Multiple receptor tyrosine kinases promote the in vitro phenotype of metastatic human osteosarcoma cell lines

A N Rettew,F W Abdul-Karim,E S Kleinerman,P J Getty,E M Greenfield,D C Lev,E D Young

doi:10.1038/oncsis.2012.34

Abstract

The survival rate for osteosarcoma patients with localized disease is 70% and only 25% for patients with metastases. Therefore, novel therapeutic and prognostic tools are needed. In this study, extensive screening and validation strategies identified Axl, EphB2, FGFR2, IGF-1R and Ret as specific receptor tyrosine kinases (RTKs) that are activated and promote the in vitro phenotype of two genetically different metastatic osteosarcoma cell lines. Initial phosphoproteomic screening identified twelve RTKs that were phosphorylated in 143B and/or LM7 metastatic human osteosarcoma cells. A small interfering RNA (siRNA) screen demonstrated that siRNA pools targeting ten of the twelve RTKS inhibited the in vitro phenotype of one or both cell lines. To validate the results, we individually tested the four siRNA duplexes that comprised each of the effective siRNA pools from the initial screen. The pattern of phenotype inhibition replicated the pattern of mRNA knockdown by the individual duplexes for seven of the ten RTKs, indicating the effects are consistent with on-target silencing. Five of those seven RTKs were further validated using independent approaches including neutralizing antibodies (IGF-1R), antisense-mediated knockdown (EphB2, FGFR2, and Ret) or small molecule inhibitors (Axl), indicating that those specific RTKs promote the in vitro behavior of metastatic osteosarcoma cell lines and are potential therapeutic targets for osteosarcoma. Immunohistochemistry demonstrated that Axl is frequently activated in osteosarcoma patient biopsy samples, further supporting our screening and validation methods to identify RTKs that may be valuable targets for novel therapies for osteosarcoma patients.

Highlights

Osteosarcoma, the most common primary bone sarcoma, predominantly affects adolescents in areas of rapid bone growth.[1,2] With the introduction of high-dose, multi-agent chemotherapy regimens in combination with surgical resection in the 1970s, the long-term survival rate increased from 20 to 70% for patients with localized disease.[2,3,4] Approximately 20% of patients present with overt metastases and 30–40% of patients diagnosed with localized disease go on to develop metastases, with the lungs being the most common sites for metastasis.[2,5] The prognosis for patients with metastases remains poor, with only 20–30% experiencing long-term survival.[6]
Validation of the small interfering RNA (siRNA) screens confirmed that five receptor tyrosine kinases (RTKs) (Axl, EphB2, FGFR2, IGF-1R and Ret) promote the in vitro behavior of the metastatic osteosarcoma cell lines
We demonstrated that Axl is frequently activated in osteosarcoma patient samples, indicating that our screening and validation methods identify RTKs that may be valuable targets for translational studies

Summary

Introduction

Osteosarcoma, the most common primary bone sarcoma, predominantly affects adolescents in areas of rapid bone growth.[1,2] With the introduction of high-dose, multi-agent chemotherapy regimens in combination with surgical resection in the 1970s, the long-term survival rate increased from 20 to 70% for patients with localized disease.[2,3,4] Approximately 20% of patients present with overt metastases and 30–40% of patients diagnosed with localized disease go on to develop metastases, with the lungs being the most common sites for metastasis.[2,5] The prognosis for patients with metastases remains poor, with only 20–30% experiencing long-term survival.[6]. Small interfering RNA (siRNA) screening identified the activated RTKs that might contribute to motility, invasion, colony formation and/ or cell growth in vitro. Validation of the siRNA screens confirmed that five RTKs (Axl, EphB2, FGFR2, IGF-1R and Ret) promote the in vitro behavior of the metastatic osteosarcoma cell lines.

Results

Conclusion