Functional Characterization Of Igfbp5 In The Inhibition Of Osteosarcoma Tumorgenicity.

10038 Background: Osteosarcoma (OS) is the most common primary malignancy of bone. At presentation, the majority of patients have metastatic disease. Therefore, there is a clinical need to identify molecular markers that provide insight into the pathogenesis OS. The goal of this investigation is to functionally characterize the novel marker, IGFBP5, in osteosarcoma tumor progression. Methods: We created adenoviral constructs expressing the unique domains of IGFBP5 (N-term, C-term, linker), full length IGFBP5, and siIGFBP5. We conducted in vitro and in vivo assays on adenovirally infected OS cell lines MG63, MG63.2, and 143B. Western blot and immunohistochemical (IHC) staining were used to assay the apoptotic marker Caspase 3. Cell migration and invasion were measured using Wound Healing and Matrigel assays. Osteoblastic differentiation was measured using alkaline phosphastase activity and IHC staining for the late osteogenic markers osteocalcin and osteopontin. Finally, clinical patient samples were stained for IGFBP5 expression in patient matched primary tumors and metastatic pulmonary lesions. Results: IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and osteogenic differentiation. Analysis of the three functional domains indicated that the N-terminal domain was primarily responsible for inhibiting cell proliferation and inducing apoptosis, while the C-terminal domain inhibited cell migration and invasiveness. In an orthotopic animal model, overexpression of IGFBP5 inhibited OS tumor growth and lung metastasis, while siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and lung metastasis. IHC staining of clinical OS samples confirmed that primary OS tumors expressed significantly lower levels of IGFBP5 than their corresponding pulmonary metastases. Conclusions: These results suggest that IGFBP5 is a novel marker in OS pathogenesis, and the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS. Furthermore, while the N-terminal domain of IGFB5 is responsible for inhibition of cell proliferation, the C-terminal domain is responsible for inhibiting cell invasion and migration.