Validation of a role for Active Beta-Catenin (ABC) in promoting Metastatic Phenotype in Osteosarcoma

Introduction: Osteosarcoma (OS) is an aggressive primary bone malignancy with peak incidence in children/young adults <25 years of age. The 5-year event-free-survival for patients with localized disease and metastatic disease is ~65% and ~25%, respectively. Despite intensive chemotherapy, metastasis of osteosarcoma occurs in 15-20% of cases. This goes to show that there is an urgent need for identifying prognostic markers to facilitate risk stratification. The Wnt/β-catenin (β-cat) pathway plays a crucial role in skeletal development and is deregulated in OS. However, its role in OS, especially in OS progression remains unknown. We investigated the role of the β-cat pathway, specifically the transcriptionally active form of β-cat, Active Beta Catenin (ABC), in OS progression. ABC constitutes a unique pool of β-cat that is dephosphorylated at Serine 37 and Threonine 41 at the N-terminal domain. Objective: The focus of our study was to determine if ABC is a prognostic biomarker that correlates with metastatic dissemination at disease progression. Methods: Two sets of paired cell lines, SaOS2/SaOS2-LM7 and HOS/HOS-143B were utilized for this study. Using a pEGFP-β-cat fusion construct plasmid, we carried out site directed mutagenesis to the N-terminal S33, S37, T41 and S45 in order to mimic endogenous ABC [GeneArt, Invitrogen]. The pEGFP-ABC and pEGFP-β-cat constructs were then transfected into SaOS2 and HOS cells and subject to Western Blot analysis and immunofluorescence. We also carried out immunohistochemical analysis (IHC) on 30 OS patient samples to determine whether nuclear ABC levels were correlated to "aggressive" disease. Results: Initial observations from both paired cell lines indicated that endogenous nuclear ABC levels, but not β-cat, increase with OS progression. SaOS2 Cells transfected with pEGFP-ABC and pEGFP-β-cat plasmid constructs via immunofluorescence showed similar activity to that of the endogenous ABC and β-cat: endogenous and pEGFP-ABC were both seen to translocate to the nucleus while both the endogenous and pEGFP-β-cat remained cytosolic or membrane bound. We analyzed whether nuclear ABC levels were correlated to aggressive OS, as determined by metastasis at diagnosis or resection (30 patients) via IHC. We observed a significantly greater number of patients with metastatic disease at diagnosis (p=0.029, two tailed) or at the time of resection (p=0.007, two tailed) showing high nuclear levels of ABC (>25% nuclear positivity) compared to samples which did not show metastasis at diagnosis or resection.

Conclusion: This strong correlation between high nuclear ABC levels and metastatic disease helps propel our research to further work with the plasmid constructs to determine whether ABC has potential to be used as a reliable prognostic marker.