Boichuk Sergei, Bikinieva Firyuza, Dunaev Pavel, Galembikova Aigul, Mikheeva Ekaterina, Valeeva Elena, Mani Shinjit, Khromova Natalia, Kopnin Pavel, Shigapova Leyla, Deviatiiarov Ruslan, Shagimardanova Elena, Ryzhkin Sergey, Sabirov Alexey
Department of Pathology, Kazan State Medical University, Kazan 420012, Russia.
Department of Radiotherapy and Radiology, Faculty of Surgery, Russian Medical Academy of Continuous Professional Education, Moscow 125993, Russia.
Int J Mol Sci. 2025 Aug 22;26(17):8138. doi: 10.3390/ijms26178138.
We previously demonstrated that the activation of FGFR signaling in GIST may be a mechanism of GIST resistance to imatinib mesylate (IM). We show here that IM-resistant GIST cells lacking secondary mutations overexpress claudin-1 on both transcriptional and translational levels. In contrast, a knockdown of or inhibition of its activity by PDS-0330 effectively restored GIST's sensitivity to IM both in vitro and in vivo. This was evidenced by the increased expression of apoptotic markers (e.g., cleaved PARP and caspase-3) and the decreased proliferation rate of IM-resistant GIST T-1R cells treated with a combination of IM and PDS-0330 (or siRNA ). In concordance with these findings, a significant synergy was observed between IM and PDS-0330 in GIST T-1R cells. Importantly, decreased tumor size and weight were observed in IM-resistant GIST xenografts treated with a combination of IM and PDS-0330. Furthermore, the combined treatment of IM-resistant tumors induced an increase in intratumoral apoptosis and other changes, as defined by the histopathologic response rate. Based on the co-immunoprecipitation and immunofluorescence microscopy data, we also demonstrated the strong interaction pattern between CLDN1 and FGFR2. Of note, the inhibition or knockdown of effectively decreased the phosphorylation of FGFR2 and FRS-2, a well-known FGFR adaptor protein, thereby illustrating CLDN1's ability to regulate FGFR-signaling and thereby promote FGFR-mediated survival in KIT-inhibited GIST. Consequently, CLDN1 inhibition in GIST effectively disrupted the FGFR-mediated pathway and re-sensitized tumor cells to IM. In concordance with these data, molecular profiling of CLDN1-inhibited GIST T-1R cells illustrated a significant decrease in the majority of FGFR transcripts, including FGFR2, 3, and 4. Additionally, several FGFR ligands (e.g., FGF14, -19, and -23) were also down-regulated in PDS-0330-treated GIST. Notably, exogenous FGF-2 increased CLDN1 expression in a time-dependent manner. In contrast, pan-FGFR inhibitors effectively reduced CLDN1 levels in IM-resistant GIST T-1R cells, thereby illustrating a cross-talk between CLDN1- and FGFR-mediated pathways in IM-resistant GIST. Based on subcellular fractionation and immunofluorescence microscopy data, we also observed partial relocalization of CLDN1 into the cytoplasm in IM-resistant GIST. Notably, PDS-0330 effectively abrogated this relocalization, suggesting that changes in CLDN1 subcellular distribution might also impact GIST resistance to IM. Lastly, based on our small cohort clinical study (n = 24), we observed the increased expression of CLDN1 in most "high-risk" primary GIST known to be associated with poor prognosis and aggressive behavior, thereby illustrating the prognostic value of increased CLDN1 expression in GIST and providing a further rationale to evaluate the effectiveness of CLDN1 inhibition for GIST therapy.
我们之前证明，胃肠道间质瘤（GIST）中FGFR信号的激活可能是GIST对甲磺酸伊马替尼（IM）耐药的一种机制。我们在此表明，缺乏二次突变的IM耐药GIST细胞在转录和翻译水平上均过表达紧密连接蛋白-1（claudin-1）。相反，通过PDS-0330敲低或抑制其活性可有效恢复GIST在体外和体内对IM的敏感性。用IM和PDS-0330（或小干扰RNA）联合处理的IM耐药GIST T-1R细胞中，凋亡标志物（如裂解的PARP和半胱天冬酶-3）表达增加，增殖率降低，证明了这一点。与这些发现一致，在GIST T-1R细胞中观察到IM和PDS-0330之间有显著的协同作用。重要的是，用IM和PDS-0330联合处理的IM耐药GIST异种移植瘤的肿瘤大小和重量均减小。此外，根据组织病理学反应率定义，IM耐药肿瘤的联合治疗诱导肿瘤内凋亡增加及其他变化。基于免疫共沉淀和免疫荧光显微镜数据，我们还证明了CLDN1与FGFR2之间有强烈的相互作用模式。值得注意的是，对其进行抑制或敲低可有效降低FGFR2和FRS-2（一种著名的FGFR衔接蛋白）的磷酸化，从而说明CLDN1调节FGFR信号传导并由此促进KIT抑制的GIST中FGFR介导的存活的能力。因此，GIST中CLDN1的抑制有效破坏了FGFR介导的途径，并使肿瘤细胞对IM重新敏感。与这些数据一致，对CLDN1抑制的GIST T-1R细胞进行分子分析表明，包括FGFR2、3和4在内的大多数FGFR转录本显著减少。此外，在PDS-0330处理的GIST中，几种FGFR配体（如FGF14、-19和-23）也下调。值得注意的是，外源性FGF-2以时间依赖性方式增加CLDN1表达。相反，泛FGFR抑制剂可有效降低IM耐药GIST T-1R细胞中的CLDN1水平，从而说明IM耐药GIST中CLDN1和FGFR介导的途径之间存在相互作用。基于亚细胞分级分离和免疫荧光显微镜数据，我们还观察到IM耐药GIST中CLDN1部分重新定位于细胞质中。值得注意的是，PDS-0330有效消除了这种重新定位，表明CLDN1亚细胞分布的变化也可能影响GIST对IM的耐药性。最后，基于我们的小型队列临床研究（n = 24），我们观察到在大多数已知与预后不良和侵袭性行为相关的“高危”原发性GIST中CLDN1表达增加，从而说明GIST中CLDN1表达增加的预后价值，并为评估CLDN1抑制对GIST治疗的有效性提供了进一步的理论依据。
