NCI-60肿瘤细胞系转录因子蛋白质组学分析及耐药转录因子ESRRA的机制研究

Transcription factors(TF) play an important role in tumor development and anti-tumor drug therapy. In the previous study, we used a synthetic DNA containing a concatenated tandem array of the consensus TF response elements (catTFRE) as an affinity reagent and combined mass spectrometry-based proteomics to construct a transcription factor proteomic map of NCI-60 cell line. Integrating proteomics data with anti-tumor drug efficacy, we found nuclear receptor ESRRA was associated with platinum drug resistance. In the data set, a total of 849 TFs was identified, with an average of 380 per cell line. Tumor cells still retain their tissue and organ source characteristics at the transcription factor level, which partly lost at transcriptome or proteome level. Knockdown of ESRRA in HT29 cell line significantly enhanced the pharmacodynamic effect of Carboplatin. However, the mechanism by which nuclear receptor ESRRA causes platinum resistance in tumor cells remains unclear. In this study, we intend to use modern molecular biology methods such as mass spectrometry, CRISPR and ChIP-seq to explore the following questions. We aim to identify co-regulatory factors interacting with ESRRA and their downstream target genes, which may explain the mechanism of drug resistance they regulated. We will also demonstrate their clinical significance in colon cancer. This study will provide a basis for understanding the characteristics of different tumor cells at the transcription factor level, find new transcription factors related to the efficacy of anticancer drugs, and guide the combination therapy of tumors.

转录因子在肿瘤发生发展及抗肿瘤药物治疗中具有重要的作用。前期研究中，我们通过串联多拷贝双链DNA结合元件(catTFRE)富集转录因子，联用基于质谱的蛋白质组学技术，构建了NCI-60细胞系转录因子蛋白质组学图谱，并筛选出与铂类药物耐药相关的核受体ESRRA。研究发现：①共鉴定到转录因子849个，平均每种细胞系380个；②肿瘤细胞在转录因子层面仍保留其组织器官来源特征；③在HT29细胞系中，敲低ESRRA能显著提高卡铂药效。但是ESRRA产生耐药性的机制仍不清楚。本研究拟利用质谱分析、CRISPR、ChIP-seq等现代分子生物学方法，旨在：①鉴定与ESRRA作用的共调节因子；②鉴定ESRRA调控的下游靶基因，明确其产生耐药性的机理；③ESRRA在结直肠癌组织中的表达临床意义。本研究将为从转录因子层面认识不同肿瘤细胞特征、寻找与抗癌药物药效相关转录因子和指导肿瘤联合用药提供依据。

乳腺癌是最常见的恶性肿瘤之一，化疗是目前治疗乳腺癌的有效手段，然而日益严重的药物耐药性是恶性肿瘤复发和致死的主要原因。转录因子在肿瘤发生发展及抗肿瘤药物治疗中具有重要的作用。课题组前期通过串联多拷贝双链DNA结合元件富集转录因子，联用基于质谱的蛋白质组学技术，构建了NCI-60细胞系转录因子蛋白质组学图谱，并筛选出与铂类药物耐药相关的核受体ESRRA。. 本项目的研究旨在探讨雌激素相关受体ESRRA促进乳腺癌发生和顺铂化疗耐药的机制。通过对63对带注释的肿瘤临床标本的病例对照队列中ESRRA表达的分析表明，ESRRA在乳腺癌组织中高表达。ESRRA的过表达促进了乳腺癌细胞在体内外的增殖和转移，增加了顺铂的化疗耐药性，增强了肿瘤细胞多能性；而ESRRA敲低导致相反的效果。我们使用 RNA-seq、ChIP-seq和双荧光素酶等实验，表明ESRRA可以通过结合其启动子区域直接诱导CCNE2转录表达，然后增强癌细胞的多能性，并测定促进肿瘤细胞对顺铂的耐药性。CCNE2的过表达可以逆转由ESRRA敲除引起的乳腺癌细胞对顺铂敏感，并促进肿瘤生长加速。总之，我们的研究表明ESRRA在乳腺癌中充当致癌基因并通过调节CCNE2的转录促进顺铂耐药，并可能作为乳腺癌的治疗靶点。