厄洛替尼与Foretinib双靶向调控EGFR/MET信号通路治疗TNBC及建立相应基于机制模型的研究

Triple negative breast cancer (TNBC) is a biologically aggressive tumor with a strong association with distant recurrence and poor prognosis. Studies have suggested Epidermal Growth Factor Receptor (EGFR) is an important target in treatment of TNBC, but the development of resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) is a major challenge in their clinical usage. Mesenchymal-epithelial transition (MET) is also a potential therapeutic target for TNBC, which plays a role in the occurrence of EGFR-TKI resistance. Targeting EGFR and MET for the therapy of TNBC and establishing the corresponding mechanism-based Pharmacokinetic/Pharmacodynamic (PK/PD) model have not yet been investigated in previous studies. In this study, we will focus on 1) in vitro studies with seven different erlotinib combined with foretinib schedules. Cell growth inhibition, protein and mRNA modulation in relative signaling pathway, as well as cell cycle distribution will be analyzed to evaluate the pharmacological effects of different schedules, and combination index will be calculated to assess the interaction between two drugs. 2) a preclinical in vivo study to compare the tumor inhibition, the effects on tumor markers and EGFR-TKI resistance between erlotinib monotherapy, foretinib monotherapy, erlotinib and foretinib used simultaneously, erlotinib and foretinib used with interval in MDA-MB-231 cells xenograft tumor model in female BALB/c nude mice. 3) an indirect response model (IDR model) to describe EGFR level in tumor and a non-mechanism based model to describe EGFR-TKI resistance had been established in the preliminary study. Based on the established models, a mechanism-based PK/PD model will be developed to quantitatively describe the relationships between plasma concentration of erlotinib and/or foretinib, EGFR, MET and tumor inhibition. This mechanism-based PK/PD modeling we proposed can explicitly distinguish between drug-specific and biological system-specific parameters. Drug-specific parameters describe the interaction between the drug and the biological system in terms of target affinity and target activation, whereas system-specific parameters describe the functioning of tumor growth and the occurrence of resistance. The mechanism-based PK/PD model together with TNBC patient-level system-specific parameters can be used to simulate and predict the schedule-dependent interaction between erlotinib and foretinib in clinic. It is our expectation that the results from conducting the proposed research projects could contribute greatly to 1) providing scientific basis and predictable PK/PD model for targeting EGFR/MET for TNBC treatment and EGFR-TKI resistance inhibition; 2) the discovery of new drug candidates of TNBC and 3) optimizing the sequencing of erlotinib and foretinib combination therapy for clinical use.

三阴性乳腺癌（TNBC）是一类复发率高、预后差的乳腺癌。EGFR是TNBC治疗的一个重要位点，但其抑制剂TKI易产生耐药现象；而MET作为TNBC另一潜在治疗靶点，与EGFR-TKI耐药密切相关。目前，双靶向调控EGFR/MET信号通路治疗TNBC并抑制EGFR-TKI耐药发生，以及建立相应基于机制PK/PD模型的研究尚属空白。本项目将在体外考察厄洛替尼和Foretinib单独及联合给药对MED-MB-231乳腺癌细胞增殖的抑制作用，对EGFR、MET两条信号通路与细胞周期的调节作用，以及对于EGFR-TKI耐药性产生的影响。在荷瘤鼠模型中通过前期开发的检测方法评价两药联用的药动学性质，并考察其对肿瘤生长、肿瘤标志物及耐药性的影响。申请者已建立基于EGFR的链式模型与非机制耐药模型，据此发展基于机制的模型用以定量描述和预测两药联用对肿瘤、EGFR/MET通路与耐药性的影响，并优化治疗方案。

三阴性乳腺癌（TNBC）是一类复发率高、预后差的乳腺癌。MET作为TNBC重要的治疗靶点，与EGFR-TKI耐药密切相关。因此，双靶向调控EGFR/MET信号通路及建立相应的PK/PD模型对于治疗TNBC及相应抗肿瘤药物的研发具有重要意义。本项目拟考察福瑞替尼及厄洛替尼在体内外单独及联合给药对MDA-MB-231细胞增殖的抑制作用和对相应肿瘤标志物的影响，及其在荷瘤鼠体内的药动学特征并建立相应的模型。在本研究中，建立了福瑞替尼及厄洛替尼在小鼠血浆中的质谱分析方法。同时，采用SRB法考察了单独及联合给药下对MDA-MB-231细胞体外增殖的抑制作用。结果显示，两药联合用药的协同指数并未达到协同效应的标准，而单独给予福瑞替尼对于MDA-MB-231细胞即可达到明显的抑制作用，其最大抑制率为91.3%，IC50为21.83 μM。随后，采用流式细胞法考察了福瑞替尼对细胞凋亡和细胞周期的影响，结果显示福瑞替尼能够显著诱导MDA-MB-231细胞凋亡，最大凋亡率为14.6±2.70%。此外，能使得肿瘤细胞分裂停滞于G2/M期，从而抑制其转移。建立了荷瘤鼠模型，考察了福瑞替尼的体内抗肿瘤药效。通过Western印迹法检测其在细胞及肿瘤组织中对于p-MET及HGF的蛋白表达的影响，并采用免疫组化染色法加以验证。通过Real-time PCR测定福瑞替尼在细胞及肿瘤组织中对于p-MET及HGF的RNA表达的影响。实验结果表明，福瑞替尼能够显著抑制体内肿瘤的生长，且药效与剂量呈正相关。给药组荷瘤鼠的体重并未显著下降，显示其具有良好的安全性。对于HGF/MET信号通路的抑制作用可能对于福瑞替尼抗三阴性乳腺癌发挥着重要的作用，而其对于肿瘤耐药的影响还需要进一步深入研究。此外，还建立了福瑞替尼的PK模型。本研究对于全面了解靶向于HGF/MET信号通路治疗三阴性乳腺癌及相关药物的开发具有一定意义。