Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT–PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

[1]  R. Baxter,et al.  Insulin-like Growth Factor-binding Protein-5 Inhibits the Growth of Human Breast Cancer Cells in Vitro and in Vivo* , 2003, Journal of Biological Chemistry.

[2]  C. Nanni,et al.  Preclinical In vivo Study of New Insulin-Like Growth Factor-I Receptor–Specific Inhibitor in Ewing's Sarcoma , 2007, Clinical Cancer Research.

[3]  A. Davis,et al.  Prognostic factors in osteosarcoma: a critical review. , 1994, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[4]  R. Tibshirani,et al.  Significance analysis of microarrays applied to the ionizing radiation response , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. He,et al.  S100A6 Expression and Function in Human Osteosarcoma , 2008, Clinical orthopaedics and related research.

[6]  M. L. Le Beau,et al.  Identification of a novel metastasis-suppressor region on human chromosome 12. , 1998, Cancer research.

[7]  T. He,et al.  Hey1 Basic Helix-Loop-Helix Protein Plays an Important Role in Mediating BMP9-induced Osteogenic Differentiation of Mesenchymal Progenitor Cells* , 2009, Journal of Biological Chemistry.

[8]  G. Siegal,et al.  Cytogenetics and Molecular Biology of Osteosarcoma , 2002, Laboratory Investigation.

[9]  Wei Jiang,et al.  Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing. , 2007, Gene.

[10]  P. Rotwein,et al.  Insulin-like growth factor binding protein-5 in osteogenesis: facilitator or inhibitor? , 2007, Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society.

[11]  J. Szatkowski,et al.  Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery , 2004, Gene Therapy.

[12]  David W. Johnson,et al.  Altered messenger RNA and protein expressions for insulin‐like growth factor family members in clear cell and papillary renal cell carcinomas , 2005, International journal of urology : official journal of the Japanese Urological Association.

[13]  R. Baxter,et al.  Nuclear Import of Insulin-like Growth Factor-binding Protein-3 and -5 Is Mediated by the Importin β Subunit* , 2000, The Journal of Biological Chemistry.

[14]  G. Ottaviani,et al.  The etiology of osteosarcoma. , 2009, Cancer treatment and research.

[15]  M. Gebhardt,et al.  Survival Data for 648 Patients with Osteosarcoma Treated at One Institution , 2004, Clinical orthopaedics and related research.

[16]  M. Mison,et al.  Osteosarcoma , 1985, The Lancet.

[17]  J. Shand,et al.  The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain. , 2001, Journal of molecular endocrinology.

[18]  C. Heizmann,et al.  Increased expression of S100A6 is associated with decreased metastasis and inhibition of cell migration and anchorage independent growth in human osteosarcoma. , 2005, Cancer letters.

[19]  S. Avnet,et al.  Insulin receptor isoform A and insulin-like growth factor II as additional treatment targets in human osteosarcoma. , 2009, Cancer research.

[20]  L. Reis,et al.  Differential expression of IGFBP-5 and two human ESTs in thyroid glands with goiter, adenoma and papillary or follicular carcinomas. , 2003, Cancer letters.

[21]  T. He,et al.  Establishment and characterization of a new highly metastatic human osteosarcoma cell line , 2009, Clinical & Experimental Metastasis.

[22]  A. Hoeflich,et al.  IGF-binding protein-5: flexible player in the IGF system and effector on its own. , 2002, The Journal of endocrinology.

[23]  K. Kinzler,et al.  A simplified system for generating recombinant adenoviruses. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  A. Montag,et al.  An Orthotopic Model of Human Osteosarcoma Growth and Spontaneous Pulmonary Metastasis , 2005, Clinical & Experimental Metastasis.

[25]  R. Baxter,et al.  Enhancement of tumor necrosis factor-alpha-induced growth inhibition by insulin-like growth factor-binding protein-5 (IGFBP-5), but not IGFBP-3 in human breast cancer cells. , 2005, Endocrinology.

[26]  G. Fuller,et al.  Insulin-like growth factor-binding protein 2 and 5 are differentially regulated in ovarian cancer of different histologic types , 2006, Modern Pathology.

[27]  Bin Tean Teh,et al.  Altered expression of members of the IGF-axis in clear cell renal cell carcinoma. , 2005, International journal of oncology.

[28]  A. Hoeflich,et al.  Insulin-like growth factor-binding protein-5 inhibits growth and induces differentiation of mouse osteosarcoma cells. , 2001, Biochemical and biophysical research communications.

[29]  S. Kaste,et al.  Metastases detected at the time of diagnosis of primary pediatric extremity osteosarcoma at diagnosis , 1999, Cancer.

[30]  C. Muro-Cacho,et al.  Genetic and molecular abnormalities in tumors of the bone and soft tissues. , 2001, Cancer control : journal of the Moffitt Cancer Center.

[31]  T. He,et al.  A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. , 2009, Stem cells and development.

[32]  Wei Zhang,et al.  Multifunctional roles of insulin-like growth factor binding protein 5 in breast cancer , 2008, Breast Cancer Research.

[33]  A. Montag,et al.  Connective Tissue Growth Factor (CTGF) Is Regulated by Wnt and Bone Morphogenetic Proteins Signaling in Osteoblast Differentiation of Mesenchymal Stem Cells* , 2004, Journal of Biological Chemistry.

[34]  S. Rosenzweig.  What's new in the IGF-binding proteins? , 2004, Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society.

[35]  K. Kinzler,et al.  A protocol for rapid generation of recombinant adenoviruses using the AdEasy system , 2007, Nature Protocols.

[36]  S. Mohan,et al.  Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) Interacts with a Four and a Half LIM Protein 2 (FHL2)* , 2002, The Journal of Biological Chemistry.

[37]  H. Kimura,et al.  Prognosis of Osteosarcoma With Pulmonary Metastases at Initial Presentation Is Not Dismal , 1998, Clinical orthopaedics and related research.

[38]  Wei Jiang,et al.  Inhibitor of DNA Binding/Differentiation Helix-Loop-Helix Proteins Mediate Bone Morphogenetic Protein-induced Osteoblast Differentiation of Mesenchymal Stem Cells* , 2004, Journal of Biological Chemistry.

[39]  Denise E. Mauldin,et al.  Regulation of global gene expression in the bone marrow microenvironment by androgen: Androgen ablation increases insulin‐like growth factor binding protein‐5 expression , 2007, The Prostate.