CRISPR筛选转录的超级增强剂确定了三阴性乳腺癌进展的驱动因素
CRISPR Screening of Transcribed Super-Enhancers Identifies Drivers of Triple-Negative Breast Cancer Progression
影响因子:16.60000
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
摘要
三阴性乳腺癌（TNBC）是乳腺癌治疗最常的顽固性形式，这部分是由于靶向疗法的缺乏。对超出蛋白质编码基因的调节元件的系统分析可能会发现治疗干预的途径。为此，我们分析了TNBC特异性转录增强剂的调节机制以及其非编码增强子RNA（ERNA）转录本。使用高通量的CRISPR-ISPRPRINEPREDS ASS与RNA测序结合的高通量CRISPR-INTERPRANION ASS系统探测了前30个ERNA产生的超增强剂的功能，从而使靶基因全基因组无偏见。每种超级增强剂的直接靶基因的高分辨率HI-C染色质相互作用图的产生，这突出了它们对TNBC患者中临床结果更差的基因的倾向。说明了该数据集的效用，删除了控制附近PODXL基因的超级增强剂或其ERNAS的特定降解，从而对靶基因表达，细胞增殖和迁移产生了深远的抑制作用。此外，这种超级增强剂的丧失抑制了TNBC小鼠异种移植模型中的肿瘤生长和转移。单细胞RNA测序和针对转座酶可访问的染色质与高通量测序分析的分析表明，与非绝对细胞类型相比，TNBC肿瘤标本的恶性细胞在TNBC肿瘤标本的恶性细胞中的活性增强。总的来说，这项工作研究了有关如何编码在Erna产生的超级增强剂中的几个基本问题，该信息驱动了基因表达网络，这些网络是TNBC生物学的基础。意义：对ERNA产生的超级增强剂的综合分析定义了控制基因表达的全球模式的分子机制，这些模式调节了乳腺癌中临床结局的临床结局，从而突出了增强子作为生物标志物和治疗靶标的潜力。
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.