BackgroundOsteosarcoma is the most common pediatric bone cancer in the United States. Advances in surgery and chemotherapy have resulted in a cure rate of 65% for children who present with localized disease. However, patients who have metastatic disease have only a 25% survival rate. Attempts to improve this outcome using various intensification regimens have not improved survival. Unfortunately, these results demonstrate the inability to target the tumor using intensification of chemotherapy to salvage patients with metastatic disease. Therefore, we have turned our efforts to identifying biological elements in this disorder that are amenable to modulation.HOS is a human osteosarcoma cell line that does not form primary tumors and does not form metastatic lung nodules in immunodeficient mice. However, treatment of HOS cells with the chemical N-methyl-N-nitro-N-nitrosoguanidine (MNNG) resulted in a transformed cell line, MNNG-HOS, that is able to form primary tumors and also metastatic nodules in the lung. Further characterization of the chemically transformed cell line resulted in the identification of the Met oncogene as the likely mediator of metastasis.Met is a cell surface receptor that binds a single ligand, hepatocyte growth factor, and has tyrosine kinase activity. Met is mutated or over-expressed in a variety of tumors, including many carcinomas and soft tissue sarcomas. In addition, a study in large dogs with osteosarcoma has shown that 10% of these tumors harbor mutations in Met. These findings led us to study Met and its possible role in osteosarcoma metastasis.We showed that Met is highly expressed in patient samples by immunohistochemistry. We also collaborated with members of Donald Bottaros laboratory in the Urological Oncology Branch to optimize a dual antibody electrochemiluminescence (ECL) assay to obtain quantitative values of expression, to show that Met was highly expressed in the vast majority of osteosarcoma cell lines and tumor samples, with a subset also expressing phosphorylated, activated Met. We went on to test various osteosarcoma cell lines to determine if they were responsive to a Met inhibitor, PHA-665752. ECL assays demonstrated significantly lower levels of phospho-Met levels in cells treated with PHA-665752. These levels returned to their high baseline levels when examined 96 hours after treatment, demonstrating that the inhibitor was targeting Met. We then tested the ability of PHA-665752 to inhibit the following parameters: proliferation (MTT analysis), motility (scratch assay), migration (through an 8 micron pore) and invasion (migration through a matrigel matrix). PHA-665752 had no effect on proliferation in any of the osteosarcoma cell lines tested. However, it was effective in inhibiting one or more of the other parameters in three osteosarcoma cell lines. These included LR, MG63.2 and MNNG-HOS. LR and MG63.2 express the highest levels of phospho-Met and MNNG-HOS has activated Met due to a translocation. These results demonstrated that only cells that have activated Met have a decrease in the metastatic phenotype in vitro when Met is inhibited.We then utilized a genetic approach to complement the pharmacological experiments. Specifically, we utilized Met specific sh-RNA constructs to demonstrate that lentiviral mediated infection of these constructs into high Met expressing osteosarcoma cell lines resulted in inhibition of in vitro parameters of metastases. We proceeded to test the above constructs in a murine model of metastases and demonstrated that mice injected with sh-MET infected osteosarcoma cell lines had prolonged survival compared to control mice. With the plan to translate these findings for clinical use, we then obtained an orally available small molecule Met specific inhibitor, PF-04217903. This compound is currently in clinical trials. Utilization of this compound in osteosarcoma cell lines affected parameters of metastasis in vitro. Inaddition, treatment of mice with this compound dramatically reduced the number of resulting metastatic lung nodules.Plans for 2012In 2012, we are preparing a manuscript detailing the above findings for submission to the journal Cancer Research. We would also like to identify the mechanism by which Met is activated in osteosarcoma.Significance and GoalsThe successful completion of the above objectives may reveal the mechanism, or mechanisms, of action by which Met is activated in osteosarcoma cell lines. Several Met-specific inhibitors are currently in clinical trials, which allows for rapid translation into the clinic, if we are able to successfully complete our objectives.
