Inhibition of FGFR2-Signaling Attenuates a Homology-Mediated DNA Repair in GIST and Sensitizes Them to DNA-Topoisomerase II Inhibitors

Abstract

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.

FGF/FGFR-Dependent Molecular Mechanisms Underlying Anti-Cancer Drug Resistance

“…The administration of BGJ398 reduced cell viability and enhanced apoptosis in GIST (gastrointestinal stromal tumor) T-1 cell line resistant to imatinib, but not in parental cells [ 46 ]. In GIST cells resistant to doxorubicin, the administration of BGJ398 resulted in a delay in DNA repair [ 212 ]. BGJ398 was also found to reduce cell viability, induce apoptosis, and increase the cytotoxicity of 5-fluorouracil or oxaliplatin in CRC cells [ 52 ].…”

Angiogenesis in gastrointestinal stromal tumors: From bench to bedside

“…The FGF2/R2 signaling has been extensively studied in GIST as a drug resistance mechanism. Sergei et al [ 54 ] and Boichuk et al [ 55 ] demonstrated that the blockage of FGFR2 signaling could enhance the responsiveness to DNA-Topoisomerase II inhibitors[ 54 ] while the downregulation of FGF2 signaling might stimulate the response to imatinib[ 55 ]. It’s contribution in GIST progression has been reviewed[ 56 ] but data about potential effects in GIST vascularization process are missing.…”

Inhibition of AKT-Signaling Sensitizes Soft Tissue Sarcomas (STS) and Gastrointestinal Stromal Tumors (GIST) to Doxorubicin via Targeting of Homology-Mediated DNA Repair

“…Despite the sensitivity of STS and (in less extent) GIST to DNA-damaging agents was shown in multiple reports [20][21][22][23], however, to date, a little is known about the role of AKT in DNA DSB repair (in particular HR) in STS and GIST. Given that inhibition of FGF-signaling in GIST effectively sensitized them to Topo II inhibitors via attenuating HR-mediated DNA DSB repair [35], we sought to examine the downstream signaling pathways responsible for this phenomenon. We present here the novel data illustrating that AKT-but not a MEK-signaling pathway regulates an efficiency of homology-mediated DNA damage repair in STS and GIST.…”