靶向FoxM1c结合肽P201的优化及抗肝癌分子作用机制

FoxM1 protein plays key roles in the occurrence, development, metastasis and drug resistance of various cancers, it was regarded as the Achilles’heel of cancer and therefore the important target for anticancer drug discovery and the candidate molecular marker for cancers. In our previous work, by prokaryotic expression and purification of the DNA binding domain of human FoxM1c(FoxM1c-DBD) protein and the phage random peptide library selection against it, we had obtained the P201 peptide with independent intellectual property rights which has strong killing effects on cancer cells and is especially selective for liver cancer cells. Here in order to overcome the shortcomings of P201 peptide in its relatively higher concentration in cancer cell inhibition and less knowledge of its molecular mechanisms for cancers, we plan to integrate multiple methods and high-throughput ways to obtain the rationalized P201im with greatly lowed IC50 for cancers. Alanine scanning mutagenesis, biased phage combinatory peptide library construction and selection as well as molecular simulation and virtual screening, peptide modification will all be utilized in the study. More importantly, we will study and disclose the inhibitory molecular mechanism of P201im for especially the liver cancers based on FoxM1 signal transduction passway both in vitro and in vivo. On the one hand, the work would obtain the anticancer lead peptides targeting FoxM1 with creativities, raised efficiencies and especially confirmed mechanisms, and on the other hand, find new and specific peptide markers for the diagnosis and therapy of cancers including liver cancers.

FoxM1在多种癌症的发生发展、转移与耐药中有着重要的作用，被认为是癌症的致命伤，是抗癌药物筛选的重要靶标和候选分子标记。我们实验室前期通过FoxM1c的DNA结合区（FoxM1c-DBD）重组表达和噬菌体随机肽库筛选，获得了具有自主知识产权、强抗癌活性和尤其是肝癌细胞选择性的抗癌多肽P201。本申请书拟针对P201尚存在癌细胞杀伤浓度偏高、分子作用机制欠深入等不足，一方面，通过Ala扫描突变、偏性噬菌体组合肽库的构建与筛选、分子模拟与虚拟筛选、多肽修饰等多尺度、高通量的分析与研究，获得有效降低IC50优化的P201im；更重要地，以肝癌为主要研究对象，体内外研究P201im多肽对肝癌的靶向抑制作用和基于FoxM1信号通路的抗癌分子作用机制。本研究可望获得具有原始创新及提高作用效果与明确分子机制的抗癌多肽先导药物，也可望为肝癌等恶性肿瘤的诊断与治疗提供一种新的分子标记。

项目背景与主要研究内容：FoxM1被认为是癌症的致命伤。本项目是在我们实验室前期通过噬菌体随机肽库筛选获得的靶向FoxM1结合肽P201的基础上，主要开展了Ala扫描突变、噬菌体偏性二级肽库的构建与高通量筛选、筛选多肽的分子模拟与对接、多肽9R-修饰和优化多肽的确定，以及以肝癌HCCLM3细胞为主要研究对象，通过RNA转录组测序、qRT-PCR、WB、IHC等多种方法，体内外研究优化多肽及化药索拉菲尼（Sorafenib）联合用药的靶向抑制作用和基于FoxM1信号通路的抗癌分子作用机制的研究工作。同时，本项目增加开展了靶向FoxM1-DBD蛋白的噬菌体随机环七肽库的高通量筛选、目的多肽的9R-/氟链化学修饰与合成，以及目的多肽的药效和初步作用机制研究。重要结果与关键数据：1）确定了 P201多肽的5个关键氨基酸及位点；2）获得了相比于9R-P201对肝癌HCCLM3细胞更强抑制率的优化多肽9R-P49，以及具有强抗癌活性的9R-CP29L多肽及其氟链修饰化学合成肽8r-cp29C-F13；3）发现和确定9R-P49多肽对癌细胞的抑制效率与FoxM1的蛋白表达呈正相关；4）在HCCLM3移植瘤裸鼠模型中，9R-P49、9R-CP29L均可显著抑制肿瘤的生长，且P49与Sorafenib联合用药具有协同增效/增敏作用；5）确定了9R-P49、9R-CP29L对FoxM1的特异性亲和作用以及其主要通过降低FOXM1及其通路下游相关基因（CDC25B等）的表达而抑制肿瘤的分子作用机制；6）初步研究发现，9R-P49、9R-CP29L对HCCLM3肝癌细胞的杀伤作用也与细胞铁死亡相关，并提出了多肽- FoxM1-ALOXE3-铁死亡-肿瘤抑制的创新假设。科学意义：9R-P49、9R-CP29L所呈现的对HCCLM3等多种癌细胞的强抑制作用，以及这种抑制作用与癌细胞FoxM1表达水平正相关特征的揭示，无论单独还是联合用药，为靶向FoxM1的优化多肽9R-P49及9R-CP29L、8r-cp29C-F13多肽的未来抗癌开发，提供了多种新的先导多肽药物选择和治疗策略。同时，鉴于9R-P49、9R-CP29L处理显著上调ALOXE3，提示细胞铁死亡可能参与其抗癌活性，从而为9R-P49、9R-CP29L多肽的抗癌作用提供了一条新的作用机制。