Abstract

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma.

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References

  1. 1.

    & Molecular pathogenesis of osteosarcoma. DNA Cell Biol. 26, 1–18 (2007).

  2. 2.

    et al. A meta-analysis of osteosarcoma outcomes in the modern medical era. Sarcoma 2012, 704872 (2012).

  3. 3.

    , & Osteosarcoma: evolution of treatment paradigms. Sarcoma 2013, 203531 (2013).

  4. 4.

    et al. Effect of timing of pulmonary metastases identification on prognosis of patients with osteosarcoma: the Japanese Musculoskeletal Oncology Group study. J. Clin. Oncol. 20, 3470–3477 (2002).

  5. 5.

    et al. Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols. J. Clin. Oncol. 21, 2011–2018 (2003).

  6. 6.

    et al. Osteogenic sarcoma with clinically detectable metastasis at initial presentation. J. Clin. Oncol. 11, 449–453 (1993).

  7. 7.

    Breed-predispositions to cancer in pedigree dogs. ISRN Vet. Sci. 2013, 2013:941275 (2013).

  8. 8.

    et al. The presence of p53 mutations in human osteosarcomas correlates with high levels of genomic instability. Proc. Natl. Acad. Sci. USA 100, 11547–11552 (2003).

  9. 9.

    , , & Translational biology of osteosarcoma. Nat. Rev. Cancer 14, 722–735 (2014).

  10. 10.

    et al. Identification of osteosarcoma driver genes by integrative analysis of copy number and gene expression data. Genes Chromosom. Cancer 51, 696–706 (2012).

  11. 11.

    et al. Identification of interactive networks of gene expression associated with osteosarcoma oncogenesis by integrated molecular profiling. Hum. Mol. Genet. 18, 1962–1975 (2009).

  12. 12.

    et al. Integrative analysis reveals relationships of genetic and epigenetic alterations in osteosarcoma. PLoS ONE 7, e48262 (2012).

  13. 13.

    & Harnessing transposons for cancer gene discovery. Nat. Rev. Cancer 10, 696–706 (2010).

  14. 14.

    et al. Recurrent somatic structural variations contribute to tumorigenesis in pediatric osteosarcoma. Cell Rep. 7, 104–112 (2014).

  15. 15.

    Osteosarcomagenesis: modeling cancer initiation in the mouse. Sarcoma 2011, 694136 (2011).

  16. 16.

    & Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 133, 3231–3244 (2006).

  17. 17.

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

  18. 18.

    , , , & Knockdown of Akt sensitizes osteosarcoma cells to apoptosis induced by cisplatin treatment. Int. J. Mol. Sci. 12, 2994–3005 (2011).

  19. 19.

    , & A review of clinical and molecular prognostic factors in osteosarcoma. J. Cancer Res. Clin. Oncol. 134, 281–297 (2008).

  20. 20.

    et al. Caprin-1, a novel Cyr61-interacting protein, promotes osteosarcoma tumor growth and lung metastasis in mice. Biochim. Biophys. Acta 1832, 1173–1182 (2013).

  21. 21.

    et al. Expression of bone morphogenetic proteins and receptors in sarcomas. Clin. Orthop. Relat. Res. (365), 175–183 (1999).

  22. 22.

    et al. CDH11 expression is associated with survival in patients with osteosarcoma. Cancer Genomics Proteomics 5, 37–42 (2008).

  23. 23.

    et al. Bufalin-inhibited migration and invasion in human osteosarcoma U-2 OS cells is carried out by suppression of the matrix metalloproteinase-2, ERK, and JNK signaling pathways. Environ. Toxicol. 29, 21–29 (2014).

  24. 24.

    et al. miR-16 inhibits cell proliferation by targeting IGF1R and the Raf1-MEK1/2-ERK1/2 pathway in osteosarcoma. FEBS Lett. 587, 1366–1372 (2013).

  25. 25.

    et al. Evaluation of eIF4E expression in an osteosarcoma-specific tissue microarray. J. Pediatr. Hematol. Oncol. 33, 524–528 (2011).

  26. 26.

    , , & Tumor-suppressing effects of miR-141 in human osteosarcoma. Cell Biochem. Biophys. 69, 319–325 (2014).

  27. 27.

    , , , & Aberrant ADAM10 expression correlates with osteosarcoma progression. Eur. J. Med. Res. 19, 9 (2014).

  28. 28.

    et al. Bone deposition, bone resorption, and osteosarcoma. J. Orthop. Res. 28, 1142–1148 (2010).

  29. 29.

    et al. Rapamycin increases pCREB, Bcl-2, and VEGF-A through ERK under normoxia. Acta Biochim. Biophys. Sin. (Shanghai) 45, 259–267 (2013).

  30. 30.

    , , & Critical role of Notch signaling in osteosarcoma invasion and metastasis. Clin. Cancer Res. 14, 2962–2969 (2008); retracted 15 September 2013.

  31. 31.

    , , , & Methotrexate in pediatric osteosarcoma: response and toxicity in relation to genetic polymorphisms and dihydrofolate reductase and reduced folate carrier 1 expression. J. Pediatr. 154, 688–693 (2009).

  32. 32.

    et al. Proteomic identification of 14-3-3ɛ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells. J. Orthop. Res. 32, 848–854 (2014).

  33. 33.

    et al. YY1 overexpression is associated with poor prognosis and metastasis-free survival in patients suffering osteosarcoma. BMC Cancer 11, 472 (2011).

  34. 34.

    et al. Cell cycle regulator gene CDC5L, a potential target for 6p12-p21 amplicon in osteosarcoma. Mol. Cancer Res. 6, 937–946 (2008).

  35. 35.

    et al. Overexpression of fibroblast activation protein and its clinical implications in patients with osteosarcoma. J. Surg. Oncol. 108, 157–162 (2013).

  36. 36.

    et al. Repression of p53-dependent sequence-specific transactivation by MEF2c. Biochem. Biophys. Res. Commun. 214, 468–474 (1995).

  37. 37.

    , , & Elevated expression of AKT2 correlates with disease severity and poor prognosis in human osteosarcoma. Mol. Med. Rep. 10, 737–742 (2014).

  38. 38.

    et al. microRNA-194 suppresses osteosarcoma cell proliferation and metastasis in vitro and in vivo by targeting CDH2 and IGF1R. Int. J. Oncol. 45, 1437–1449 (2014).

  39. 39.

    & Inhibition of SENP5 suppresses cell growth and promotes apoptosis in osteosarcoma cells. Exp. Ther. Med. 7, 1691–1695 (2014).

  40. 40.

    et al. miRNA signatures associate with pathogenesis and progression of osteosarcoma. Cancer Res. 72, 1865–1877 (2012).

  41. 41.

    , , & miR-17 inhibitor suppressed osteosarcoma tumor growth and metastasis via increasing PTEN expression. Biochem. Biophys. Res. Commun. 444, 230–234 (2014).

  42. 42.

    et al. Downregulation of microRNA-26a is associated with metastatic potential and the poor prognosis of osteosarcoma patients. Oncol. Rep. 31, 1263–1270 (2014).

  43. 43.

    et al. MAPK7 and MAP2K4 as prognostic markers in osteosarcoma. Hum. Pathol. 43, 994–1002 (2012).

  44. 44.

    et al. High WT1 expression is associated with very poor survival of patients with osteogenic sarcoma metastasis. Clin. Cancer Res. 12, 4237–4243 (2006).

  45. 45.

    et al. Molecular subtypes of osteosarcoma identified by reducing tumor heterogeneity through an interspecies comparative approach. Bone 49, 356–367 (2011).

  46. 46.

    et al. Copy number gains in EGFR and copy number losses in PTEN are common events in osteosarcoma tumors. Cancer 113, 1453–1461 (2008).

  47. 47.

    et al. Somatic induction of Pten loss in a preclinical astrocytoma model reveals major roles in disease progression and avenues for target discovery and validation. Cancer Res. 65, 5172 (2005).

  48. 48.

    et al. Molecular characterization of the pediatric preclinical testing panel. Clin. Cancer Res. 14, 4572–4583 (2008).

  49. 49.

    , & ErbB-2 and Met reciprocally regulate cellular signaling via plexin-B1. J. Biol. Chem. 283, 1893–1901 (2008).

  50. 50.

    et al. CLDN3 inhibits cancer aggressiveness via Wnt-EMT signaling and is a potential prognostic biomarker for hepatocellular carcinoma. Oncotarget. 5, 7663–7676 (2014).

  51. 51.

    et al. Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4, 209–221 (2003).

  52. 52.

    & SNARE-dependent interaction of Src, EGFR and β1 integrin regulates invadopodia formation and tumor cell invasion. J. Cell Sci. 127, 1712–1725 (2014).

  53. 53.

    et al. MicroRNA let-7c inhibits migration and invasion of human non–small cell lung cancer by targeting ITGB3 and MAP4K3. Cancer Lett. 342, 43–51 (2014).

  54. 54.

    et al. Inactivation of Rho GTPases by p190 RhoGAP reduces human pancreatic cancer cell invasion and metastasis. Cancer Sci. 97, 848–853 (2006).

  55. 55.

    et al. Expression of p114RhoGEF predicts lymph node metastasis and poor survival of squamous-cell lung carcinoma patients. Tumour Biol. 34, 1925–1933 (2013).

  56. 56.

    & Frequent genetic alterations and reduced expression of the Axin1 gene in oral squamous cell carcinoma: involvement in tumor progression and metastasis. Oncol. Rep. 17, 73–79 (2007).

  57. 57.

    et al. PHLPP is a negative regulator of RAF1, which reduces colorectal cancer cell motility and prevents tumor progression in mice. Gastroenterology 146, 1301–1312 (2014).

  58. 58.

    & Knockdown of ubiquitin associated protein 2–like inhibits the growth and migration of prostate cancer cells. Oncol. Rep. 32, 1578–1584 (2014).

  59. 59.

    et al. A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat. Biotechnol. 27, 264–274 (2009).

  60. 60.

    et al. Clonal selection drives genetic divergence of metastatic medulloblastoma. Nature 482, 529–533 (2012).

  61. 61.

    Parallel progression of primary tumours and metastases. Nat. Rev. Cancer 9, 302–312 (2009).

  62. 62.

    et al. Dissecting the PI3K signaling axis in pediatric solid tumors: novel targets for clinical integration. Front. Oncol. 3, 93 (2013).

  63. 63.

    , , , & ERK5 silencing inhibits invasion of human osteosarcoma cell via modulating the Slug/MMP-9 pathway. J. Clin. Exp. Pathol. 4, 2640–2647 (2014).

  64. 64.

    & Critical signaling pathways in bone sarcoma: candidates for therapeutic interventions. Curr. Oncol. Rep. 11, 446–453 (2009).

  65. 65.

    Forward genetic screen for malignant peripheral nerve sheath tumor formation identifies novel genes and genetic pathways driving tumorigenesis. Nat. Genet. 45, 756–766 (2013).

  66. 66.

    et al. A transposon-based genetic screen in mice identifies genes altered in colorectal cancer. Science 323, 1747–1750 (2009).

  67. 67.

    et al. A modified Sleeping Beauty transposon system that can be used to model a wide variety of human cancers in mice. Cancer Res. 69, 8150 (2009).

  68. 68.

    , & Neurofibromatosis type 1, hyperparathyroidism, and osteosarcoma: interplay? Eur. Arch. Otorhinolaryngol. 259, 540–542 (2002).

  69. 69.

    , & Management of childhood malignant peripheral nerve sheath tumor. Paediatr. Drugs 9, 239–248 (2007).

  70. 70.

    et al. Bone sarcomas arising in patients with neurofibromatosis type 1. J. Bone Joint Surg. Br. 91, 1223 (2009).

  71. 71.

    et al. Mice heterozygous for a mutation at the Nf2 tumor suppressor locus develop a range of highly metastatic tumors. Genes Dev. 12, 1121–1133 (1998).

  72. 72.

    et al. The NF2 gene and merlin protein in human osteosarcomas. Neurogenetics 2, 73–74 (1998).

  73. 73.

    et al. Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell 96, 701–712 (1999).

  74. 74.

    & The role of semaphorins and their receptors in gliomas. J. Signal Transduct. 2012, 902854 (2012).

  75. 75.

    , , , & The plexin-A1 receptor activates vascular endothelial growth factor–receptor 2 and nuclear factor–κB to mediate survival and anchorage-independent growth of malignant mesothelioma cells. Cancer Res. 69, 1485–1493 (2009).

  76. 76.

    et al. Suppression of bone formation by osteoclastic expression of semaphorin 4D. Nat. Med. 17, 1473–1480 (2011).

  77. 77.

    et al. MET overexpression turns human primary osteoblasts into osteosarcomas. Cancer Res. 66, 4750–4757 (2006).

  78. 78.

    et al. ErbB2 expression is correlated with increased survival of patients with osteosarcoma. Cancer 94, 1397–1404 (2002).

  79. 79.

    , , , & Semaphorin 4D cooperates with VEGF to promote angiogenesis and tumor progression. Angiogenesis 15, 391–407 (2012).

  80. 80.

    , & Metastasis: from dissemination to organ-specific colonization. Nat. Rev. Cancer 9, 274–284 (2009).

  81. 81.

    , , & Sarcomas in TP53 germline mutation carriers. Cancer 118, 1387–1396 (2012).

  82. 82.

    , , , & TAPDANCE: an automated tool to identify and annotate transposon insertion CISs and associations between CISs from next generation sequence data. BMC Bioinformatics 13, 154 (2012).

  83. 83.

    et al. Novel molecular and computational methods improve the accuracy of insertion site analysis in Sleeping Beauty–induced tumors. PLoS ONE 6, e24668 (2011).

  84. 84.

    & A comprehensive guide to Sleeping Beauty–based somatic transposon mutagenesis in the mouse. Curr. Protoc. Mouse Biol. 347–368 (2011).

  85. 85.

    , , , & Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436, 272–276 (2005).

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Acknowledgements

B.S.M. was funded by US National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases Musculoskeletal Training Grant AR050938. This research was funded by the Sobiech Osteosarcoma Fund Award, the Children's Cancer Research Fund, an American Cancer Center Research Professor Grant (123939) and National Cancer Institute grant R01 CA113636 (to D.A.L.). We extend our thanks to the University of Minnesota resources involved in our project. The University of Minnesota Genomics Center provided services for RNA sequencing, oligonucleotide preparation and Sanger sequencing. The Minnesota Supercomputing Institute maintains the Galaxy software platform, as well as provides data management services and training. The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center (US National Institutes of Health grant P30 CA077598).

Author information

Affiliations

  1. Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA.

    • Branden S Moriarity
    • , George M Otto
    • , Eric P Rahrmann
    •  & David A Largaespada
  2. Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.

    • Branden S Moriarity
    • , George M Otto
    • , Eric P Rahrmann
    •  & David A Largaespada
  3. Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.

    • Branden S Moriarity
    • , George M Otto
    • , Eric P Rahrmann
    • , Susan K Rathe
    • , Natalie K Wolf
    • , Madison T Weg
    • , Rebecca S LaRue
    • , Nuri A Temiz
    • , Aaron L Sarver
    • , Milcah C Scott
    • , Jaime F Modiano
    •  & David A Largaespada
  4. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA.

    • George M Otto
    • , Eric P Rahrmann
    • , Natalie K Wolf
    • , Madison T Weg
    • , Luke A Manlove
    • , Kevin J Holly
    •  & David A Largaespada
  5. Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA.

    • Rebecca S LaRue
  6. Ontario Cancer Institute, Toronto, Ontario, Canada.

    • Sam D Molyneux
    •  & Rama Khokha
  7. Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

    • Kwangmin Choi
  8. Department of Veterinary Clinical Sciences, University of Minnesota, St. Paul, Minnesota, USA.

    • Milcah C Scott
    •  & Jaime F Modiano
  9. BioNet, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, USA.

    • Colleen L Forster
  10. Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA.

    • Jaime F Modiano
  11. Tumor and Metastasis Biology Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland, USA.

    • Chand Khanna
  12. Tissue Array Research Program (TARP), Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland, USA.

    • Stephen M Hewitt
  13. Department of Orthopedic Surgery, Musculoskeletal Tumor Center, People's Hospital, Peking University, Beijing, China.

    • Yi Yang
  14. Department of Pediatrics, Albert Einstein College of Medicine and Children's Hospital at Montefiore, Bronx, New York, USA.

    • Richard Gorlick
  15. Department of Molecular Pharmacology, Albert Einstein College of Medicine and Children's Hospital at Montefiore, Bronx, New York, USA.

    • Richard Gorlick
  16. Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

    • Michael A Dyer

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Contributions

B.S.M., G.M.O., E.P.R., S.K.R., N.K.W., M.T.W., L.A.M. and K.J.H. performed laboratory experiments and/or analyzed the data. N.A.T. and K.C. performed bioinformatic data analysis of RNA sequencing, methylome and copy number analysis data. M.A.D. provided RNA sequencing and methylation data for human osteosarcoma samples. C.L.F. performed immunohistochemistry staining on osteosarcoma tumor microarrays. M.C.S. and J.F.M. provided data on canine osteosarcoma gene expression and outcome. A.L.S. analyzed the deep sequencing data for CIS analysis. R.K. and S.D.M. acquired and analyzed data from COSMIC and CGC. R.S.L. performed comparative analysis of CIS genes among SB screens. S.M.H. and C.K. assessed the histology of mouse tumors. R.G. and Y.Y. generated the immortalized osteoblast cells. D.A.L. supervised laboratory experiments and assisted in writing the manuscript. B.S.M. wrote the manuscript.

Competing interests

D.A.L. is a founder of Discovery Genomics, Inc., and holds stock. D.A.L. is a founder of NeoClone Biotechnology, Inc., and holds stock. Some research in the Largaespada laboratory, not related to this work, is funded by Genentech, Inc.

Corresponding author

Correspondence to David A Largaespada.

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https://doi.org/10.1038/ng.3293

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