BRG1介导化疗期间乳腺癌CTC升高及耐药的机制研究

Several clinical trials have recently demonstrated that increased circulating tumor cell (CTC) number in response to chemotherapy was highly associated with chemoresistance in breast cancer. However, the mechanism underline this phenomena remain unclear. It has been reported that EMT have closed connection with CTC number changes and chemoresistance as well. Our previous works found that inhibition of chromatin remodeling factor BRG1 in breast cancer cell leads to a series of biological changes including down-regulation of Snail (An EMT marker gene), reduced cell migration ability and enhanced cell sensitivity to chemotherapy; furthermore, we also found a protein-protein interaction between Snail and p53. In view of these findings, we aim to uncover the mechanism of BRG1-Snail-p53 axis regulated variation of CTC cell number and chemosensitivity during breast cancer chemotherapy by design overdrive and rescuer experiments targeting each factor in BGR1-Snail-p53 axis. Peptide-magnetic nanoparticles based CTC enrichment technology was used in present study to collect the CTC numbers of patients and to test certain gene expression subsequently. This new method possess similar specificity and better sensitivity in capturing CTC as compared with conventional testing system (CellsearchTM), and thus provide a high quality of exploring platform for our mechanism research; meanwhile, we also applied high throughput simple western charge technology (ProteinSimple NanoProTM system) in this study, thus allowed us to acquire substantial information such as abundance and modification of certain proteins in mircoscale clinical sample. In all, the expected achievement of our study will provide a better understanding and usage of CTC number variation after chemotherapy, and improve refinement of current clinical intervention strategy against metastatic breast cancer.

临床研究证实化疗阶段乳腺癌患者血循环肿瘤细胞（CTC）增多预示耐药，然而其机制尚不明确。既往报道EMT与CTC数量变化及耐药密切相关。本课题组已发现：抑制乳腺癌细胞BRG1表达后EMT标志基因Snail下调、细胞迁移能力减弱、药敏性增强；同时我们还发现Snail与经典耐药基因p53存在互作关系。据此，本项目拟采用最新的多肽-纳米磁珠专利技术获取CTC并检测目的基因表达；并利用乳腺癌细胞系在体、内外条件下对BRG1-Snail-p53通路各环节设计正、反实验，明确该通路调控乳腺癌EMT及耐药；本研究还将采用最新的NanoPro技术通量筛选p53可能的下游靶基因，从而完整阐述BRG1-Snail-p53通路调控乳腺癌患者化疗后CTC数量及药敏性变化的机制；项目中的新技术有效克服了CTC样本稀缺而导致的机制研究壁垒，预期成果可为CTC检测技术的深化发展以及制定更为合理的乳腺癌化疗策略提供理论基础

临床研究证实化疗阶段乳腺癌患者血循环肿瘤细胞（CTC）增多预示耐药，然而其机制尚不明确。既往报道EMT与CTC数量变化及耐药密切相关。课题组前期已通过体外实验证明：抑制乳腺癌细胞BRG1表达后EMT标志基因Snail下调、细胞迁移能力减弱、药敏性增强；同时我们还发现Snail与经典耐药基因p53存在互作关系。进一步的，课题组采用多肽-纳米磁珠技术获取CTC并进行计数，并通过前瞻性研究证实该CTC分选技术在临床中的应用价值，该成果已发表在《The breast》上。在后续研究中、课题组分析CTC中BRG1的表达与患者无病生存期（PFS）未见显著差异，因此从临床水平的证据上，不支持体外实验结果。因此，CTC中BRG1表达水平不能作为预后因子，其原因尚待进一步探讨。但是在临床试验过程中我们发现，乳腺癌外周血淋巴细胞亚群中的自然杀伤（NK）细胞与CTC的联合计数可以有效地预测患者PFS。NK细胞具有不依赖于MHC分子，独立识别肿瘤细胞特性；同时我们发现，NK细胞维持或高于正常水平时，CTC依旧高于阈值的患者PFS较短。我们推测，这类患者由于其CTC能够发生免疫逃逸，因而其恶性程度更高，是导致患者预后差的原因之一。针对此类患者的CTC进行分子特征研究将拓展我们对乳腺癌恶性生物学行为的认知，相关研究工作具有潜在的临床应用价值。该部分成果目前已发表在《Chinese Journal of Cancer Research》上