Abstract
Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome neurofibromatosis type 1. To identify genetic drivers of MPNST development, we used the Sleeping Beauty (SB) transposon-based somatic mutagenesis system in mice with somatic loss of transformation-related protein p53 (Trp53) function and/or overexpression of human epidermal growth factor receptor (EGFR). Common insertion site (CIS) analysis of 269 neurofibromas and 106 MPNSTs identified 695 and 87 sites with a statistically significant number of recurrent transposon insertions, respectively. Comparison to human data sets identified new and known driver genes for MPNST formation at these sites. Pairwise co-occurrence analysis of CIS-associated genes identified many cooperating mutations that are enriched in Wnt/β-catenin, PI3K-AKT-mTOR and growth factor receptor signaling pathways. Lastly, we identified several new proto-oncogenes, including Foxr2 (encoding forkhead box R2), which we functionally validated as a proto-oncogene involved in MPNST maintenance.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ducatman, B.S., Scheithauer, B.W., Piepgras, D.G., Reiman, H.M. & Ilstrup, D.M. Malignant peripheral nerve sheath tumors. A clinicopathologic study of 120 cases. Cancer 57, 2006–2021 (1986).
Evans, D.G. et al. Malignant peripheral nerve sheath tumours in neurofibromatosis 1. J. Med. Genet. 39, 311–314 (2002).
Watson, M.A. et al. Gene expression profiling reveals unique molecular subtypes of neurofibromatosis type I–associated and sporadic malignant peripheral nerve sheath tumors. Brain Pathol. 14, 297–303 (2004).
Holtkamp, N. et al. Subclassification of nerve sheath tumors by gene expression profiling. Brain Pathol. 14, 258–264 (2004).
Miller, S.J. et al. Large-scale molecular comparison of human schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues. Cancer Res. 66, 2584–2591 (2006).
Zou, C. et al. Clinical, pathological, and molecular variables predictive of malignant peripheral nerve sheath tumor outcome. Ann. Surg. 249, 1014–1022 (2009).
Messiaen, L.M. et al. Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum. Mutat. 15, 541–555 (2000).
Leppig, K.A. et al. Familial neurofibromatosis 1 microdeletions: cosegregation with distinct facial phenotype and early onset of cutaneous neurofibromata. Am. J. Med. Genet. 73, 197–204 (1997).
De Raedt, T. et al. Elevated risk for MPNST in NF1 microdeletion patients. Am. J. Hum. Genet. 72, 1288–1292 (2003).
Ballester, R. et al. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell 63, 851–859 (1990).
Sherman, L.S., Atit, R., Rosenbaum, T., Cox, A.D. & Ratner, N. Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts. J. Biol. Chem. 275, 30740 (2000).
Basu, T.N. et al. Aberrant regulation of Ras proteins in malignant-tumor cells from type-1 neurofibromatosis patients. Nature 356, 713–715 (1992).
Cichowski, K. & Jacks, T. NF1 tumor suppressor gene function: narrowing the GAP. Cell 104, 593–604 (2001).
Forus, A. et al. Comparative genomic hybridization analysis of human sarcomas: I. occurrence of genomic imbalances and identification of a novel major amplicon at 1q21-q22 in soft tissue sarcomas. Genes Chromosom. Cancer 14, 8–14 (1995).
Lothe, R.A. et al. Gain of 17q24-qter detected by comparative genomic hybridization in malignant tumors from patients with von Recklinghausen's neurofibromatosis. Cancer Res. 56, 4778–4781 (1996).
Mechtersheimer, G. et al. Analysis of chromosomal imbalances in sporadic and NF1-associated peripheral nerve sheath tumors by comparative genomic hybridization. Genes Chromosom. Cancer 25, 362–369 (1999).
Mertens, F. et al. Cytogenetic findings in malignant peripheral nerve sheath tumors. Int. J. Cancer 61, 793–798 (1995).
Plaat, B.E. et al. Computer-assisted cytogenetic analysis of 51 malignant peripheral-nerve-sheath tumors: sporadic vs. neurofibromatosis-type-1-associated malignant schwannomas. Int. J. Cancer 83, 171–178 (1999).
Schmidt, H. et al. Genomic imbalances of 7p and 17q in malignant peripheral nerve sheath tumors are clinically relevant. Genes Chromosom. Cancer 25, 205–211 (1999).
Schmidt, H. et al. Gains in chromosomes 7, 8q, 15q and 17q are characteristic changes in malignant but not in benign peripheral nerve sheath tumors from patients with Recklinghausen's disease. Cancer Lett. 155, 181–190 (2000).
Schmidt, H. et al. Cytogenetic characterization of six malignant peripheral nerve sheath tumors: comparison of karyotyping and comparative genomic hybridization. Cancer Genet. Cytogenet. 128, 14–23 (2001).
Birindelli, S. et al. Rb and TP53 pathway alterations in sporadic and NF1-related malignant peripheral nerve sheath tumors. Lab. Invest. 81, 833–844 (2001).
Legius, E. et al. TP53 mutations are frequent in malignant NFI tumors. Genes Chromosom. Cancer 10, 250–255 (2006).
Perry, A. et al. Differential NF1, p16, and EGFR patterns by interphase cytogenetics (FISH) in malignant peripheral nerve sheath tumor (MPNST) and morphologically similar spindle cell neoplasms. J. Neuropathol. Exp. Neurol. 61, 702–709 (2002).
Menon, A.G. et al. Chromosome 17p deletions and p53 gene mutations associated with the formation of malignant neurofibrosarcomas in von Recklinghausen neurofibromatosis. Proc. Natl. Acad. Sci. USA 87, 5435 (1990).
Kourea, H.P., Orlow, I., Scheithauer, B.W., Cordon-Cardo, C. & Woodruff, J.M. Deletions of the INK4A gene occur in malignant peripheral nerve sheath tumors but not in neurofibromas. Am. J. Pathol. 155, 1855 (1999).
Nielsen, G.P. et al. Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation. Am. J. Pathol. 155, 1879 (1999).
Mantripragada, K.K. et al. High-resolution DNA copy number profiling of malignant peripheral nerve sheath tumors using targeted microarray-based comparative genomic hybridization. Clin. Cancer Res. 14, 1015 (2008).
Mawrin, C. et al. Immunohistochemical and molecular analysis of p53, RB, and PTEN in malignant peripheral nerve sheath tumors. Virchows Arch. 440, 610–615 (2002).
Holtkamp, N. et al. Mutation and expression of PDGFRA and KIT in malignant peripheral nerve sheath tumors, and its implications for imatinib sensitivity. Carcinogenesis 27, 664 (2006).
Storlazzi, C.T. et al. Identification of a novel amplicon at distal 17q containing the BIRC5/SURVIVIN gene in malignant peripheral nerve sheath tumours. J. Pathol. 209, 492–500 (2006).
Badache, A. & De Vries, G.H. Neurofibrosarcoma-derived Schwann cells overexpress platelet-derived growth factor (PDGF) receptors and are induced to proliferate by PDGF BB. J. Cell. Physiol. 177, 334–342 (1998).
Badache, A., Muja, N. & De Vries, G.H. Expression of Kit in neurofibromin-deficient human Schwann cells: role in Schwann cell hyperplasia associated with type 1 neurofibromatosis. Oncogene 17, 795–800 (1998).
Keng, V.W. et al. A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat. Biotechnol. 27, 264–274 (2009).
Dupuy, A.J. 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, 8150–8156 (2009).
Wu, X. et al. Clonal selection drives genetic divergence of metastatic medulloblastoma. Nature 482, 529–533 (2012).
Quintana, R.M. et al. A transposon-based analysis of gene mutations related to skin cancer development. J. Invest. Dermatol. 133, 239–248 (2013).
Lappe-Siefke, C. et al. Disruption of Cnp1 uncouples oligodendroglial functions in axonal support and myelination. Nat. Genet. 33, 366–374 (2003).
Tabone-Eglinger, S. et al. Frequent EGFR positivity and overexpression in high-grade areas of human MPNSTs. Sarcoma 2008, 849156 (2008).
Holtkamp, N. et al. MMP-13 and p53 in the progression of malignant peripheral nerve sheath tumors. Neoplasia 9, 671–677 (2007).
Ling, B.C. et al. Role for the epidermal growth factor receptor in neurofibromatosis-related peripheral nerve tumorigenesis. Cancer Cell 7, 65–75 (2005).
de Vries, A. et al. Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function. Proc. Natl. Acad. Sci. USA 99, 2948–2953 (2002).
Carli, M. et al. Pediatric malignant peripheral nerve sheath tumor: the Italian and German soft tissue sarcoma cooperative group. J. Clin. Oncol. 23, 8422–8430 (2005).
Stucky, C.C.H. et al. Malignant peripheral nerve sheath tumors (MPNST): the Mayo Clinic experience. Ann. Surg. Oncol. 19, 878–885 (2012).
Pytel, P., Taxy, J.B. & Krausz, T. Divergent differentiation in malignant soft tissue neoplasms: the paradigm of liposarcoma and malignant peripheral nerve sheath tumor. Int. J. Surg. Pathol. 13, 19–28 (2005).
Magro, G. et al. Multinucleated floret–like giant cells in sporadic and NF1-associated neurofibromas: a clinicopathologic study of 94 cases. Virchows Arch. 456, 71–76 (2010).
Rodriguez, F.J., Folpe, A.L., Giannini, C. & Perry, A. Pathology of peripheral nerve sheath tumors: diagnostic overview and update on selected diagnostic problems. Acta Neuropathol. 123, 295–319 (2012).
Collier, L.S., Carlson, C.M., Ravimohan, S., Dupuy, A.J. & Largaespada, D.A. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436, 272–276 (2005).
Sarver, A.L., Erdman, J., Starr, T., Largaespada, D.A. & Silverstein, K.A. 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).
Brett, B.T. et al. Novel molecular and computational methods improve the accuracy of insertion site analysis in Sleeping Beauty–induced tumors. PLoS ONE 6, e24668 (2011).
Gregorian, C. et al. PTEN dosage is essential for neurofibroma development and malignant transformation. Proc. Natl. Acad. Sci. USA 106, 19479–19484 (2009).
Largaespada, D.A. & Collier, L.S. Transposon-mediated mutagenesis in somatic cells: identification of transposon–genomic DNA junctions. Methods Mol. Biol. 435, 95–108 (2008).
Feber, A. et al. Comparative methylome analysis of benign and malignant peripheral nerve sheath tumors. Genome Res. 21, 515–524 (2011).
Yang, J. et al. Genomic and molecular characterization of malignant peripheral nerve sheath tumor identifies the IGF1R pathway as a primary target for treatment. Clin. Cancer Res. 17, 7563–7573 (2011).
Forbes, S.A. et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 39, D945–D950 (2011).
Ghadimi, M.P. et al. Targeting the PI3K/mTOR axis, alone and in combination with autophagy blockade, for the treatment of malignant peripheral nerve sheath tumors. Mol. Cancer Ther. 11, 1758–1769 (2012).
Saito, T. et al. Nuclear β-catenin correlates with cyclin D1 expression in spindle and pleomorphic sarcomas but not in synovial sarcoma. Hum. Pathol. 37, 689–697 (2006).
Mo, W. et al. CXCR4/CXCL12 mediate autocrine cell-cycle progression in NF1-associated malignant peripheral nerve sheath tumors. Cell 152, 1077–1090 (2013).
Keng, V.W. et al. PTEN and NF1 inactivation in Schwann cells produces a severe phenotype in the peripheral nervous system that promotes the development and malignant progression of peripheral nerve sheath tumors. Cancer Res. 72, 3405–3413 (2012).
Starr, T.K. et al. A transposon-based genetic screen in mice identifies genes altered in colorectal cancer. Science 323, 1747–1750 (2009).
Jessen, K.R. Glial cells. Int. J. Biochem. Cell Biol. 36, 1861–1867 (2004).
Pérez-Mancera, P.A. et al. The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma. Nature 486, 266–270 (2012).
Subramanian, S. et al. Genome-wide transcriptome analyses reveal p53 inactivation mediated loss of miR-34a expression in malignant peripheral nerve sheath tumours. J. Pathol. 220, 58–70 (2010).
Je, E.M., An, C.H., Yoo, N.J. & Lee, S.H. Mutational analysis of PIK3CA, JAK2, BRAF, FOXL2, IDH1, AKT1 and EZH2 oncogenes in sarcomas. APMIS 120, 635–639 (2012).
Jacks, T. et al. Tumour predisposition in mice heterozygous for a targeted mutation in Nf1. Nat. Genet. 7, 353–361 (1994).
Cichowski, K. et al. Mouse models of tumor development in neurofibromatosis type 1. Science 286, 2172–2176 (1999).
Zhu, Y., Ghosh, P., Charnay, P., Burns, D.K. & Parada, L.F. Neurofibromas in NF1: Schwann cell origin and role of tumor environment. Science 296, 920–922 (2002).
Wu, J. et al. Plexiform and dermal neurofibromas and pigmentation are caused by Nf1 loss in desert hedgehog-expressing cells. Cancer Cell 13, 105–116 (2008).
Katoh, M. Identification and characterization of human FOXN6, mouse Foxn6, and rat Foxn6 genes in silico. Int. J. Oncol. 25, 219 (2004).
Santo, E.E. et al. Oncogenic activation of FOXR1 by 11q23 intrachromosomal deletion-fusions in neuroblastoma. Oncogene 31, 1571–1581 (2012).
Uhlen, M. et al. Towards a knowledge-based human protein atlas. Nat. Biotechnol. 28, 1248–1250 (2010).
Rhodes, D.R. et al. ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia 6, 1 (2004).
Rhodes, D.R. et al. Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia 9, 166 (2007).
Myatt, S.S. & Lam, E.W.F. The emerging roles of forkhead box (Fox) proteins in cancer. Nat. Rev. Cancer 7, 847–859 (2007).
Stemmer-Rachamimov, A.O. et al. Comparative pathology of nerve sheath tumors in mouse models and humans. Cancer Res. 64, 3718–3724 (2004).
Dai, M. et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 33, e175 (2005).
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc., B 57, 289–300 (1995).
Irizarry, R.A. et al. The human colon cancer methylome shows similar hypo-and hypermethylation at conserved tissue-specific CpG island shores. Nat. Genet. 41, 178–186 (2009).
Yang, J. et al. Deletion of the WWOX gene and frequent loss of its protein expression in human osteosarcoma. Cancer Lett. 291, 31–38 (2010).
Olshen, A.B., Venkatraman, E., Lucito, R. & Wigler, M. Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics 5, 557–572 (2004).
van de Wiel, M.A. et al. CGHcall: calling aberrations for array CGH tumor profiles. Bioinformatics 23, 892–894 (2007).
Sun, W. et al. Integrated study of copy number states and genotype calls using high-density SNP arrays. Nucleic Acids Res. 37, 5365–5377 (2009).
DeRycke, M.S. et al. S100A1 expression in ovarian and endometrial endometrioid carcinomas is a prognostic indicator of relapse-free survival. Am. J. Clin. Pathol. 132, 846–856 (2009).
Rizzardi, A.E. et al. Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn. Pathol. 7, 42 (2012).
Daniel, A.R., Faivre, E.J. & Lange, C.A. Phosphorylation-dependent antagonism of sumoylation derepresses progesterone receptor action in breast cancer cells. Mol. Endocrinol. 21, 2890–2906 (2007).
Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector–based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).
Wood, A.J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).
Mussolino, C. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39, 9283–9293 (2011).
Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).
Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).
Doyon, Y. et al. Transient cold shock enhances zinc-finger nuclease–mediated gene disruption. Nat. Methods 7, 459–460 (2010).
Acknowledgements
We would like to thank the Biomedical Genomics Center at the University of Minnesota for performing Illumina deep sequencing. We would like to thank the Biological Materials Procurement Network (BioNet), specifically S. Schmechel, A. Rizzardi, C. Forster and S. Bowell, for the construction, immunohistochemical staining and scanning of the TMA. We also acknowledge the following shared resources of the Masonic Cancer Center at the University of Minnesota: The Mouse Genetics Laboratory, Biostatistics and Bioinformatics, Comparative Pathology and the Tissue Procurement Facility. We thank the Minnesota Supercomputing Institute for computational resources. We thank the Research Animal Resources at the University of Minnesota and specifically A. Aliye for his technical support in mouse maintenance. This work received funding from the US National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (NINDS) through grant P50 N5057531, the Margaret Harvey Schering Trust, The Zachary Neurofibromatosis Research Fund and The Jacqueline Dunlap Neurofibromatosis Research Fund. Work performed by A.L.W. was supported by Children's Tumor Foundation Young Investigators Award 2011-01-018.
Author information
Authors and Affiliations
Contributions
E.P.R., A.L.W., B.S.M., D.A.B., N.K.W. and V.W.K. performed laboratory experiments and/or analyzed the data. K.C. performed bioinformatic data analysis of microarray expression, methylome and CNA data. A.S. analyzed deep sequencing data for CIS analysis. M.H.C. assessed histology and graded mouse tumors. C.L.M. provided magnetic resonance images (MRIs) of human MPNSTs and data analysis. M.R.W. generated the iHSCs. B.G. and E.S. generated and analyzed SNP array data. N.R. and D.A.L. supervised laboratory experiments and assisted in writing the manuscript. E.P.R. wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
D.A.L. has ownership interest (including patents) in Discovery Genomics, Inc. and NeoClone Biotechnologies International. He is also a consultant/Advisory Board member of Discovery Genomics, Inc. and NeoClone Biotechnologies International.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–11 (PDF 46133 kb)
Supplementary Tables 1–12
Supplementary Tables 1–12 (XLSX 129 kb)
Supplementary Data 1
Supplementary Data 1 (TXT 2099 kb)
Supplementary Data 2
Supplementary Data 2 (TXT 9090 kb)
Rights and permissions
About this article
Cite this article
Rahrmann, E., Watson, A., Keng, V. et al. Forward genetic screen for malignant peripheral nerve sheath tumor formation identifies new genes and pathways driving tumorigenesis. Nat Genet 45, 756–766 (2013). https://doi.org/10.1038/ng.2641
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ng.2641
This article is cited by
-
FOXM1 promotes neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor progression in a NUF2-dependent manner
Cancer Gene Therapy (2023)
-
CRISPR and transposon in vivo screens for cancer drivers and therapeutic targets
Genome Biology (2020)
-
A functional genetic screen defines the AKT-induced senescence signaling network
Cell Death & Differentiation (2020)
-
SEMA4C is a novel target to limit osteosarcoma growth, progression, and metastasis
Oncogene (2020)
-
The Ras-related gene ERAS is involved in human and murine breast cancer
Scientific Reports (2018)