Osteosarcoma (OSA) is the most common malignant bone tumor in children and dogs. Currently, 30-40% of children and greater than 90% of dogs succumb to the disease following treatment. The receptor tyrosine kinase (RTK) Met has emerged as a potential target for therapeutic intervention for OSA. Met overexpression in human OSA is associated with a more aggressive phenotype and poor survival, and aberrant expression in normal human osteoblasts induces malignant transformation. The purpose of this work was to evaluate Met as a target for therapeutic intervention in OSA.
The first objective was to determine if Met interacts with heat shock protein 90 (Hsp90) in OSA and evaluate a novel Hsp90 inhibitor, STA-1474, for OSA treatment. Hsp90 associated with co-chaperones in canine OSA cells but not normal canine osteoblasts, indicating formation of an active superchaperone complex in malignant versus normal tissue. STA-1474 promoted loss of cell proliferation, apoptosis, and induction of Hsp70. STA-1474 treatment also resulted in the down-regulation of HGF (hepatocyte growth factor) induced p-Met/Met, p-STAT3, and p-Akt/Akt. These data suggest that STA-1474 may be useful for the treatment of OSA.
The second objective was to determine whether RTKs Met, epidermal growth factor receptor (EGFR), and Recepteur d'origine nantais (Ron) interact in OSA and explore the functional consequences of such interactions. EGFR and Ron phosphorylation was present in canine OSA tumor tissues, and Met was associated with EGFR and Ron in canine OSA cell lines. High Ron expression was prognostic for survival. Gefitinib (small molecule EGFR inhibitor) and crizotinib (small molecule Met inhibitor) inhibited OSA cell proliferation in an additive manner. Prolonged TGF alpha exposure promoted Met phosphorylation. Co-activation of EGFR and Met with their ligands resulted in amplified ERK1/2 and STAT3 phosphorylation. These data indicate that Met, Ron, and EGFR functionally interact in canine OSA, altering the manner in which these cells respond to growth factor stimulation.
The final objective involved characterization of a germline Met point mutation (G966S) identified in 75% of Rottweilers, a breed at high risk for OSA. NIH3T3 cells stably transduced with G966S canine Met demonstrated greater scattering which was enhanced with HGF and blocked by a Met inhibitor, indicating these changes were directly related to Met expression and signaling. These cells also exhibited nonstimulated basal Met autophosphorylation. As reagents to study canine signal transduction pathways are limited, NIH3T3 cells were stably transduced with the equivalent mouse Met mutation (G963S). The G963S transduced cells exhibited enhanced matrix metalloproteinase 2 (MMP2) activity. Genomic integration of the Met construct was evident, but not gene expression based on cDNA analysis. CMV promoter methylation was suspected. Stably transduced 293TN cells expressing the Met construct based on analysis of cDNA were generated.
In summary, these studies support targeting Met for the treatment of canine OSA. Interaction between Met and the RTK’s EGFR and Ron represents a novel finding in canine OSA and indicates that simultaneous targeting of all of these receptors may be beneficial. Further work will assess the G966S canine Met mutation contribution to OSA development in the Rottweiler.