Abstract 2194: Novel receptor tyrosine kinases in osteosarcoma identified by phosphoproteomic and functional genomic screening

Abstract

Abstract Osteosarcoma is the most common primary bone malignancy and is largely attributed to rapidly growing bones of adolescents. Approximately one-third of patients succumb to pulmonary metastasis despite high-dose neoadjuvant chemotherapy protocols. Survival rates have not improved over the last 30 years therefore novel therapeutic agents are needed. Tyrosine kinases have emerged as targets for the development of new cancer therapies since their activation leads to enhanced proliferation, survival, cell adhesion and metastasis. Unlike other cancers, the role of tyrosine kinases has not been well established in osteosarcoma. In this study, we used two families of genetically related human osteosarcoma cell lines with varying potentials for tumorigenesis and metastasis in mice. Phosphoproteomic and siRNA screening were performed to identify specific receptor tyrosine kinases that are activated and contribute to the in vitro behavior of the metastatic osteosarcoma cell lines. In the metastatic LM7 cells, 12 of the 42 RTKs on the phospho-receptor tyrosine kinase antibody array were phosphorylated. All except Axl had increased levels of phosphorylation compared to the parental, non-metastatic Saos-2 cells. Nine of the 12 RTKs were also phosphorylated in the metastatic 143B cells. Of these, FGFR3, PDGFRα and c-Ret had increased levels of phosphorylation when compared to the parental, non-metastatic TE85 cells. siRNAs targeting the 12 RTKs found to be activated in the metastatic cell lines were screened to identify those RTKs that are important for motility, invasion, colony formation and/or cell growth. In the 143B cell line, 9 of the 12 siRNAs inhibited at least one assay by ≥ 35%. Eph Kinase B2 (EphB2) knockdown had the greatest effect resulting in > 85% inhibition of motility, invasion, colony formation and cell growth compared to cells treated with luciferase control siRNA. In the LM7 cell line, 7 of the 12 siRNAs inhibited at least one of the assays by ≥ 35%. IGF-1R silencing had the greatest effect by inhibiting motility by 85%, colony formation by 58% and cell growth by 32%. Validation of the siRNA screening results have so far indicated that the effects from IGF-1R, EphB2, FGFR2, FGFR3 and c-Ret knockdown are consistent with on-target silencing in LM7 cells. Studies are underway to validate the remaining siRNAs. From our screens, we identified specific activated receptor tyrosine kinases that contribute to the in vitro behavior of osteosarcoma and may be valuable therapeutic targets. Furthermore, unlike IGF-1R, which has been widely studied in osteosarcoma, this is the first known report demonstrating that EphB2, FGFR2, FGFR3 and c-Ret may be important to the progression of osteosarcoma. Further studies will enable us to identify the underlying role of these receptor tyrosine kinases in ostesoarcoma. 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 2194.