Epigenetic Mechanisms of Gene Expression in Thoracic Malignancies

胸部恶性肿瘤基因表达的表观遗传机制

• 批准号: 10014536
• 负责人: DAVID SCHRUMP
• 金额: $ 65.06万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014536
• 关键词: Adolescent and Young Adult; Aerodigestive Tract; Affect; Asbestos; Biological; Cell Line; Cells; Chemoprevention; Cigarette; Cigarette Smoker; Clinical; DNA Methylation; Databases; Development; Dose; Environmental Carcinogens; Epidemiology; Epidermal Growth Factor Receptor; Epigenetic Process; Epiregulin; Epithelial Cells; Evaluation; Event; Gene Expression; Growth; Health Hazards; Histology; Human; Immunohistochemistry; In Vitro; Individual; Lung; Malignant Neoplasms; Malignant Pleural Mesothelioma; Malignant neoplasm of esophagus; Malignant neoplasm of lung; Malignant neoplasm of thorax; Manuscripts; Mediating; Mesothelial Cell; Mesothelioma; Neoplasms; Non-Small-Cell Lung Carcinoma; Normal tissue morphology; Oncogenic; Pathway interactions; Patients; Pharmacology; Phase; Phenocopy; Plants; Pleura; Plicamycin; Prevention; Primary Neoplasm; Proteins; Publications; Publishing; Quantitative Reverse Transcriptase PCR; Regimen; Repression; Ring Finger Domain; Risk Assessment; Series; Signal Transduction; Smoke; Smoker; Sp1 Transcription Factor; Specimen; Stream; Structure of respiratory epithelium; TP53 gene; Tobacco; Transcript; Tumor Suppressor Genes; Ubiquitin; Up-Regulation; Xenograft procedure; base; cancer cell; carcinogenesis; chemotherapeutic agent; cigarette smoke; cigarette smoking; epigenomics; experimental study; filamin; homeodomain; hookah; in vitro Model; in vivo; inhibitor/antagonist; knock-down; new therapeutic target; novel; novel strategies; overexpression; pre-clinical; promoter; respiratory; tissue culture; transcriptome; transcriptome sequencing; transcriptomics; treatment strategy

Eighty percent of lung and 50-80% of esophageal cancers are directly attributable to cigarette smoking. Nearly 70% of malignant pleural mesotheliomas (MPM) arise in smokers or former smokers. Although numerous epigenetic alterations have been identified in tobacco-associated thoracic malignancies, limited information is available regarding the sequence of epigenomic events by which tobacco components establish and maintain the "DNA methylation paradox" during aerodigestive tract carcinogenesis. Conceivably, identification of epigenomic mechanisms that mediate initiation and progression of thoracic malignancies may hasten the development of novel strategies for treatment and prevention of these neoplasms. The specific aims of this project include: 1. Elucidation of epigenomic alterations mediated by cigarette smoke during aerodigestive tract carcinogenesis. 2. Identification of novel epigenomic targets in thoracic malignancies. Novel in-vitro models and correlative experiments with primary tumor/normal tissue specimens have been utilized to identify epigenomic alterations which contribute to initiation and progression of lung and esophageal cancers and malignant pleural mesotheliomas. A steady stream of manuscripts have been published by our group describing the epigenomic effects of cigarette smoke and other environmental carcinogens in normal aerodigestive tract epithelial cells and thoracic cancer cells in-vitro and in-vivo. Presently, an estimated 10-20% adolescents and young adults in the US smoke hookah, which they perceive to be a safe alternative to cigarettes. As such hookah is an emerging health hazard. Recently, a series of experiments have been conducted to examine the epigenenomic and transcriptomic effects of hookah smoke relative to conventional cigarette smoke in normal and immortalized human respiratory epithelial cells. Briefly, primary normal human small airway epithelial cells (SAEC) from three donors, as well as cdk4/hTERT-immortalized SAEC and human bronchial epithelial cells (HSAEC and HBEC, respectively) were cultured for 5 days (short-term) or 12 months (long term) in normal media with or without cigarette smoke condensate (CSC) or water pipe condensate (WPC). CSC and WPC mediated dose-dependent growth inhibitory effects in cultured respiratory epithelia cells, which coincided with decreased levels of global H4K16Ac and H4K20Me3. CSC and WPC mediated distinct as well as overlapping transcriptome signatures, and pathway modulations that were cell line and dose-dependent. 211 and 329, vs 161 and 261 transcripts were commonly modulated across all five lines by low concentration and high concentration CSC, and low concentration and high concentration WPC, respectively. 61 and 96 transcripts were commonly modulated across all five lines by low concentration CSC and WPC, and high concentration CSC and WPC, respectively. Epiregulin (EREG) encoding a master regulator of EGFR signaling which has been implicated in promotion and progression of lung cancers was up-regulated, whereas Filamin A Interacting Protein 1-Like (FILIP1L) encoding an inhibitor of lung cancer invasion was down-regulated in the five cell lines following CSC and WPC exposures. Induction of EREG and repression of FILIP1L by CSC and WPC coincided with DNA methylation changes within the respective promoters. Given the relatively limited overlap between transcriptome signatures mediated by CSC and WPC in respiratory epithelial cells despite large numbers of cancer-associated transcripts and common pathways affected by these condensates, additional experiments were performed to examine if CSC and WPC preferentially modulated cancer-associated transcriptomes in a histology-specific manner. Briefly, cancer associated transcripts uniquely modulated by CSC or WPC or commonly regulated by CSC and WPC in SAEC or immortalized respiratory epithelial cells were compared to expression levels of these transcripts in 10 NSCLC and 10 SCLC lines relative to SAEC using publicly accessible RNA-seq databases. Notably, transcriptome signatures uniquely associated with CSC or WPC exposures or common to both segregated based on NSCLC vs SCLC histology. Subsets of transcripts that were uniquely up-regulated by either CSC, WPC, or both in SAEC were similarly up-regulated in NSCLC lines but down-regulated in SCLC lines and vice versa, whereas subsets of transcripts that were down-regulated by CSC, WPC or both in SAEC were down-regulated in NSCLC yet up-regulated in SCLC lines and vice versa. A similar phenomenon was observed following analysis of CSC and or WPC transcriptome signatures in HBEC and HSAEC. Further studies are necessary to determine the biologic significance of these provocative findings, and to ascertain if these signatures can prove useful in epidemiologic analyses of lung cancers arising in hookah or cigarette smokers, risk assessment in smokers with no clinical evidence of malignancy, and chemoprevention trials in these individuals. A comprehensive manuscript describing these findings has been submitted for publication. Additional studies have focused on delineation of epigenetic mechanisms contributing to malignant pleural mesotheliomas (MPM), and identification of novel therapeutic targets in these neoplasms. Micro-array, qRT-PCR, immunoblot, and immunohistochemistry experiments demonstrated that expression of ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1)- a master regulator of DNA methylation was significantly higher in MPM lines/specimens relative to normal mesothelial cells/ normal pleura. Tissue culture experiments demonstrated that asbestos induced UHRF1 expression in normal mesothelial cells. Analysis of two independent databases demonstrated that UHRF1 up-regulation correlated inversely with overall survival in MPM patients. Knockdown of UHRF1 reversed global hypomethylation and up-regulated tumor suppressor gene expression, inhibiting proliferation, invasion, and clonogenicity of MPM cells, as well as growth of MPM xenografts. Constitutive over-expression of p53 significantly reduced UHRF1 expression in MPM cells. These effects were phenocopied by exposure of MPM cells/xenografts to the repurposed chemotherapeutic agent, mithramycin, which we have previously shown to deplete the oncogenic transcription factor SP1, and up-regulate and activate p53 in MPM cells. These results provide further preclinical rationale for our recently initiated phase I/II evaluation of mithramycin in patients with thoracic malignancies. Collectively, our findings provide the first evidence that UHRF1 is a novel, druggable epigenetic driver in malignant pleural mesotheliomas, and warrant further studies to optimize pharmacologic regimens that inhibit UHRF1 expression/activity for mesothelioma therapy. A manuscript pertaining to these studies has been submitted for publication.

肺中有80％和50-80％的食管癌直接归因于吸烟。吸烟者或以前的吸烟者出现了近70％的恶性胸膜间皮瘤（MPM）。尽管在与烟草相关的胸部恶性肿瘤中鉴定出了许多表观遗传改变，但是在体验型癌症中，烟草成分建立并维持“ DNA甲基化悖论”的表观基因组事件的序列有限的信息有限。可以想象，介导胸腔恶性肿瘤开始和进展的表观基因组机制可能会加快治疗和预防这些肿瘤的新策略的发展。该项目的具体目的包括：1。阐明在机动道癌发生过程中香烟烟雾介导的表观基因组改变。 2。鉴定胸腔恶性肿瘤中新型表观基因组靶标。已经利用与原发性肿瘤/正常组织样本的新型体外模型和相关实验来鉴定表观基因组学的改变，这有助于肺和食管癌的起始和进展，以及恶性胸膜间心瘤。我们的小组已经发表了稳定的手稿，描述了香烟烟雾和其他环境致癌物在正常的机场消化道上皮细胞和胸腔癌细胞中的表观基因组作用。目前，估计有10-20％的青少年和年轻人在美国烟熏水烟粉，他们认为这是香烟的安全替代品。因此，水烟是一种新兴的健康危害。最近，已经进行了一系列实验，以检查正常和永生的人呼吸性上皮细胞中，水烟烟雾相对于常规香烟烟的表观遗传和转录组作用。简而言之，将三个供体的原发性人类小气道上皮细胞（SAEC）以及CDK4/HTERT-IMMORTALIZED SAEC和人支气管上皮细胞（HSAEC和HBEC分别培养5天（短期）或12个月（长期）或12个月（长期）或无烟浓烟（Concect condect condens condens condens condens condens condence）或12个月（长期）。 CSC和WPC介导的剂量依赖性生长抑制作用在培养的呼吸性上皮细胞中，这与全局H4K16AC和H4K20ME3的水平降低相吻合。 CSC和WPC介导了不同的和重叠的转录组特征，以及依赖细胞系和剂量依赖的途径调制。通常通过低浓度和高浓度CSC以及低浓度和高浓度WPC在所有五个线上调节211和329，VS 161和261转录本。低浓度CSC和WPC和高浓度CSC和WPC通常在所有五个线上调节61和96个转录本。编码EGFR信号的主要调节剂的环保蛋白（EREG）与肺癌的促进和进展有关，而filamin a filamin是编码肺癌抑制剂的1-类蛋白1类（FILIP1L）的相互作用的蛋白质，在CSC和WPC的五个细胞系中下调了肺癌的抑制剂。 CSC和WPC对EREG的诱导和FILIP1L的抑制与相应启动子内的DNA甲基化变化一致。鉴于尽管大量癌症相关的转录本和受这些冷凝物影响的共同途径，但CSC和WPC在呼吸道上皮细胞中介导的转录组特征之间的重叠相对有限，但仍进行了其他实验，以检查CSC和WPC优先调节的癌症调节型在组织学跨越的组织学方式中是否优先调节癌症。简而言之，将通过CSC或WPC唯一调节的癌症相关转录本在SAEC或不朽的呼吸上皮细胞中通常由CSC和WPC调节，与10 NSCLC和10个SCLC线的这些转录物的表达水平相对于SAEC使用公共访问的RNA-SESEQ DATABASE中的10 NSCLC和10个SCLC线的表达水平进行了比较。值得注意的是，转录组特征与CSC或WPC暴露唯一相关，或基于NSCLC与SCLC组织学分离的共有。由CSC，WPC或SAEC唯一上调的转录本的子集类似地在NSCLC线上上调，但在SCLC线中被下调，反之亦然，而在NSCL中，在NSCL和SCLC中被下调的转录本下调，而CSC，WPC或两者都下调了。在HBEC和HSAEC中对CSC和或WPC转录组特征分析后，观察到了类似的现象。对于确定这些挑衅性发现的生物学意义，需要进一步的研究，并确定这些特征是否可以证明对在水烟中或吸烟者中产生的肺癌的流行病学分析有用，在这些人中没有临床证据的吸烟者和这些人的化学措施试验的吸烟者的风险评估。一项描述这些发现的全面手稿已提交出版。其他研究集中在描述有助于恶性胸膜间皮瘤（MPM）的表观遗传机制，并鉴定了这些肿瘤中新型治疗靶标。微阵列，QRT-PCR，免疫印迹和免疫组织化学实验表明，泛素蛋白样在植物同源域和环指域1（UHRF1）的表达 - 与正常梅皮细胞/正常细胞/正常pleura相比，MPM的DNA甲基化的主调节剂明显更高。组织培养实验表明，石棉诱导正常间皮细胞中的UHRF1表达。对两个独立数据库的分析表明，UHRF1上调与MPM患者的总体存活率成反比。 UHRF1的敲低逆转了全局低甲基化和上调肿瘤抑制基因表达，抑制了MPM细胞的增殖，侵袭和克隆性以及MPM异种移植物的生长。 p53的组成性过表达显着降低了MPM细胞中的UHRF1表达。通过将MPM细胞/异种移植物暴露于重新利用的化学治疗剂Mithramycin中，我们以前已证明我们以前证明会耗尽致癌转录因子SP1，并在MPM细胞中上调和激活p53。这些结果为我们最近开始的胸腔恶性肿瘤患者对毛霉素的I/II期评估提供了进一步的临床前原理。总的来说，我们的发现提供了第一个证据，即UHRF1是恶性胸膜间皮瘤中新型的可药物表观遗传驱动因素，并有必要进一步研究以优化抑制间皮瘤治疗UHRF1表达/活性的药理方案。与这些研究有关的手稿已提交出版。