CRISPR筛选转录超增强子驱动三阴性乳腺癌进展的分子机制

CRISPR Screening of Transcribed Super-Enhancers Identifies Drivers of Triple-Negative Breast Cancer Progression

CANCER RESEARCH

影响因子:16.6

分区:医学1区 Top / 肿瘤学1区

发表日期:2024 Nov 04

作者: Michael W Lewis, Caitlin M King, Kamila Wisniewska, Matthew J Regner, Alisha Coffey, Michael R Kelly, Raul Mendez-Giraldez, Eric S Davis, Douglas H Phanstiel, Hector L Franco

DOI: 10.1158/0008-5472.CAN-23-3995

摘要

三阴性乳腺癌（TNBC）是乳腺癌中治疗最顽固的类型，部分原因在于缺乏靶向治疗。系统分析超越蛋白编码基因的调控元件，有望发现新的治疗途径。为此，我们分析了TNBC特异性转录增强子的调控机制及其非编码增强子RNA（eRNA）转录产物。采用高通量CRISPR干扰结合RNA测序的方法系统检测了30个最高产eRNA的超增强子的功能，从而无偏向地识别了全基因组的靶基因。高分辨率Hi-C染色质相互作用图谱的生成，帮助注释了每个超增强子的直接靶基因，显示其偏好与预后差的TNBC患者相关的基因相互作用。利用该数据集，敲除调控邻近PODXL基因的超增强子或特异性降解其eRNA，明显抑制靶基因表达、细胞增殖和迁移。此外，失去该超增强子在TNBC小鼠异种移植模型中抑制肿瘤生长和转移。单细胞RNA测序和高通量转座酶-开放染色质分析显示，该超增强子在TNBC肿瘤样本的恶性细胞中活性增强，明显高于非恶性细胞类型。总体而言，本研究探讨了eRNA产生的超增强子编码的调控信息如何驱动基因表达网络，进而影响TNBC的生物学特性。意义：对eRNA产生的超增强子进行整合分析，揭示控制基因表达全局模式的分子机制，为乳腺癌的生物标志物和治疗靶点开发提供新思路。

Abstract

Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer, which is due in part to the paucity of targeted therapies. A systematic analysis of regulatory elements that extend beyond protein-coding genes could uncover avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their noncoding enhancer RNA (eRNA) transcripts. The functions of the top 30 eRNA-producing super-enhancers were systematically probed using high-throughput CRISPR-interference assays coupled to RNA sequencing that enabled unbiased detection of target genes genome-wide. Generation of high-resolution Hi-C chromatin interaction maps enabled annotation of the direct target genes for each super-enhancer, which highlighted their proclivity for genes that portend worse clinical outcomes in patients with TNBC. Illustrating the utility of this dataset, deletion of an identified super-enhancer controlling the nearby PODXL gene or specific degradation of its eRNAs led to profound inhibitory effects on target gene expression, cell proliferation, and migration. Furthermore, loss of this super-enhancer suppressed tumor growth and metastasis in TNBC mouse xenograft models. Single-cell RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses demonstrated the enhanced activity of this super-enhancer within the malignant cells of TNBC tumor specimens compared with nonmalignant cell types. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of TNBC. Significance: Integrative analysis of eRNA-producing super-enhancers defines molecular mechanisms controlling global patterns of gene expression that regulate clinical outcomes in breast cancer, highlighting the potential of enhancers as biomarkers and therapeutic targets.