Deregulation of receptor tyrosine kinase (RTK)-signaling is frequently observed in many human malignancies, making activated RTKs the promising therapeutic targets. In particular, activated RTK-signaling has a strong impact on tumor resistance to various DNA damaging agents, e.g., ionizing radiation and chemotherapeutic drugs. We showed recently that fibroblast growth factor receptor (FGFR)-signaling might be hyperactivated in imatinib (IM)-resistant gastrointestinal stromal tumors (GIST) and inhibition of this pathway sensitized tumor cells to the low doses of chemotherapeutic agents, such as topoisomerase II inhibitors. Here, we report that inhibition of FGFR-signaling in GISTs attenuates the repair of DNA double-strand breaks (DSBs), which was evidenced by the delay in γ-H2AX decline after doxorubicin (Dox)-induced DNA damage. A single-cell gel electrophoresis (Comet assay) data showed an increase of tail moment in Dox-treated GIST cells cultured in presence of BGJ398, a selective FGFR1-4 inhibitor, thereby revealing the attenuated DNA repair. By utilizing GFP-based reporter constructs to assess the efficiency of DSBs repair via homologous recombination (HR) and non-homologous end-joining (NHEJ), we found for the first time that FGFR inhibition in GISTs attenuated the homology-mediated DNA repair. Of note, FGFR inhibition/depletion did not reduce the number of BrdU and phospho-RPA foci in Dox-treated cells, suggesting that inhibition of FGFR-signaling has no impact on the processing of DSBs. In contrast, the number of Dox-induced Rad51 foci were decreased when FGFR2-mediated signaling was interrupted/inhibited by siRNA FGFR2 or BGJ398. Moreover, Rad51 and -H2AX foci were mislocalized in FGFR-inhibited GIST and the amount of Rad51 was substantially decreased in -H2AX-immunoprecipitated complexes, thereby illustrating the defect of Rad51 recombinase loading to the Dox-induced DSBs. Finally, as a result of the impaired homology-mediated DNA repair, the increased numbers of hypodiploid (i.e., apoptotic) cells were observed in FGFR2-inhibited GISTs after Dox treatment. Collectively, our data illustrates for the first time that inhibition of FGF-signaling in IM-resistant GIST interferes with the efficiency of DDR signaling and attenuates the homology-mediated DNA repair, thus providing the molecular mechanism of GIST’s sensitization to DNA damaging agents, e.g., DNA-topoisomerase II inhibitors.

Activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway is well documented for a broad spectrum of human malignancies supporting their growth and progression. Accumulating evidence has also implicated AKT as a potent modulator of anti-cancer therapies via regulation of DNA damage response and repair (DDR) induced by certain chemotherapeutic agents and ionizing radiation (IR). In the present study, we examined the role of AKT signaling in regulating of Rad51 turnover and cytotoxic effects of topoisomerase II inhibitor, doxorubicin (Dox) in soft tissue sarcomas (STS) and gastrointestinal stromal tumors (GIST) in vitro. Blocking of AKT signaling (MK-2206) enhanced cytotoxic and pro-apoptotic effects of Dox in vast majority of STS and GIST cell lines. The phosphorylated form of Akt co-immunoprecipitates with Rad51 after Dox-induced DNA damage, whereas Akt inhibition interrupts this interaction and decreases Rad51 protein level by enhancing protein instability via proteasome-dependent degradation. Inhibition of Akt signaling in Dox-treated cells was associated with the increased number of γ-H2AX-positive cells, decrease of Rad51 foci formation and its colocalization with γ-H2AX foci, thereby revealing unsuccessful DDR events. This was also in consistency with an increase of tail moment (TM) and olive tail moment (OTM) in Dox-treated GIST and STS cells cultured in presence of Akt inhibitor after Dox washout. Altogether, our data illustrates that inhibition of AKT signaling is STS and GIST might potentiate the cytotoxic effect of topoisomerase II inhibitors via attenuating the homology-mediated DNA repair.