特异靶向FGFR2膜外域的变构抑制肽抗胃癌活性与初步机制研究

Gastric cancer is a highly heterogeneous disease, cytotoxic chemotherapy is still widely used as standard treatment paradigms with few other targeted therapies available. Novel and high efficient targeted therapies for gastric cancer are highly needed. The high incidence of FGFR2 amplification in gastric cancer is closely related to poor prognosis and chemotherapy resistance, which proved to be a potential drug target for gastric cancer. So far, there are no approved inhibitors targeted towards FGFR2 has been found due to insufficient isoform selectivity and toxicity issues. Based on previous work on antagonistic peptides targeted to FGFR, our group has successfully identified a novel FGFR2 antagonistic peptide T15, which shows highly selectivity and excellent stability. We have demonstrated that T15 possesses good biocompatibility and potent anti-gastric cancer activities associated with inhibition of FGFR2 in vitro. These preliminary data indicate that the antagonist peptide T15 displayed some characteristics as allosteric inhibitor, the in-depth mechanism remains unclear and thoroughly study is needed. In the present project, we plan to investigate the gastric cancer targeted mechanism of T15 by gene silencing and site-directed mutagenesis. Firstly, FGFR2-binding specificity of T15 will be confirmed by Surface Plasmon Resonance technique, gene silencing and plasmid transfection. Secondly, the molecular mechanism of allosteric inhibition by T15 is further clarified by computer simulation, site-directed mutagenesis, and co-crystallization experiments. Furthermore, the anti-gastric cancer activity and chemotherapy sensitization effects of T15 will be evaluated on FGFR2-silenced gastric cancer cell lines and nude mice model of gastric cancer xenograft. The present project will provide a molecular probe for studying the oncogenic FGFR2 signaling networks in gastric cancer as well as new views of potential antineoplastic mechanisms.

胃癌异质性高，虽有靶向HER2和VEGFR2的个别药物，但一线药物仍为顺铂等细胞毒药物，亟待新靶向药物。FGFR2在胃癌中扩增发生率高，且与不良预后和化疗耐药密切相关，是胃癌治疗的重要靶点。目前暂无高效低毒的FGFR2抑制剂用于胃癌治疗。在课题组前期拮抗肽研究的基础上，我们发现了靶向选择性好、稳定性高的FGFR2拮抗肽T15，其毒性小且具有较强的体外抑制胃癌细胞生长活性。前期结果表明该拮抗肽具有变构抑制剂的某些特征，但作用机制尚不明确。本项目拟通过定点突变、基因沉默等手段探究T15特异靶向FGFR2的抗胃癌机制。通过SPR、基因沉默、质粒转染等确证T15的结合特异性；通过计算模拟、定点突变、共结晶等阐明T15变构FGFR2的分子机制；通过基因沉默、异种移植瘤、WB等研究T15体内外抗胃癌及增敏胃癌化疗的药理药效。本项目将为FGFR2药理研究提供分子探针，为抗胃癌作用机制研究提供新思路。

胃癌是一种异质性非常高的肿瘤，目前仅有个别抗胃癌的靶向药物，临床一线药物仍为顺铂等细胞毒药物，因此，针对不同分子亚型的胃癌，研发低毒高效的靶向性药物克服胃癌异质性是实现精准治疗的有效手段。异常表达的成纤维细胞生长因子受体（FGFR）是胃癌发生发展与化疗耐药的关键驱动因素，可作为胃癌治疗的潜在靶标。本项目通过SPR和质粒转染技术发现短肽P72（即T15）可与FGFR2膜外域（FGFR2D2D3）相结合。分子对接和分子动力学模拟结果显示复合物FGFR2D2D3-P72体系中以氢键相互作用为主，且P72可通过改变内源性配体结合位点处蛋白质的二级结构影响内源性配体FGF1与FGFR2膜外域的结合，从而起到抑制FGFR2活化的作用。随后，细胞实验发现P72对FGF-2诱导的胃癌细胞生长有明显的抑制作用，且可将细胞阻滞于G0/G1期；通过建立裸小鼠胃癌SGC-7901细胞异位瘤模型，发现P72能有效抑制移植瘤的生长速度，降低肿瘤负荷，且比阳性药具有更低的毒性。进一步，western blot和免疫组化结果显示，P72能显著下调肿瘤组织中p-FGFR、p-FRS2、p-AKT、p-ERK1/2和Ki-67蛋白的表达。以上结果表明P72能有效结合FGFR2，下调FGFR2及其下游通路蛋白FRS2α、AKT、ERK1/2的磷酸化水平，抑制胃癌细胞的增殖，从而发挥良好的抗胃癌作用。此外，本项目以天然产物去甲二氢愈创木酸（NDGA）和其对称性类似物Af23为先导物，在保留骨架结构（双芳基-1,4-二烯-3-酮）和单边的活性功能基团（3,4-二羟基）的基础上，设计、合成了一系列不对称的NDGA类似物（Y1-Y18）。迁移率变动分析实验发现，Y14对FGFR1具有良好的抑制作用。药理实验结果表明Y14可显著抑制FGFR1及其下游激酶AKT和ERK的磷酸化，从而抑制胃癌细胞的生长、存活和迁移。进一步MTT和细胞克隆实验发现，与5-FU单独给药相比，Y14与5-FU联合给药后可显著下调胃癌细胞中FGFR1的磷酸化水平，增强5-FU的抗胃癌活性。以上结果表明Y14可通过抑制FGFR1的磷酸化及其下游信号通路，发挥良好的抗胃癌活性和胃癌化疗增敏效果。该项目研究结果为胃癌的靶向治疗提供了候选药物，为胃癌化疗增敏提供了新途径。.