Effects of chondroitin sulfate proteoglycan 4 (NG2/CSPG4) on soft-tissue sarcoma growth depend on tumor developmental stage

Sarcomas, and the mesenchymal precursor cells from which they arise, express chondroitin sulfate proteoglycan 4 (NG2/CSPG4). However, NG2/CSPG4's function and its capacity to serve as a therapeutic target in this tumor type are unknown. Here, we used cells from human tumors and a genetically engineered autochthonous mouse model of soft-tissue sarcomas (STSs) to determine NG2/CSPG4's role in STS initiation and growth. Inhibiting NG2/CSPG4 expression in established murine and human STSs decreased tumor volume by almost two-thirds and cell proliferation rate by 50%. NG2/CSPG4 antibody immunotherapy in human sarcomas established as xenografts in mice similarly decreased tumor volume, and expression of a lentivirus blocking NG2/CSPG4 expression inhibited tumor cell proliferation and increased the latency of engraftment. Gene profiling showed that Ng2/Cspg4 deletion altered the expression of genes regulating cell proliferation and apoptosis. Surprisingly, Ng2/Cspg4 deletion at the time of tumor initiation resulted in larger tumors. Gene expression profiling indicated substantial down-regulation of insulin-like growth factor binding protein (Igfbp) genes when Ng2/Cspg4 is depleted at tumor initiation, but not when Ng2/Cspg4 is depleted after tumor initiation. Such differences may have clinical significance, as therapeutic targeting of a signaling pathway such as NG2/CSPG4 may have different effects on cell behavior with tumor progression. NG2/CSPG4 depletion has divergent effects, depending on the developmental stage of sarcoma. In established tumors, IGF signaling is active, and NG2 inhibition targets cell proliferation and apoptosis.

[1]  B. Alman,et al.  Mesenchymal Tumors Can Derive from Ng2/Cspg4-Expressing Pericytes with β-Catenin Modulating the Neoplastic Phenotype. , 2016, Cell reports.

[2]  Gary D Bader,et al.  Identification of CD146 as a marker enriched for tumor-propagating capacity reveals targetable pathways in primary human sarcoma , 2015, Oncotarget.

[3]  D. Kirsch,et al.  The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis , 2015, Disease Models & Mechanisms.

[4]  Pier Andrea Nicolosi,et al.  Theranostic Impact of NG2/CSPG4 Proteoglycan in Cancer , 2015, Theranostics.

[5]  Raphael Gottardo,et al.  Orchestrating high-throughput genomic analysis with Bioconductor , 2015, Nature Methods.

[6]  W. Stallcup,et al.  Shedding of NG2 by MMP-13 attenuates anoikis. , 2014, DNA and cell biology.

[7]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[8]  Hyo-Jong Lee,et al.  Transcriptional and posttranslational regulation of insulin-like growth factor binding protein-3 by Akt3. , 2014, Carcinogenesis.

[9]  C. Fletcher.  Recently characterized soft tissue tumors that bring biologic insight , 2014, Modern Pathology.

[10]  K. Sakimura,et al.  NG2 proteoglycan promotes tumor vascularization via integrin-dependent effects on pericyte function , 2013, Angiogenesis.

[11]  Pier Andrea Nicolosi,et al.  NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion. , 2013, Journal of molecular cell biology.

[12]  U. Cavallaro,et al.  Multivalent proteoglycan modulation of FGF mitogenic responses in perivascular cells , 2013, Angiogenesis.

[13]  B. Alman,et al.  A High Throughput Screen Identifies Nefopam as Targeting Cell Proliferation in β-Catenin Driven Neoplastic and Reactive Fibroproliferative Disorders , 2012, PloS one.

[14]  H. Pass,et al.  CSPG4 as a Target of Antibody-Based Immunotherapy for Malignant Mesothelioma , 2012, Clinical Cancer Research.

[15]  Jiayi Hu,et al.  Hedgehog and Notch signaling regulate self-renewal of undifferentiated pleomorphic sarcomas. , 2012, Cancer research.

[16]  A. Roberts,et al.  Ablation of NG2 Proteoglycan Leads to Deficits in Brown Fat Function and to Adult Onset Obesity , 2012, PloS one.

[17]  Gautier Koscielny,et al.  Ensembl Genomes: an integrative resource for genome-scale data from non-vertebrate species , 2011, Nucleic Acids Res..

[18]  H. Immervoll,et al.  Expression of the progenitor marker NG2/CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma , 2011, Acta Neuropathologica.

[19]  H. Immervoll,et al.  Targeting the NG2/CSPG4 Proteoglycan Retards Tumour Growth and Angiogenesis in Preclinical Models of GBM and Melanoma , 2011, PloS one.

[20]  Xinhui Wang,et al.  The CSPG4-specific monoclonal antibody enhances and prolongs the effects of the BRAF inhibitor in melanoma cells , 2011, Immunologic research.

[21]  T. Jacks,et al.  Uncoupling cancer mutations reveals critical timing of p53 loss in sarcomagenesis. , 2011, Cancer research.

[22]  W. Barry,et al.  CSPG4 protein as a new target for the antibody-based immunotherapy of triple-negative breast cancer. , 2010, Journal of the National Cancer Institute.

[23]  M. Graf,et al.  Use of NG2 (7.1) in AML as a tumor marker and its association with a poor prognosis. , 2010, Cancer genomics & proteomics.

[24]  David G. Kirsch,et al.  Cross Species Genomic Analysis Identifies a Mouse Model as Undifferentiated Pleomorphic Sarcoma/Malignant Fibrous Histiocytoma , 2009, PloS one.

[25]  B. Alman,et al.  Gli2 and p53 cooperate to regulate IGFBP-3- mediated chondrocyte apoptosis in the progression from benign to malignant cartilage tumors. , 2009, Cancer cell.

[26]  Carlos Cordon-Cardo,et al.  MFH classification: differentiating undifferentiated pleomorphic sarcoma in the 21st Century , 2009, Expert review of anticancer therapy.

[27]  G. Carrera,et al.  Soft-tissue Sarcoma Metastases Identified on Abdomen and Pelvis CT Imaging , 2009, Clinical orthopaedics and related research.

[28]  A. Daigeler,et al.  MFH revisited: outcome after surgical treatment of undifferentiated pleomorphic or not otherwise specified (NOS) sarcomas of the extremities—an analysis of 140 patients , 2009, Langenbeck's Archives of Surgery.

[29]  P. Picci,et al.  NG2 expression predicts the metastasis formation in soft‐tissue sarcoma patients , 2009, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[30]  H. Pehamberger,et al.  Reduction of Human Melanoma Tumor Growth in Severe Combined Immunodeficient Mice by Passive Transfer of Antibodies Induced by a High Molecular Weight Melanoma-Associated Antigen Mimotope Vaccine , 2008, Clinical Cancer Research.

[31]  S. Ferrone,et al.  Expression of high molecular weight-melanoma associated antigen (HMW-MAA) in primary ALM lesions is associated with a poor prognosis. , 2008, Pigment cell research.

[32]  P. Lønning,et al.  The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling , 2008, Oncogene.

[33]  T. Watanabe,et al.  p27Kip1 nuclear localization and cyclin-dependent kinase inhibitory activity are regulated by glycogen synthase kinase-3 in human colon cancer cells , 2008, Cell Death and Differentiation.

[34]  Jan Grimm,et al.  A spatially and temporally restricted mouse model of soft tissue sarcoma , 2007, Nature Medicine.

[35]  T. Mustelin,et al.  Differential phosphorylation of NG2 proteoglycan by ERK and PKCα helps balance cell proliferation and migration , 2007, The Journal of cell biology.

[36]  T. Jacks,et al.  Restoration of p53 function leads to tumour regression in vivo , 2007, Nature.

[37]  E. Ghigo,et al.  Dual effects of IGFBP‐3 on endothelial cell apoptosis and survival: Involvement of the sphingolipid signaling pathways , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[38]  S. Ferrone,et al.  Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms , 2004, The Journal of cell biology.

[39]  J. Downward.  PI 3-kinase, Akt and cell survival. , 2004, Seminars in cell & developmental biology.

[40]  C. Beaudry,et al.  Migrating glioma cells activate the PI3-K pathway and display decreased susceptibility to apoptosis , 2003, Journal of Cell Science.

[41]  P. Meltzer,et al.  Mechanisms of sarcoma development , 2003, Nature Reviews Cancer.

[42]  M. Daly,et al.  PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes , 2003, Nature Genetics.

[43]  P. Meltzer,et al.  Soft tissue sarcomas of adults: state of the translational science. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[44]  W. Stallcup.  The NG2 proteoglycan: Past insights and future prospects , 2002, Journal of neurocytology.

[45]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[46]  M. King,et al.  Insulin-like Growth Factor-binding Protein-3 Modulates Expression of Bax and Bcl-2 and Potentiates p53-independent Radiation-induced Apoptosis in Human Breast Cancer Cells* , 2000, The Journal of Biological Chemistry.

[47]  D. Alderson,et al.  IGFBp‐3 prolongs the p53 response and enhances apoptosis following UV irradiation , 2000, International journal of cancer.

[48]  M. Liimatta,et al.  Insulin-like Growth Factor (IGF)-I Regulates IGF-binding Protein-5 Gene Expression through the Phosphatidylinositol 3-Kinase, Protein Kinase B/Akt, and p70 S6 Kinase Signaling Pathway* , 1999, The Journal of Biological Chemistry.

[49]  T Ochiya,et al.  PDGF (alpha)-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from the NG2 knockout mouse. , 1999, Journal of cell science.

[50]  M. Welters,et al.  Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues. , 1997, British Journal of Cancer.

[51]  A. Klippel,et al.  Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt , 1997, Molecular and cellular biology.

[52]  P. Lollini,et al.  Insulin-like growth factor I receptor-mediated circuit in Ewing's sarcoma/peripheral neuroectodermal tumor: a possible therapeutic target. , 1996, Cancer research.

[53]  I. Bernstein,et al.  The human homologue of rat NG2, a chondroitin sulfate proteoglycan, is not expressed on the cell surface of normal hematopoietic cells but is expressed by acute myeloid leukemia blasts from poor-prognosis patients with abnormalities of chromosome band 11q23. , 1996, Blood.

[54]  C. Heldin,et al.  Co‐localization of NG2 proteoglycan and PDGF α‐receptor on O2A progenitor cells in the developing rat brain , 1996, Journal of neuroscience research.

[55]  S. Ferrone,et al.  Human high molecular weight-melanoma associated antigen mimicry by mouse anti-idiotypic monoclonal antibody MK2-23: modulation of the immunogenicity in patients with malignant melanoma. , 1995, Clinical cancer research : an official journal of the American Association for Cancer Research.

[56]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[57]  S. Ferrone,et al.  Human high molecular weight-melanoma-associated antigen (HMW-MAA): a melanoma cell surface chondroitin sulfate proteoglycan (MSCP) with biological and clinical significance. , 2004, Critical reviews in immunology.

[58]  C. Paraskeva,et al.  Increased p53-dependent apoptosis by the insulin-like growth factor binding protein IGFBP-3 in human colonic adenoma-derived cells. , 2000, Cancer research.

[59]  D. Engelhardt,et al.  Characterization of Insulin-Like Growth Factor Binding Proteins (IGFBPs) Secreted by Bovine Adrenocortical Cells in Primary Culture: Regulation by Insulin-Like Growth Factors (IGFs) and Adrenocorticotropin (ACTH)* , 1999, Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme.

[60]  T. Ochiya,et al.  PDGF α-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from the NG 2 knockout mouse , 1999 .

[61]  D. Hill,et al.  Distinct Sites of Insulin-like Growth Factor (igf)-ii Expression and Localization in Lesioned Rat Brain: Possible Roles of Igf Binding Proteins (igfbps) in the Mediation of Igf-ii Activity* , 2022 .