靶向β-catenin/Lef-1相互作用小分子抑制剂的发现及其抑制非小细胞肺癌转移的分子机制研究

Lung cancer is the leading cause of cancer death in China due to its aggressive clinical pathogenesis. Non-small-cell lung cancer (NSCLC), a heterogeneous class of tumors, represents approximately 85% of all new lung cancer cases. Because of a rapid distant metastasis to multiple organs within months of diagnosis, the average of 5-year survival rate for NSCLC is only 16%. Because of the shortage of effective anti-metastatic agents in NSCLC clinical practice, relapse and metastasis could greatly account for these therapeutic failures. Hyperactivation of the canonical Wnt/β-catenin/Lef-1 pathway has been to be essential for metastasis to brain and bone during NSCLC progression. Importantly, fucosytransferase 8 (FUT8) is greatly up-regulated during epithelial-mesenchymal transition (EMT), a critical process for malignant transformation of tumor, via trans-activation of β-catenin/Lef1 signaling. Moreover, this signaling pathway contributes to promote NSCLC recurrence and resistance by maintaining cancer stem cells (CSC) dormancy. Therefore, suppressing β-catenin/Lef1 signaling pathway is an important anti-metastatic strategy for NSCLC therapeutics. In this study, we will use FITC-Lef1 as β-catenin aptamer to first develop a fluorescence polarization-based high throughput screening (HTS) method to identify novel antagonists specially targeting β-catenin/Lef1 interaction. Furthermore, we will further elucidate the molecular basis for the anti-metastatic effect using a cell-based assay and xenograft nude mice. Our proposed research is expected to shed light on the development of novel antagonists targeting β-catenin/Lef1 interaction for NSCLC therapeutics by inhibiting metastasis.

我国肺癌的发病率和死亡率高居恶性肿瘤之首，非小细胞肺癌（Non-Small Cell Lung Cancer, NSCLC）约占所有肺癌的85%，其5年生存率仅为16%，复发和转移仍是NSCLC高死亡率的重要原因。Wnt/β-catenin/Lef1信号通路在NSCLC发生与发展中高度活化，通过增强FUT8的表达而促进上皮-间质转化和NSCLC转移；另外，通过维持NSCLC干细胞休眠体的形成而促进肿瘤复发和耐药。因此，β-catenin/Lef1相互作用可能成为NSCLC分子靶向治疗的新靶点。本研究拟以β-catenin/Lef1相互作用为靶点，以FITC-Lef1作为β-catenin结合配体，建立基于荧光偏振原理的高通量筛选模型理性化发现靶向β-catenin/Lef1相互作用小分子抑制剂，并进一步探索其抑制NSCLC转移的分子机制，为其在NSCLC分子靶向治疗中的应用奠定基础。

在经典Wnt信号通路中，β-catenin/LEF1（lymphoid enhancer factor 1, LEF1）相互作用在非小细胞肺癌（non-small cell lung cancer, NSCLC）的生长分化、化疗耐药、转移复发等过程中发挥着重要的促进作用，已成为新型靶向性抗NSCLC转移药物开发的理想靶标之一。为了高效筛选抑制β-catenin/LEF1相互作用的苗头化合物，本研究旨在应用荧光偏振（fluorescence polarization, FP）原理，成功地建立了FP高通量筛选模型，并筛选到血根碱（sanguinarine）具有良好的抑制活性。MTT实验结果表明，血根碱对A459、H1299等NSCLC细胞具有明显的细胞毒性。TOPFlash实验结果证实，血根碱对转染的HEK293细胞内β-catenin介导的转录活性具有明显的抑制作用。表面等离子共振（surface plasmon resonance, SPR）实验证实，血根碱与β-catenin的解离常数值（dissociation constant, KD）为50.8 µM，说明了β-catenin/LEF1相互作用是血根碱抗癌活性的潜在分子靶标之一。文中以β-catenin/LEF1相互作用为靶标，通过系统的实验优化方案，成功地建立了适用于药物高通量筛选的FP筛选模型，为新型Wnt抑制剂的高效筛选和以血根碱为先导化合物的新型抗癌药物分子设计奠定了坚实基础。

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