Abstract 719: Role of secreted frizzled-related proteins in inhibiting growth and invasion in human osteosarcoma
Authors: Pay Chin Leow, Zheng Yang, Pui Lai Rachel Ee
Journal: Cancer Research (2010)

Abstract Aberrant activation of the canonical Wnt/β-catenin signaling pathway promotes osteosarcoma tumorigenesis and metastasis. Secreted frizzled-related proteins (SFRPs), a family of five glycoproteins (SFRP1-5), antagonize this pathway by competing with Frizzled receptors for Wnt ligands. Down-regulation of SFRPs through epigenetic silencing was commonly found in various malignancies. Specifically, in osteosarcoma, the loss of SFRP-3 expression has previously been observed in osteogenic sarcoma biopsy specimens and several osteosarcoma cells lines. Despite strong evidence of their status as tumor suppressors, the implication of SFRP-1, −2, −4 and −5 in the pathogenesis of osteosarcoma has yet been reported. The objective of this study, therefore, is to understand the role of SFRPs as Wnt antagonists and their functions in affecting key anti-cancer processes such as the suppression of growth and invasion, induction of apoptosis and cell cycle disruption, as well as down-regulation of various Wnt target oncogenes in osteosarcoma. In this study, we first performed western blot analysis and detected abundant levels of β-catenin protein in a panel of metastatic osteosarcoma cell lines, suggesting frequent activation of canonical Wnt/β-catenin signaling in osteosarcoma. Effective inhibitions of the Wnt/β-catenin pathway by restoration of SFRPs in U2OS cells were subsequently shown by marked suppression of both β-catenin/Tcf transcriptional activities and β-catenin protein using luciferase reporter assays and western blot analysis respectively. Immunofluorescence confocal microscopy analysis revealed an abnormal decrease in β-catenin accumulations in the nuclei and a corresponding increase in β-catenin localization to the cell membranes in U2OS cells over-expressing SFRPs. We next performed wound healing and Matrigel invasion assays and observed that ectopic expression of SFRPs significantly decreased osteosarcoma cell migration and invasion. Western blotting showed that the reduced U2OS cell invasion and migration correlated with the protein expression of matrix metalloproteinases-2 and −9. Furthermore, using MTT proliferation and colony formation assays, we found that restoration of SFRPs significant inhibited both anchorage-dependent and -independent growth in U2OS cells. These anti-proliferative effects were attributed to GO/G1 phase arrest in U2OS/SFRP-1, U2OS/SFRP-2 and U2OS/SFRP-5 cells and G2/M phase arrest in U2OS/SFRP-4 cells. At the same time, these anti-proliferative effects correlated with the suppression of major Wnt target oncogenes such as cyclin D1, c-Myc and survivin. Collectively, our findings strongly suggest that SFRPs may be exploited as a therapeutic target in the treatment of osteosarcoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 719.