Abstract 828: Src plays a role in chemoresistance and migration in chondrosarcoma cell lines
Authors: van Oosterwijk Jolieke G. , de Bruijn Inge H. Briaire- , van Ruler Maayke A.J.H. , Herpers Bram , Gelderblom Hans , van de Water Bob , Bovee Judith V.M.G. 
Published: August 2012

Abstract
Chondrosarcomas (CS) are malignant cartilage-forming tumors of bone. High grade chondrosarcomas show metastasis formation in 71% of cases, for which currently no treatment strategies exist. Kinome profiling revealed high GSK3β, AKT, and Src pathway activity. These kinases play a role in either cell survival, migration, or both. Our aim was to identify the roles of these pathways in chemoresistance and migration and investigate downstream effects while pinpointing the most important kinase in chondrosarcoma. We used 5 conventional chondrosarcoma cell lines and 3 dedifferentiated chondrosarcoma cell lines for the experimental procedures. Kinase inhibition was performed with enzastaurin (GSK3β and AKT inhibition) and dasatinib (Src inhibition). WST and Live cell imaging with AnnexinV staining (BD Pathway 855 Bioimager) were performed to investigate cell viability and apoptosis formation. Proliferation and migration assays were performed with the RTCA xCelligence System (Roche). Tissue microarrays (TMAs) were constructed containing 8 EC, 92 central CS (grade I n=42, grade II n=35, grade III n=14), 11 OC and 45 peripheral CS (grade I n=31, grade II n=11, grade III n=3). Overexpression of Src family members can lead to excess Nrf2 translocation from the nucleus. Immunohistochemistry was performed for the Src family members and GPX3, the target of the transcription factor Nrf2. Western blot was performed for Src family members, Nrf2, and GPX3. Inhibition of GSK3β and Akt with enzastaurin in chondrosarcoma cell lines was ineffective, also when combined with Src inhibition (dasatinib) or doxorubicin. Combination treatment of dasatinib with doxorubicin, however, showed synergistic loss in cell viability and apoptosis formation, with cleaved caspase3 activation, which was not observed after dasatinib single treatment. Migration assays in cell lines (n=6) revealed that at low concentrations of dasatinib (200nM), migration was successfully inhibited. TMA staining revealed sporadic expression of Yes and Lck (5%), expression of Src (57%) and abundant expression of Fyn (78%) throughout our panel of CS tissues. Fyn is described to be associated with metastasis formation and is a good candidate to play a role in chondrosarcoma progression. Overall pSrc (active Src) approached 100% in all grades. Western blot analysis showed nuclear localization of Nrf2 and restoration of GPX3 transcription after dasatinib treatment of cell lines. We show that Src family kinases rather than GSK3β and Akt kinases contribute to chemoresistance in chondrosarcoma. Src kinase inhibition successfully prepared cells for chemotherapy and acts synergistically with doxorubicin, and moreover was able to completely inhibit chondrosarcoma cell migration and restore GPX3 transcription. These results strongly indicate Src family kinases, and in particular Fyn, to be a potential target for the treatment of inoperable and metastatic chondrosarcomas.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 828. doi:1538-7445.AM2012-828