The E-F Hand Calcium-Binding Protein S100A4 Regulates the Proliferation, Survival and Differentiation Potential of Human Osteosarcoma Cells

Background/Aims: Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults. Molecular mechanisms underlying the pathogenesis of OS remain to be fully understood. Several members of the E-F hand calcium-binding S100 protein family are differentially expressed in human cancers. We previously showed that S100A6 is highly expressed in OS tumors. In this study, we investigated the role of S100A4 in regulating OS proliferation and osteogenic differentiation. Methods/Results: Endogenous S100 expression was examined by semi-quantitative PCR in human OS lines. Adenoviral vector-mediated overexpression and RNAi knockdown of S100A4 were used to assess S100A4's effects on cell proliferation, migration and invasion and osteogenic differentiation. Apoptosis was assessed by using anti-caspase-3 immunostaining and flow cytometry with annexin V staining. Early osteogenic marker alkaline phosphatase (ALP) and late markers osteocalcin (OCN) and osteopontin (OPN) were assessed to determine the status of osteogenic differentiation. We found that S100A4 was elevated in metastatic MG63.2 cells. S100A4 knockdown inhibited cell proliferation, prolonged cell doubling time, and induced significant apoptosis. Silencing S100A4 expression in OS cells delayed cell wounding closure and diminished the numbers of migrated OS cells in transwell invasion assay. Furthermore, silencing S100A4 expression stimulated ALP activity, as well as late markers OPN and OCN, in both OS cells and mesenchymal stem cells. Conclusion: Our results strongly suggest that S100A4 may promote OS tumor growth by regulating the cell cycle, reducing apoptosis, and inhibiting osteogenic differentiation. Thus, S100A4 may serve as a marker for tumorigenic potential, as well as a therapeutic target.

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