The Activation of the WNT Signaling Pathway Is a Hallmark in Neurofibromatosis Type 1 Tumorigenesis

Purpose: The hallmark of neurofibromatosis type 1 (NF1) is the onset of dermal or plexiform neurofibromas, mainly composed of Schwann cells. Plexiform neurofibromas can transform into malignant peripheral nerve sheath tumors (MPNST) that are resistant to therapies. Experimental Design: The aim of this study was to identify an additional pathway in the NF1 tumorigenesis. We focused our work on Wnt signaling that is highly implicated in cancer, mainly in regulating the proliferation of cancer stem cells. We quantified mRNAs of 89 Wnt pathway genes in 57 NF1-associated tumors including dermal and plexiform neurofibromas and MPNSTs. Expression of two major stem cell marker genes and five major epithelial–mesenchymal transition marker genes was also assessed. The expression of significantly deregulated Wnt genes was then studied in normal human Schwann cells, fibroblasts, endothelial cells, and mast cells and in seven MPNST cell lines. Results: The expression of nine Wnt genes was significantly deregulated in plexiform neurofibromas in comparison with dermal neurofibromas. Twenty Wnt genes showed altered expression in MPNST biopsies and cell lines. Immunohistochemical studies confirmed the Wnt pathway activation in NF1-associated MPNSTs. We then confirmed that the knockdown of NF1 in Schwann cells but not in epithelial cells provoked the activation of Wnt pathway by functional transfection assays. Furthermore, we showed that the protein expression of active β-catenin was increased in NF1-silenced cell lines. Wnt pathway activation was strongly associated to both cancer stem cell reservoir and Schwann–mesenchymal transition. Conclusion: We highlighted the implication of Wnt pathway in NF1-associated tumorigenesis. Clin Cancer Res; 20(2); 358–71. ©2013 AACR.

[1]  S. Ivy,et al.  Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways , 2011, Nature Reviews Clinical Oncology.

[2]  K. Leroy,et al.  Malignant peripheral nerve sheath tumors associated with neurofibromatosis type 1: a clinicopathologic and molecular study of 17 patients. , 2001, Archives of dermatology.

[3]  D. Burns,et al.  Cell of origin and microenvironment contribution for NF1-associated dermal neurofibromas. , 2009, Cell stem cell.

[4]  Sharmistha Sarkar,et al.  A Two-Step Model for Colon Adenoma Initiation and Progression Caused by APC Loss , 2009, Cell.

[5]  M. Schumacher,et al.  Lithium enhances remyelination of peripheral nerves , 2012, Proceedings of the National Academy of Sciences.

[6]  Ivan Radovanovic,et al.  HEDGEHOG-GLI1 Signaling Regulates Human Glioma Growth, Cancer Stem Cell Self-Renewal, and Tumorigenicity , 2007, Current Biology.

[7]  J. Zeller,et al.  Association between benign and malignant peripheral nerve sheath tumors in NF1 , 2005, Neurology.

[8]  D. Burns,et al.  Induction of abnormal proliferation by nonmyelinating schwann cells triggers neurofibroma formation. , 2008, Cancer cell.

[9]  L. R. Howe,et al.  Twist is up-regulated in response to Wnt1 and inhibits mouse mammary cell differentiation. , 2003, Cancer research.

[10]  W. Hahn,et al.  Roots and stems: stem cells in cancer , 2006, Nature Medicine.

[11]  E. Pasmant,et al.  Unravelling the genetic basis of variable clinical expression in neurofibromatosis 1. , 2009, Human molecular genetics.

[12]  Jun Liu,et al.  Diagnostic and prognostic value of the methylation status of secreted frizzled-related protein 2 in colorectal cancer. , 2011, Clinical and investigative medicine. Medecine clinique et experimentale.

[13]  Y. Iwamoto,et al.  Prognostic Significance of AKT/mTOR and MAPK Pathways and Antitumor Effect of mTOR Inhibitor in NF1-Related and Sporadic Malignant Peripheral Nerve Sheath Tumors , 2012, Clinical Cancer Research.

[14]  J. Carey,et al.  Double inactivation of NF1 in tibial pseudarthrosis. , 2006, American journal of human genetics.

[15]  R. Huang,et al.  Epithelial-Mesenchymal Transitions in Development and Disease , 2009, Cell.

[16]  Michael D. Cole,et al.  c-Myc Transforms Human Mammary Epithelial Cells through Repression of the Wnt Inhibitors DKK1 and SFRP1 , 2007, Molecular and Cellular Biology.

[17]  I. Bièche,et al.  Molecular profiling of malignant peripheral nerve sheath tumors associated with neurofibromatosis type 1, based on large-scale real-time RT-PCR , 2004, Molecular Cancer.

[18]  Mikael Wiberg,et al.  Characterisation of human mesenchymal stem cells following differentiation into Schwann cell-like cells , 2009, Neuroscience Research.

[19]  J. Friedman.  Epidemiology of neurofibromatosis type 1. , 1999, American journal of medical genetics.

[20]  Randall T. Moon,et al.  Proximal events in Wnt signal transduction , 2009, Nature Reviews Molecular Cell Biology.

[21]  D. Burns,et al.  Neurofibromas in NF1: Schwann Cell Origin and Role of Tumor Environment , 2002, Science.

[22]  M. Schumacher,et al.  Wnt/β-Catenin Signaling Is an Essential and Direct Driver of Myelin Gene Expression and Myelinogenesis , 2011, The Journal of Neuroscience.

[23]  N. Ratner,et al.  Nf1 mutation expands an EGFR-dependent peripheral nerve progenitor that confers neurofibroma tumorigenic potential. , 2008, Cell stem cell.

[24]  A. Clarke,et al.  K-ras and Wnt signaling synergize to accelerate prostate tumorigenesis in the mouse. , 2009, Cancer research.

[25]  J. Beckmann,et al.  The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/β-catenin pathway , 2009, Oncogene.

[26]  G. Benvenuto,et al.  Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models. , 2000, The Journal of clinical investigation.

[27]  B. Zbar,et al.  Oncogenic Mutants of RON and MET Receptor Tyrosine Kinases Cause Activation of the β-Catenin Pathway , 2001, Molecular and Cellular Biology.

[28]  I. Bièche,et al.  Identification of Genes Potentially Involved in the Increased Risk of Malignancy in NF1-Microdeleted Patients , 2011, Molecular medicine.

[29]  N. Socci,et al.  Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway. , 2007, The Journal of clinical investigation.

[30]  I. Bièche,et al.  Differential Expression of CCN1/CYR61, CCN3/NOV, CCN4/WISP1, and CCN5/WISP2 in Neurofibromatosis Type 1 Tumorigenesis , 2010, Journal of neuropathology and experimental neurology.

[31]  M. Watson,et al.  Large-scale molecular comparison of human schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues. , 2006, Cancer research.

[32]  S. Marley,et al.  Influence of PI‐3K/Akt pathway on Wnt signalling in regulating myeloid progenitor cell proliferation. Evidence for a role of autocrine/paracrine Wnt regulation , 2009, British journal of haematology.

[33]  H. Clevers,et al.  Wnt signalling in stem cells and cancer , 2005, Nature.

[34]  D. Louis,et al.  Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation. , 1999, The American journal of pathology.

[35]  J. Behrens,et al.  The Wnt connection to tumorigenesis. , 2004, The International journal of developmental biology.

[36]  J. Kantelip,et al.  Autocrine regulation of cord blood-derived human mast cell activation by IL-10. , 2001, The Journal of allergy and clinical immunology.

[37]  J. Szatkowski,et al.  Tyrosine kinase inhibitor STI-571/Gleevec down-regulates the beta-catenin signaling activity. , 2003, Cancer letters.

[38]  T. Rosenbaum,et al.  Long‐term culture and characterization of human neurofibroma‐derived Schwann cells , 2000, Journal of neuroscience research.

[39]  G. Page,et al.  Integrative genomic analyses of neurofibromatosis tumours identify SOX9 as a biomarker and survival gene , 2009, EMBO molecular medicine.

[40]  C. Cordon-Cardo,et al.  Expression of p27(kip) and other cell cycle regulators in malignant peripheral nerve sheath tumors and neurofibromas: the emerging role of p27(kip) in malignant transformation of neurofibromas. , 1999, The American journal of pathology.

[41]  L. Neckers,et al.  Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription. , 2001, Cancer research.

[42]  R. Barouki,et al.  A natural sequence consisting of overlapping glucocorticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression. , 2000, The Biochemical journal.

[43]  Jeng-Shin Lee,et al.  Chromosome 17p deletions and p53 gene mutations associated with the formation of malignant neurofibrosarcomas in von Recklinghausen neurofibromatosis. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Stephen M Hewitt,et al.  Wnt5A regulates expression of tumor-associated antigens in melanoma via changes in signal transducers and activators of transcription 3 phosphorylation. , 2008, Cancer research.

[45]  S. Morrison,et al.  The loss of Nf1 transiently promotes self-renewal but not tumorigenesis by neural crest stem cells. , 2008, Cancer cell.

[46]  A. Van Evercooren,et al.  In vitro and in vivo behaviour of NDF‐expanded monkey Schwann cells , 1998, The European journal of neuroscience.

[47]  Denys Fontaine,et al.  Efficient myelin repair in the macaque spinal cord by autologous grafts of Schwann cells. , 2005, Brain : a journal of neurology.

[48]  M. Pierotti,et al.  p15INK4b, p14ARF, and p16INK4a inactivation in sporadic and neurofibromatosis type 1-related malignant peripheral nerve sheath tumors. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[49]  J. Hauw,et al.  Establishment and characterization of a mouse Schwann cell line which produces myelin in vivo , 1992, Journal of neuroscience research.

[50]  A. Bosserhoff,et al.  Expression of Dickkopf genes is strongly reduced in malignant melanoma , 2006, Oncogene.

[51]  W. Birchmeier,et al.  Wnt signalling and its impact on development and cancer , 2008, Nature Reviews Cancer.

[52]  O. Debeir,et al.  Exploring the Distinctive Biological Characteristics of Pilocytic and Low-Grade Diffuse Astrocytomas Using Microarray Gene Expression Profiles , 2006, Journal of neuropathology and experimental neurology.