S6K基因通过mTOR通路介导乳腺癌CDK4/6抑制剂耐药的机制研究

Estrogen receptor positive breast cancer represents the largest subtype of the disease, accounting for 60 to 65% of all malignant neoplasms of the breast. Cyclin-dependent kinases 4/6 (CDK4/6) inhibitors combined with endocrine therapy are the most effective therapeutic method in these patients. However, resistance to CDK4/6 inhibitors frequently develops and affects the prognosis of patients seriously. The molecular mechanism of CDK4/6 inhibitor resistance remains largely unknown, thus lacking prediction and basis of treatment. Here we show that based on our previous work on circulation tumor DNA, amplification of S6K gene is associated with resistance to CDK4/6 inhibitors in patients with breast cancer. Patient-derived breast cancer organoids were successfully established, and a representative organoid with S6K gene amplification confirmed resistance to CDK4/6 inhibitor. We have further investigated potential mechanisms of resistance using cell line models and bioinformatics analysis, showing that higher expression of S6K could activate the mTOR pathway and elevated levels of cyclin D1, which lead to CDK4/6 inhibitors resistance. Here we propose for the first time that S6K gene is the key molecule in the mechanism of resistance to CDK4/6 inhibitor. We plan to utilize plasmid transfection, RNA interference and CRISPR technique to construct cells with elevated or low expression of S6K gene. We would explore the molecular mechanism of S6K gene in the resistance of CDK4/6 inhibitor on levels of cell, mice and organoid, in order to provide theoretical basis of resistance prediction and target exploration.

雌激素受体阳性乳腺癌占全部乳腺癌的60-65%，CDK4/6抑制剂联合抗雌激素治疗是这类患者最重要的治疗方案。然而，CDK4/6抑制剂的耐药问题极大地影响了患者的预后，其耐药机制尚不清楚，因而缺乏有效的预测手段和治疗依据。申请人基于前期在乳腺癌患者外周血ctDNA筛查中发现的S6K基因扩增与CDK4/6抑制剂耐药相关的独特现象，通过人源乳腺癌类器官模型及细胞实验证实S6K基因扩增可以激活mTOR通路、上调cyclinD1水平，导致CDK4/6抑制剂耐药，进而提出“S6K基因可能通过促进乳腺癌细胞G1/S周期转换介导CDK4/6抑制剂耐药”的科学假说。拟通过质粒转染、RNA干扰和CRISPR技术构建S6K基因异常表达的乳腺癌细胞，从细胞、动物和类器官模型三个水平探索S6K基因扩增介导乳腺癌CDK4/6抑制剂耐药的分子机制，为预测治疗疗效和探寻新的治疗靶点提供理论依据。

自2015年至今，CDK4/6抑制剂已经在临床上，成为晚期激素受体阳性乳腺癌患者的标准的一线治疗。然而，大约20%-30%的患者存在原发性耐药，从治疗一开始就无法从CDK4/6抑制剂治疗中获益。目前临床上还未找到任何明确的生物标记物，可以预测CDK4/6抑制剂的耐药或提示疗效。近年来，液体活检，尤其是ctDNA技术被广泛应用于临床肿瘤患者的耐药性分析。为了寻找CDK4/6抑制剂耐药相关的基因，我们在乳腺癌患者外周血ctDNA中，利用二代测序技术，检测了基于中国乳腺癌患者群体的与肿瘤发生发展相关的1021个基因的突变情况谱。发现RPS6KB1基因，也就是S6K基因扩增，与激素受体阳性乳腺癌患者CDK4/6抑制剂的原发耐药密切相关。在此基础上，我们在另一个超过一百例患者的独立队列中，利用免疫组化的方法检测患者肿瘤组织中S6K的表达情况，发现S6K基因高表达患者，在接受CDK4/6抑制剂治疗后，无进展生存期显著缩短。因此，S6K有望用于预测CDK4/6抑制剂的原发耐药，该成果已获得国家发明专利授权1项。同时，本项目开展了一系列细胞和分子生物学实验，探索S6K基因扩增介导CDK4/6抑制剂原发耐药的分子机制。之后在患者来源的类器官模型，以及移植瘤小鼠的动物模型中，寻找克服耐药的策略。我们发现S6K扩增可能是通过增加cMyc的翻译，提高myc蛋白表达水平，从而促进cyclinE的转录，使得G1/S周期转换的旁路被激活，导致了CDK4/6抑制剂的原发耐药。因此，对于存在S6K扩增的乳腺癌患者，可以考虑直接加用mTOR抑制剂，从而进一步提高抗肿瘤活性。上述研究成果已在国内外高水平期刊发表SCI论文2篇，阐明了ER+乳腺癌细胞对CDK4/6 抑制剂原发耐药的机制，拓展乳腺癌耐药机制的知识领域；并为S6K 作为疗效预测标志物在临床广泛应用、筛选可能从治疗中获益的患者奠定基础，为其作为新的治疗靶点提供理论依据。