背景:关于介导干性的机制的最新见解,异质性,肺癌的转移潜力尚未完全转化为治疗这些恶性肿瘤的有效方案。这项研究试图确定肺癌治疗的新靶点。
方法:将14个SCLC和10个NSCLC细胞系的转录组和DNA甲基化组和来自SAEC的正常人小气道上皮细胞(SAEC)和诱导多能干细胞(iPSC)克隆进行比较。SCLC线路,肺iPSC(Lu-iPSC),和SAEC进一步通过DNaseI超敏反应(DHS-seq)进行评估。使用基因组足迹的双变量分析定量染色质可及性和转录因子(TF)足迹深度的变化。使用标准技术来检查核因子1C(NFIC)敲低后SCLC细胞的生长和致瘤性以及转录组和葡萄糖代谢的变化,并检查暴露于BET抑制剂后SCLC细胞中的NFIC表达。
结果:在Lu-iPSC和SCLC之间观察到转录组和DNA甲基化组的显著共性;然而,这项分析没有提供关于肺癌特有通路的信息.将DNase-seq的结果与RNA-seq的链接使得能够鉴定先前与SCLC无关的网络。当与足迹深度结合时,NFIC,以前与SCLC无关的转录因子,在开放染色质位点的占有率最高。NFIC敲低受损的葡萄糖代谢,减少干性,并在体外和体内抑制SCLC细胞的生长。ChIP-seq分析鉴定了NFIC启动子区域中包含溴结构域的蛋白4(BRD4)占据的许多位点。在SCLC细胞和SCLCPDX模型中,BRD4的敲低或用Bromodomain和末端外结构域(BET)抑制剂(BETi)处理显著降低NFIC表达。在SCLC中,约有8%的被BETi处理下调的基因被NFIC敲低抑制,虽然在NFIC敲低后被抑制的基因中,34%在BETi处理后的SCLC细胞中也被下调。
结论:NFIC是SCLC中BET家族蛋白转录调控的关键TF和可能介导因子。我们的发现强调了全基因组染色质可及性分析在阐明肺癌发生机制和确定肺癌治疗新靶点方面的潜力。
BACKGROUND: Recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to identify novel targets for lung cancer therapy.
METHODS: Transcriptomes and DNA methylomes of 14 SCLC and 10 NSCLC lines were compared with normal human small airway epithelial cells (SAECs) and induced pluripotent stem cell (iPSC) clones derived from SAEC. SCLC lines, lung iPSC (Lu-iPSC), and SAEC were further evaluated by DNase I hypersensitive site sequencing (DHS-seq). Changes in chromatin accessibility and depths of transcription factor (TF) footprints were quantified using Bivariate analysis of Genomic Footprint. Standard techniques were used to evaluate growth, tumorigenicity, and changes in transcriptomes and glucose metabolism of SCLC cells after NFIC knockdown and to evaluate NFIC expression in SCLC cells after exposure to BET inhibitors.
RESULTS: Considerable commonality of transcriptomes and DNA methylomes was observed between Lu-iPSC and SCLC; however, this analysis was uninformative regarding pathways unique to lung cancer. Linking results of DHS-seq to RNA sequencing enabled identification of networks not previously associated with SCLC. When combined with footprint depth, NFIC, a transcription factor not previously associated with SCLC, had the highest score of occupancy at open chromatin sites. Knockdown of NFIC impaired glucose metabolism, decreased stemness, and inhibited growth of SCLC cells in vitro and in vivo. ChIP-seq analysis identified numerous sites occupied by BRD4 in the NFIC promoter region. Knockdown of BRD4 or treatment with Bromodomain and extra-terminal domain (BET) inhibitors (BETis) markedly reduced NFIC expression in SCLC cells and SCLC PDX models. Approximately 8% of genes down-regulated by BETi treatment were repressed by NFIC knockdown in SCLC, whereas 34% of genes repressed after NFIC knockdown were also down-regulated in SCLC cells after BETi treatment.
CONCLUSIONS: NFIC is a key TF and possible mediator of transcriptional regulation by BET family proteins in SCLC. Our findings highlight the potential of genome-wide chromatin accessibility analysis for elucidating mechanisms of pulmonary carcinogenesis and identifying novel targets for lung cancer therapy.