We showed previously that inhibition of KIT signaling in GISTs activates FGFR-signaling pathway rendering cancer cells resistant to receptor tyrosine kinase inhibitor (RTKi) imatinib mesylate (IM) (Gleevec) despite of absence of secondaryKITmutations and thereby illustrating a rationale for the combined (e.g., KIT- and FGFR-targeted) therapies. We show here that long-term culture of IM-resistant GISTs (GIST-R1) with IM substantially down-regulates KIT expression and induces activation of the FGFR-signaling cascade, evidenced by increased expression of total and phosphorylated forms of FGFR1 and 2, FGF-2, and FRS-2, the well-known adaptor protein of the FGF-signaling cascade. This resulted in activation of both AKT- and MAPK-signaling pathways shown on mRNA and protein levels, and rendered cancer cells highly sensitive to pan-FGFR-inhibitors (BGJ 398, AZD 4547, and TAS-120). Indeed, we observed a significant decrease of IC50 values for BGJ 398 in the GIST subclone (GIST-R2) derived from GIST-R1 cells continuously treated with IM for up to 12 months. An increased sensitivity of GIST-R2 cells to FGFR inhibition was also revealed on the xenograft models, illustrating a substantial (>70%) decrease in tumor size in BGJ 398-treated animals when treated with this pan-FGFR inhibitor. Similarly, an increased intra-tumoral apoptosis as detected by immunohistochemical (IHC)-staining for cleaved caspase-3 on day 5 of the treatment was found. As expected, both BGJ 398 and IM used alone lacked the pro-apoptotic and growth-inhibitory activities on GIST-R1 xenografts, thereby revealing their resistance to these TKis when used alone. Important, the knockdown ofFGFR2, and, in much less content,FGF-2, abrogated BGJ 398′s activity against GIST-R2 cells both in vitro and in vivo, thereby illustrating the FGF-2/FGFR2-signaling axis in IM-resistant GISTs as a primary molecular target for this RTKi. Collectively, our data illustrates that continuous inhibition of KIT signaling in IM-resistant GISTs lacking secondaryKITmutations induced clonal heterogeneity of GISTs and resulted in accumulation of cancer cells with overexpressed FGF-2 and FGFR1/2, thereby leading to activation of FGFR-signaling. This in turn rendered these cells extremely sensitive to the pan-FGFR inhibitors used in combination with IM, or even alone, and suggests a rationale to re-evaluate the effectiveness of FGFR-inhibitors in order to improve the second-line therapeutic strategies for selected subgroups of GIST patients (e.g., IM-resistant GISTs lacking secondaryKITmutations and exhibiting the activation of the FGFR-signaling pathway).
我们先前研究表明，在胃肠道间质瘤（GIST）中抑制KIT信号通路会激活FGFR信号通路，导致癌细胞对受体酪氨酸激酶抑制剂（RTKi）甲磺酸伊马替尼（IM，商品名格列卫）产生耐药性，即使不存在继发性KIT突变。这为联合靶向治疗（如同时靶向KIT和FGFR）提供了理论依据。本研究进一步发现，对IM耐药的GIST细胞（GIST-R1）进行长期IM培养会显著下调KIT表达，并诱导FGFR信号级联的激活，具体表现为FGFR1和FGFR2的总蛋白及磷酸化形式、FGF-2以及FGFR信号级联的关键衔接蛋白FRS-2的表达增加。这导致AKT和MAPK信号通路在mRNA和蛋白水平上均被激活，并使癌细胞对泛FGFR抑制剂（BGJ 398、AZD 4547和TAS-120）高度敏感。事实上，我们观察到，在持续用IM处理长达12个月的GIST-R1细胞衍生的亚克隆（GIST-R2）中，BGJ 398的IC50值显著降低。在异种移植模型中也证实了GIST-R2细胞对FGFR抑制的敏感性增加，表现为使用泛FGFR抑制剂BGJ 398治疗的动物肿瘤体积显著缩小（>70%）。同样，在治疗第5天通过免疫组化（IHC）检测切割的caspase-3，发现肿瘤内凋亡增加。正如预期，单独使用BGJ 398或IM对GIST-R1异种移植瘤均缺乏促凋亡和生长抑制活性，从而揭示了这些细胞对单独使用这些TKI的耐药性。重要的是，敲低FGFR2（以及程度较轻的FGF-2）在体外和体内均消除了BGJ 398对GIST-R2细胞的活性，从而表明FGF-2/FGFR2信号轴是IM耐药GIST中该RTKi的主要分子靶点。总之，我们的数据表明，在缺乏继发性KIT突变的IM耐药GIST中持续抑制KIT信号，会诱导GIST的克隆异质性，导致过表达FGF-2和FGFR1/2的癌细胞积累，从而激活FGFR信号通路。这反过来使这些细胞对与IM联合使用甚至单独使用的泛FGFR抑制剂极其敏感，并为重新评估FGFR抑制剂的有效性提供了依据，以改善特定亚组GIST患者（例如，缺乏继发性KIT突变且表现出FGFR信号通路激活的IM耐药GIST）的二线治疗策略。
肿瘤(癌症)患者之家