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

Boichuk Sergei,Bikinieva Firyuza,Dunaev Pavel,Mustafin Ilshat,Shagimardanova Elena,Nurgatina Ilmira,Gorbunova Vera,Andriutsa Natalia,Galembikova Aigul,Kurtasanov Refat,Aukhadieva Aida

doi:10.3390/ijms21010352

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.

Highlights

Receptor tyrosine kinase (RTK) signaling is known to be activated upon DNA damage and in turn, activates DNA damage response (DDR) signaling networks to restore genome integrity and maintain cellular homeostasis
Based on our previous data illustrating that inhibition of Fibroblast growth factors (FGF)-signaling in gastrointestinal stromal tumors (GIST) effectively sensitized them to DNA damaging agents [18], we sought to examine whether it might be due to decreased efficiency of DDR mechanisms involved in repair of the double-strand breaks (DSBs)
To assess whether inhibition of fibroblast growth factor receptor (FGFR)-signaling has an impact on DDR in GISTs, IM-naïve (T-1) vs. resistant (T-1R) cells were treated with Dox (0.5 μg/mL) for 2 h, after which the drug-containing media was removed and cells were further cultured without drug in absence or presence of BGJ398, a selective FGFR1-4 inhibitor, for 48 h to assess an efficiency of DNA repair of Dox-induced DNA damage

Summary

Introduction

Receptor tyrosine kinase (RTK) signaling is known to be activated upon DNA damage and in turn, activates DNA damage response (DDR) signaling networks to restore genome integrity and maintain cellular homeostasis. Epidermal growth factor receptor (EGFR) became phosphorylated at residues Y845 and Y1173 after exposure to ionizing radiation, became internalized into the nuclear compartment (as a component of lipid rafts) to interact and modulate activity of DNA-PK, a key component of non-homologous end-joining (NHEJ) pathway involved in DNA double-strand breaks (DSBs) repair [5]. Besides the EGFR-mediated pathway, insulin-like growth factor 1 receptor (IGF-1R) - signaling was shown to be actively involved in the modulation of multiple DDR pathways, involved in DNA DSBs repair. Activation of IGF-1R signaling was observed in cancer cells exposed to ionizing radiation and depletion of IGF-1R or pharmacological inhibition of IGF-signaling increased cellular radiosensitivity by attenuating DSBs repair by homologous recombination (HR) and NHEJ, as well [4,8,9]. Inhibition/depletion of IGF-1R attenuates the radiation-induced Ku86 binding to DNA, thereby enhancing the radiosensitivity of lung cancer cells due to the interruption of NHEJ-mediated DSBs repair mechanisms [4]

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Conclusion