We showed previously that the autocrine activation of the FGFR-mediated pathway in GIST lacking secondaryKITmutations was a result of the inhibition of KIT signaling. We show here that the FGF2/FGFR pathway regulates VEGF-A/VEGFR signaling in IM-resistant GIST cells. Indeed, recombinant FGF2 increased the production of VEGF-A by IM-naive and resistant GIST cells. VEGF-A production was also increased in KIT-inhibited GIST, whereas the neutralization of FGF2 by anti-FGF2 mAb attenuated VEGFR signaling. Of note, BGJ 398, pan FGFR inhibitor, effectively and time-dependently inhibited VEGFR signaling in IM-resistant GIST T-1R cells, thereby revealing the regulatory role of the FGFR pathway in VEGFR signaling for this particular GIST cell line. This also resulted in significant synergy between BGJ 398 and VEGFR inhibitors (i.e., sunitinib and regorafenib) by enhancing their pro-apoptotic and anti-proliferative activities. The high potency of the combined use of VEGFR and FGFR inhibitors in IM-resistant GISTs was revealed by the impressive synergy scores observed for regorafenib or sunitinib and BGJ 398. Moreover, FGFR1/2 and VEGFR1/2 were co-localized in IM-resistant GIST T-1R cells, and the direct interaction between the aforementioned RTKs was confirmed by co-immunoprecipitation. In contrast, IM-resistant GIST 430 cells expressed lower basal levels of FGF2 and VEGF-A. Despite the increased expression VEGFR1 and FGFR1/2 in GIST 430 cells, these RTKs were not co-localized and co-immunoprecipitated. Moreover, no synergy between FGFR and VEGFR inhibitors was observed for the IM-resistant GIST 430 cell line. Collectively, the dual targeting of FGFR and VEGFR pathways in IM-resistant GISTs is not limited to the synergistic anti-angiogenic treatment effects. The dual inhibition of FGFR and VEGFR pathways in IM-resistant GISTs potentiates the proapoptotic and anti-proliferative activities of the corresponding RTKi. Mechanistically, the FGF2-induced activation of the FGFR pathway turns on VEGFR signaling via the overproduction of VEGF-A, induces the interaction between FGFR1/2 and VEGFR1, and thereby renders cancer cells highly sensitive to the dual inhibition of the aforementioned RTKs. Thus, our data uncovers the novel mechanism of the cross-talk between the aforementioned RTKs in IM-resistant GISTs lacking secondaryKITmutations and suggests that the dual blockade of FGFR and VEGFR signaling might be an effective treatment strategy for patients with GIST-acquired IM resistance via KIT-independent mechanisms.
我们先前研究表明，在缺乏继发性KIT突变的胃肠道间质瘤中，FGFR介导通路的自分泌激活是KIT信号传导受抑制的结果。本文进一步揭示，在伊马替尼耐药性GIST细胞中，FGF2/FGFR通路调控VEGF-A/VEGFR信号传导。实验证实，重组FGF2能促进伊马替尼敏感及耐药GIST细胞的VEGF-A生成。KIT抑制状态下GIST细胞的VEGF-A产量同样增加，而抗FGF2单克隆抗体的中和作用可减弱VEGFR信号传导。值得注意的是，泛FGFR抑制剂BGJ398在伊马替尼耐药GIST T-1R细胞中能有效且呈时间依赖性抑制VEGFR信号，这揭示了FGFR通路在该特定GIST细胞系中对VEGFR信号传导的调控作用。BGJ398与VEGFR抑制剂（舒尼替尼和瑞戈非尼）联用可通过增强促凋亡和抗增殖活性产生显著协同效应。瑞戈非尼或舒尼替尼与BGJ398联用获得的优异协同评分，证实了VEGFR与FGFR抑制剂联合应用于伊马替尼耐药GIST的卓越疗效。此外，在伊马替尼耐药GIST T-1R细胞中观察到FGFR1/2与VEGFR1/2共定位，并通过免疫共沉淀验证了这些受体酪氨酸激酶间的直接相互作用。相比之下，伊马替尼耐药GIST 430细胞的基础FGF2和VEGF-A表达水平较低。尽管该细胞系中VEGFR1和FGFR1/2表达上调，但这些受体酪氨酸激酶既未共定位也未发生免疫共沉淀，且FGFR与VEGFR抑制剂未显现协同效应。综上所述，对伊马替尼耐药GIST中FGFR和VEGFR通路的双重靶向作用不仅限于协同抗血管生成疗效。双重抑制能增强相应受体酪氨酸激酶抑制剂的促凋亡和抗增殖活性。机制研究表明，FGF2诱导的FGFR通路激活通过过量产生VEGF-A启动VEGFR信号传导，促使FGFR1/2与VEGFR1相互作用，从而使癌细胞对上述受体酪氨酸激酶的双重抑制高度敏感。因此，本研究揭示了缺乏继发性KIT突变的伊马替尼耐药GIST中上述受体酪氨酸激酶间交互作用的新机制，并表明FGFR与VEGFR信号传导的双重阻断可能成为通过非KIT依赖机制获得伊马替尼耐药的GIST患者的有效治疗策略。
肿瘤(癌症)患者之家