Src kinases in chondrosarcoma chemoresistance and migration: dasatinib sensitises to doxorubicin in TP53 mutant cells

Abstract

Background: Chondrosarcomas are malignant cartilage-forming tumours of bone. Because of their resistance to conventional chemotherapy and radiotherapy, currently no treatment strategies exist for unresectable and metastatic chondrosarcoma. Previously, PI3K/AKT/GSK3β and Src kinase pathways were shown to be activated in chondrosarcoma cell lines. Our aim was to investigate the role of these kinases in chemoresistance and migration in chondrosarcoma in relation to TP53 mutation status.

Methods: We used five conventional and three dedifferentiated chondrosarcoma cell lines and investigated the effect of PI3K/AKT/GSK3β pathway inhibition (enzastaurin) and Src pathway inhibition (dasatinib) in chemoresistance using WST assay and live cell imaging with AnnexinV staining. Immunohistochemistry on tissue microarrays (TMAs) containing 157 cartilaginous tumours was performed for Src family members. Migration assays were performed with the RTCA xCelligence System.

Results: Src inhibition was found to overcome chemoresistance, to induce apoptosis and to inhibit migration. Cell lines with TP53 mutations responded better to combination therapy than wild-type cell lines (P=0.002). Tissue microarray immunohistochemistry confirmed active Src (pSrc) signalling, with Fyn being most abundantly expressed (76.1%).

Conclusion: These results strongly indicate Src family kinases, in particular Fyn, as a potential target for the treatment of inoperable and metastatic chondrosarcomas, and to sensitise for doxorubicin especially in the presence of TP53 mutations.

Figure 1

Chondrosarcoma cell lines are not sensitive to PKC inhibition. (A) Schematic representation of activation of PI3K and Src pathway by RTKs. Receptor tyrosine kinases can activate PKC, phosphatidylinositol 3-kinase (PI3K) and Src. PKC and Src can also activate the PI3K/AKT/GSK3β pathway, promoting survival, proliferation and migration. The Src pathway activates the Ras/Raf pathway. Enzastaurin is a selective PKC inhibitor also reported to inhibitin activating phosphorylation of GSK3β. Dasatinib is a Src inhibitor. Adapted from Fizazi (2007). (B) HeLa cell line showing 70% decrease in cell viability after treatment with enzastaurin. Chondrosarcoma cell lines poorly respond to enzastaurin alone, and an additive effect is observed when alternating 10 μM doxorubicin (DXR) and 10 μM enzastaurin (Enz) for 24 h each for 72 h in total. No difference is observed when order of administration is reversed. Significant difference between TP53 mutant and wild-type cell lines (P=0.002). (C) Combination of enzastaurin with Src inhibitor dasatinib (Das) showing additive effect in chondrosarcoma cell lines. No significant difference is observed between TP53 mutant and wild-type cell lines (P=0.38).

Figure 2

The Src pathway is involved in chondrosarcoma chemoresistance. (A) Immunoblotting showing AKT, pAKT, Fyn, pSrc and loading control α-tubulin (a tub) for untreated chondrosarcoma cell lines and after 24-h 1 μM dasatinib. MCF-7: breast cancer cell line, shown as positive control. Presence of all kinases in all cell lines phophorylated Src in all cell lines, although levels are very low in L835. After 24-h 1 μM dasatinib treatment, levels of pSrc (at Y418) are decreased in all cell lines, and of pAKT in OUMS27, L835, L3252, JJ012 and NDCS-1. (B) Immunoblotting showing p53, MDM2 and p21 in untreated chondrosarcoma cell lines (Mock) and after 24-h 1 μM dasatinib. U2OS (osteosarcoma) cell line is shown as positive control. MDM2 expression is low in all cell lines. TP53 wild-type cell lines are negative for p53 protein expression, with low p21 protein expression, except for CH2879. TP53 mutant cell lines show high TP53 protein expression with low p21 protein expression. No change in protein levels is observed after 24-h 1 μM dasatinib treatment. (C) Combination of dasatinib (Das) with doxorubicin (DXR) leads to synergistic loss of cell viability at concentrations, which are ineffective on their own in most cell lines. Combination treatment was more effective in TP53 mutant cell lines than in TP53 wild-type cell lines (P=0.002 for cell viability, P=0.043 for combination indices). (D) Apoptosis assay in JJ012 cell line alternating 1 μM dasatinib (Das) and 1 μM doxorubicin (DXR) demonstrates the occurrence of apoptosis during combination. Apoptosis is calculated as percentage of AnnexinV-Alexxa633-stained cells per total number of HOECHST-stained cells. (D and E) Dasatinib successfully inhibits migration in chondrosarcoma cell lines in concentrations as low as 200 nM. (E) SW1353 cell line shown as representative over the course of 4 h; (F) bar chart showing migration for all cell lines.

Figure 3

Immunohistochemistry demonstrating expression of Src family members in conventional chondrosarcoma tissue. (A) High pSrc expression in grade I chondrosarcoma. (B) High-intensity nuclear Fyn expression in grade I chondrosarcoma. (C) High-intensity cytoplasmic and nuclear Src expression in grade I chondrosarcoma. (D) Absence of Yes expression in grade II chondrosarcoma. (E) Absence of Lck in grade II chondrosarcoma. Scale bars, 50 μm. (F) Scatterplot showing distribution of staining scores among chondrosarcoma tissue samples.