结直肠癌TF/FVIIa-EGFR正反馈调控环路的分子机制及靶向治疗价值

Our previous studies have demonstrated that TF expression in CRC was significantly correlated with hepatic metastasis,TNM staging and prognosis in clinical samples. TF can promote invasion and inhibit apoptosis, and this was at least partly mediated by the activation of TF/c-Fos/MMPs and TF/unfolded protein response/PERK/eIF2α/ATF4 signaling. On the basis of TF/FVIIa up-regulating EGFR ligands mRNA and bypass activating EGFR PY845 phosphorylation site in colorectal carcinoma cancer cells, We speculate there are a positive feedback regulation loop between TF/FVIIa and EGFR. In this study, we will further investigate how TF up-regulate EREG, AREG target protein expression, promoter activity and key transcription factor. We will investigate the significance and influence of EGFR signaling pathway activation on the tissue factor pathway protein expression such as TF, PAR-2, MMPs, PARP and CHOP. We will also investigate the influence of TF-targeted inhibition combined with EGFR signal inhibition in colon cancer cells growth, invasion, apoptosis, and liver metastasis. Nude mice orthotopic implantation model and in vivo imaging techniques will be used to observe whether TF/FVIIa inhibitors has synergistic anti-tumor effects with EGFR monoclonal antibody in suppressing CRC progression and metastasis. A clinical prospective study will be done to investigate whether TF can become a molecular marker for colorectal cancer patients with anti-EGFR therapy.

前期研究表明组织因子与结直癌TNM分期、肝转移及预后相关，通过TF/c-Fos/MMPs及TF-UPR-PERK/eIF2α/ATF4信号轴促进侵袭及凋亡抑制。在发现TF/FVIIa上调大肠癌EGFR配体mRNA及旁路活化EGFR PY845磷酸化位点的基础上，我们推测存在TF/FVIIa-EGFR正反馈调控环路。本课题将进一步研究TF上调EREG、AREG等目的蛋白的表达、启动子活性及关键转录因子；研究EGFR信号通路激活对组织因子通路相关蛋白TF、PAR-2、MMPs、PARP、CHOP等表达的影响及意义；研究联合靶向抑制TF及EGFR信号通路对大肠癌细胞系生长、侵袭、凋亡及肝转移的影响，通过裸鼠原位种植模型及活体成像技术，动态观察联合TF/FVIIa抑制剂及EGFR单抗对转移性大肠癌的治疗效果及有无增敏作用；前瞻性研究观察TF等能否作为结直肠癌患者抗EGFR治疗敏感性的分子标志物。

在前期关于结直肠癌TF/FVIIa通路研究的基础上，通过基因芯片杂交及生物信息学技术、Real-Time PCR、RNAi、Western Blot等实验方法，结果显示层粘连蛋白-整合素系统、糖代谢及肿瘤相关信号通路网络与结肠癌细胞TF/FVIIa通路活化相关。研究证实了结肠癌细胞中存在TF/FVIIa-EGFR双通路正反馈调控机制：结肠癌细胞TF/FVIIa通路活化后可上调EGFR配体AREG、EREG基因、蛋白表达，可旁路磷酸化EGFR的Y845位点；EGFR通路活化后可上调TF/FVIIa通路中的TF、FVII、PAR2基因表达。AREG过表达在结肠癌TF/FVIIa-EGFR双通路交互作用中具有普遍意义。此外证实了TF/FVIIa-EGFR双通路正反馈调控机制依赖于KRAS基因突变而产生，由此推测，KRAS突变的癌细胞EGFR仍可在胞外配体刺激下与TF/FVIIa通路间发生交互作用，促进TF/FVIIa通路下游效应分子表达，诱导肿瘤恶性进展，即对于KRAS突变型的患者，EGFR靶向治疗仍有治疗作用，而其明显的抑瘤效果可能在TF-EGFR联合靶向治疗后才能凸现出来，这将扭转当前临床上认为“靶向治疗对KRAS基因突变型晚期大肠癌患者无效”的观念，并为研发TF-EGFR联合靶向药物提供强有力的理论依据。