Characterization of STAT3 Activation in Osteosarcoma

Osteosarcoma (OSA) is the most common malignant bone tumor in both children and dogs. Treatment for both involves removal of the primary tumor followed by adjuvant chemotherapy. Unfortunately, 30% of children and over 90% of dogs will die of disease post-treatment. Recent evidence suggests that the molecular biology of OSA in dogs is similar to that in children including the presence of p53 mutations, aberrant Met expression, and similar transcriptional profiles. Given the similarities in biology and clinical behavior between canine and pediatric OSA, canine OSA represents a relevant animal model in which to investigate the molecular biology of this disease. While investigating signaling pathways in OSA cell lines, we identified the presence of constitutive STAT3 activation in all lines tested. The purpose of this work was to evaluate the potential utility of STAT3 as a target for therapeutic intervention in OSA.;The first objective was to determine whether STAT3 was activated in human and canine OSA cell lines and canine OSA tissues and investigate how this activation is driven by upstream tyrosine kinases. OSA cell lines and tissues had activated STAT3 and this was found to occur through activation by upstream Src tyrosine kinase. Abrogation of STAT3 signaling by small interfering RNA or small molecule Src and STAT3 inhibitors led to apoptosis and loss of expression of targets such as VEGF and MMP2. This supported a role for STAT3 in enhancing tumor cell invasion and survival.;The second objective was to investigate the effect of oncostatin M (OSM), a cytokine that activates the Janus kinase (JAK) family in enhancing STAT3 activation. The JAK family of kinases is known to activate STAT3 in cancer. OSM and members of its signaling pathway were found to be upregulated in human and canine OSA cell lines and canine tumor tissues. OSM stimulation led to STAT3, Src, and JAK2 activation with concurrent increases in MMP2 and VEGF expression. Enhanced invasion of OSA cells occurred with OSM stimulation, suggesting a role for this pathway in tumor progression and metastasis.;The final objective was to characterize the efficacy and activity of a novel curcumin analog FLLL32 when applied to canine and human OSA cells. Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities however it does not attain sufficient blood levels to do so when ingested. This new analog was generated to have improved biochemical properties and specificity for STAT3. Treatment with FLLL32 led to decreases in STAT3 DNA binding activity followed by losses of viability, increased apoptosis, and decreased expression of VEGF, survivin, and MMP2, all STAT3 transcriptional targets.;These studies provide evidence supporting the targeting of STAT3 in the treatment of canine and human OSA. Given the role of STAT3 in enhancing MMP2 and VEGF expression in OSA, this protein may prove to be an important part of targeted therapy for metastatic disease.