The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Bioinformatics and Computational Biology Graduate Program, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Cancer Res. 2024 Nov 4;84(21):3684-3700. doi: 10.1158/0008-5472.CAN-23-3995.
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.
三阴性乳腺癌（TNBC）是治疗上最顽固的乳腺癌形式，部分原因是缺乏靶向治疗。对超出蛋白质编码基因的调控元件的系统分析可以为治疗干预提供途径。为此，我们分析了 TNBC 特异性转录增强子及其非编码增强子 RNA（eRNA）转录本的调控机制。使用高通量 CRISPR 干扰测定与 RNA 测序相结合的系统方法，对前 30 个产生 eRNA 的超级增强子的功能进行了系统探测，从而能够在全基因组范围内无偏地检测靶基因。生成高分辨率的 Hi-C 染色质相互作用图谱，使每个超级增强子的直接靶基因得以注释，突出了它们对预示 TNBC 患者临床结局较差的基因的偏好性。说明了这个数据集的实用性，删除控制附近 PODXL 基因的识别超级增强子或其 eRNAs 的特异性降解导致对靶基因表达、细胞增殖和迁移的显著抑制作用。此外，该超级增强子的缺失抑制了 TNBC 小鼠异种移植模型中的肿瘤生长和转移。单细胞 RNA 测序和高通量测序分析的转座酶可及染色质分析表明，与非恶性细胞类型相比，该超级增强子在 TNBC 肿瘤标本的恶性细胞中具有增强的活性。总之，这项工作研究了几个关于调控信息如何编码到产生 eRNA 的超级增强子中，驱动基因表达网络的基本问题，这些网络是 TNBC 生物学的基础。意义：产生 eRNA 的超级增强子的综合分析定义了控制基因表达的全局模式的分子机制，这些模式调节乳腺癌的临床结局，突出了增强子作为生物标志物和治疗靶点的潜力。
