contribute oncogenic signals. Semin. Cancer Biol. 11, 97–104 (2001).

    Article  CAS  Google Scholar 

  38. Wu, X., Li, Y., Crise, B. & Burgess, S.M. Transcription start regions in the human genome are favored targets for MLV integration. Science 300, 1749–1751 (2003).

    Article  CAS  Google Scholar 

  39. Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G. & Jenkins, N.A. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436, 221–226 (2005).

    Article  CAS  Google Scholar 

  40. Penault-Llorca, F. et al. Expression of FGF and FGF receptor genes in human breast cancer. Int. J. Cancer 61, 170–176 (1995).

    Article  CAS  Google Scholar 

  41. Sjoblom, T. et al. The consensus coding sequences of human breast and colorectal cancers. Science 314, 268–274 (2006).

    Article  Google Scholar 

  42. van der Valk, M.A. Tumor incidence of the inbred mouse strains in the Netherlands Cancer Institute. in Mammary Tumors in the Mouse 45–115 (Elsevier, Amsterdam, 1981).

    Google Scholar 

  43. Hynes, N.E., Groner, B., Diggelmann, H., van Nie, R. & Michalides, R. Genomic location of mouse mammary tumor virus proviral DNA in normal mouse tissue and in mammary tumors. Cold Spring Harb. Symp. Quant. Biol. 44, 1161–1168 (1980).

    Article  CAS  Google Scholar 

  44. Devon, R.S., Porteous, D.J. & Brookes, A.J. Splinkerettes–improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res. 23, 1644–1645 (1995).

    Article  CAS  Google Scholar 

  45. Jonkers, J. & Berns, A. Retroviral insertional mutagenesis as a strategy to identify cancer genes. Biochim. Biophys. Acta 1287, 29–57 (1996).

    PubMed  Google Scholar 

  46. Merlo, G.R. et al. Growth suppression of normal mammary epithelial cells by wild-type p53. Ann. NY Acad. Sci. 698, 108–113 (1993).

    Article  CAS  Google Scholar 

  47. Sheskin, D. Handbook of Parametric and Nonparametric Statistical Procedures (CRC Press New York, 1997).

    Google Scholar 

  48. Pavlidis, P. Using ANOVA for gene selection from microarray studies of the nervous system. Methods 31, 282–289 (2003).

    Article  CAS  Google Scholar 

  49. Roberts, C.J. et al. Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287, 873–880 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to J. Hodzic, S. Bousata and P. van Schouwenburg for help with the insertional mutagenesis assay, M. Mandjes and D. Nuyten for help with the computational analyses and M. Voorhoeve for his critical comments. Parts of this study were performed for the thesis of M. K. at the medical faculty of Ludwig-Maximilians-Universität München. This work was supported by the Dutch Cancer Society (grant 2001-2489).

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Authors and Affiliations

  1. Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands

    Vassiliki Theodorou, Melanie A Kimm, Mandy Boer, Wendy Theelen & John Hilkens

  2. Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands

    Lodewyk Wessels & Jos Jonkers

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  1. Vassiliki Theodorou
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Contributions

V.T. developed the insertional mutagenesis screening protocol; performed a large part of the screen, the computational and expression analyses and gene cloning and wrote most of the manuscript. M.A.K. contributed to the development of the screening protocol, performed a substantial part of the insertional mutagenesis screen and performed some of the DNA blots. W.T. contributed to the insertional mutagenesis screen and expression analysis. M.B. performed the animal experiments and contributed to the tissue culture work. L.W. performed the statistical analyses. J.J. gave advice, contributed in the writing of the manuscript and performed some of the DNA blotting experiments. J.H. designed the study and contributed to the retroviral transduction and animal experiments and the writing of the manuscript.

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Correspondence to John Hilkens.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Expression analysis of candidate cancer genes. (PDF 805 kb)

Supplementary Fig. 2

Oligoclonality of the Rspo3- and Wnt-induced tumors. (PDF 493 kb)

Supplementary Table 1

MMTV proviral insertion sites and associated genes. (PDF 309 kb)

Supplementary Table 2

Expression analysis of MMTV-tagged genes. (PDF 39 kb)

Supplementary Table 3

NIH-DAVID Gene Ontology analysis of MMTV-targeted genes. (PDF 62 kb)

Supplementary Table 4

Gene Ontology analysis of MULV-tagged genes in lymphomas using NIH-DAVID. (PDF 15 kb)

Supplementary Table 5

Gene Ontology analysis of MULV-tagged genes in brain tumors using NIH-DAVID. (PDF 38 kb)

Supplementary Table 6

PCR primers. (PDF 22 kb)

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Theodorou, V., Kimm, M., Boer, M. et al. MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer. Nat Genet 39, 759–769 (2007). https://doi.org/10.1038/ng2034

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