Epigenetic Alterations Induced by Tobacco Smoke

一项一致的努力正在进行中，以建立一个体外系统来检查吸烟对呼吸上皮细胞的顺序表观遗传影响。简单地说，在可能相关的暴露条件下，正常人小气道上皮细胞（SAEC）和cdk4/ htert永生化的人支气管上皮细胞（HBEC）在含或不含香烟烟雾冷凝物（CSC）的正常培养基（NM）中培养长达24个月。Western blot分析显示，CSC介导H4K16Ac和H4K20Me3的剂量依赖性和时间依赖性降低，而H3K27Me3的相对水平升高；这些组蛋白改变与DNMT1表达减少和DNMT3b表达增加相吻合。焦磷酸测序和定量RT-PCR实验揭示了D4Z4、NBL2和LINE-1重复DNA序列的时间依赖性低甲基化；上调H19、IGF2、MAGE-A1和MAGE-A3的表达，激活Wnt信号；肿瘤抑制基因如RASSF1A、RUNX3和rar - β的高甲基化，这些基因在人类肺癌中经常沉默。基于阵列的DNA甲基化分析在csc暴露的HBEC衍生的软琼脂克隆中发现了新的DNA甲基化靶点；在这些细胞中也发现了CSC基因表达特征。CSC诱导的渐进式基因组低甲基化和局部DNA高甲基化与HBEC的软琼脂克隆原性而非致瘤性显著增加相一致。最近在Oncogene上发表了一篇关于该模型（9个月的CSC暴露）的初步分析结果的手稿。目前正在使用与基线相关的样本，以及连续暴露于正常培养基或CSC的6、12、18和24个月的样本，对全球DNA甲基化和基因表达进行全面分析。我们期望找到与CSC暴露时间一致的表观遗传机制沉默或激活的新靶点。利用这种独特的模型系统进行了额外的实验，以确定是否随机随机事件而不是指导机制在肺癌细胞中建立表观遗传特征。简要地说，Illumina甲基化阵列分析显示，与对照HBEC相比，暴露于CSC 9个月的HBEC中有48个基因位点低甲基化，56个基因位点高甲基化。在csc处理的HBEC中，高甲基化而非低甲基化的基因与干细胞Polycomb靶基因（pctg）高度重叠。进一步分析显示，CSC暴露后，HBEC中干细胞pctg超甲基化的可能性是非pctg的7倍。焦磷酸测序分析显示，CSC治疗沉默的多个基因出现了进行性的DNA从头高甲基化。ChIP分析显示，被CSC处理的高甲基化和完全沉默的基因在CSC暴露之前表现出相对高水平的H3K27Me3。相比之下，抗脱氧DNA甲基化的基因在治疗前的H3K27Me3水平非常低或检测不到，而H3K4Me3水平很高。有趣的是，位点特异性DNA甲基化的逐渐增加与几种表观遗传沉默基因的H3K27Me3的逐渐减少相一致。这些新发现表明，脱氧DNA甲基化和基因沉默不是恶性转化过程中随机事件的结果。我们的数据为肺癌发生过程中脱氧DNA甲基化的指导机制提供了第一个直接证据，并强调了我们的模型在肺癌细胞中DNA甲基化悖论的表观遗传机制方面产生新见解的潜力。因为它概括了在已建立的肺癌中观察到的关键表观遗传改变，这个独特的模型可能被证明非常有用，用于评估新的染色质重塑剂，以恶性转化的替代表观遗传标记为终点，用于肺癌预防。一些研究表明，在诊断或治疗期间吸烟状况会影响肺癌患者对治疗的反应和总生存期。因此，已经进行了额外的实验来检查CSC对培养的肺癌细胞的影响。简单地说，用不同浓度和暴露时间的CSC治疗几种肺癌系。在模拟每天1包（ppd）暴露5天的条件下，CSC对体外增殖率没有影响，但显著增强了裸小鼠A549和Calu-6细胞的致瘤性。Affymetrix阵列和定量RT-PCR实验显示，CSC显著抑制Dkk-1的表达，Dkk-1是一种分泌的Wnt信号拮抗剂和推定的肿瘤抑制因子。随后的ChIP、焦磷酸测序和甲基化特异性PCR实验表明，CSC对Dkk-1表达的抑制与H3K27三甲基化的剂量依赖性增加相一致，尽管持续暴露于CSC 5-60天，但Dkk-1启动子内的DNA甲基化没有明显增加。停止CSC暴露导致Dkk-1启动子相关多梳蛋白的减少，并恢复Dkk-1在这些癌细胞中的表达。Western blot和聚焦qRT-PCR阵列实验表明，CSC介导的A549和Calu-6细胞中Wnt信号的剂量依赖性增加。在这些细胞中siRNA介导的Dkk-1敲除后也发现了类似的结果。CSC暴露也抑制了正常呼吸道上皮细胞中Dkk-1的表达。有趣的是，肺癌细胞中CSC暴露或Dkk-1的敲除显著上调了Wnt5a的表达，Wnt5a是一种与癌症干细胞信号传导有关的非规范Wnt配体。Dkk-1的敲除重现了CSC在肺癌细胞中的致瘤作用。这些研究结果发表在《癌症研究》杂志上。进一步的实验专门研究了Wnt5a在肺癌细胞中的作用。简单地说，将内源性Wnt5a水平较低的Calu-6和H841细胞以及内源性Wnt5a水平相对较高的A549细胞用表达Wnt5a、靶向Wnt5a的shRNA或对照序列的慢病毒载体进行转导。与载体对照相比，组成表达Wnt5a的Calu-6和H841-而非A549细胞的增殖、迁移和侵袭均显著增加。这种现象在亲代Calu-6细胞暴露于Wnt5a后也观察到，Wnt5a衍生的六肽Box-5部分消除了这种现象。敲除Wnt5a可抑制A549细胞的增殖、迁移和侵袭。与载体对照相比，组成性表达Wnt5a的Calu-6细胞在裸鼠中的致瘤性显著增强。Western blot、启动子报告子和集中定量RT-PCR阵列实验表明，Wnt5a增加了核孤儿受体Ror2的表达，激活了非典型Wnt信号通路。微阵列分析显示，Wnt5a组成性表达后，肺癌细胞系之间的基因或微rna表达几乎没有重叠，这表明Wnt5a在肺癌细胞中的作用是高度多效的。综上所述，这些数据表明Wnt5a增强了肺癌细胞的恶性表型，并支持开发针对Wnt5a介导的信号通路的肺癌治疗方法。有关这些实验的手稿已提交出版。

A concerted effort has been underway to establish an in-vitro system to examine sequential epigenetic effects of cigarette smoke in respiratory epithelia. Briefly, normal human small airway epithelial cells (SAEC) and cdk4/hTERT-immortalized human bronchial epithelial cells (HBEC) have been cultured in normal media (NM) with or without cigarette smoke condensate (CSC) for up to 24 months under potentially relevant exposure conditions. Western blot analysis demonstrated that CSC mediated dose- and time-dependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNMT1 and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3 as well as activation of Wnt signaling; and, hypermethylation of tumor suppressor genes such as RASSF1A, RUNX3, and RAR-beta which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft agar clonogenicity but not tumorigenicity of HBEC. A manuscript pertaining to results of the initial analysis of the model (9 months of CSC exposure) were published recently in Oncogene. Comprehensive analyses of global DNA methylation and gene expression are underway using samples pertaining to baseline, as well as 6, 12, 18 and 24 months of continuous exposure to normal media or CSC. We expect to identify novel targets that have been silenced or activated by epigenetic mechanisms coinciding with duration of CSC exposure. Additional experiments utilizing this unique model system have been performed to ascertain if random stochastic events rather than instructive mechanisms establish epigenetic signatures in lung cancer cells. Briefly, Illumina methylation array analysis revealed 48 gene loci to be hypomethylated whereas 56 were hypermethylated in HBEC exposed to CSC for nine months relative to control HBEC. Hypermethylated- but not hypomethylated genes in CSC-treated HBEC were highly overlapped with stem cell Polycomb target genes (PCTGs). Further analysis revealed that stem cell PCTGs were seven-fold more likely to be hypermethylated than non-PCTGs in HBEC following CSC exposure. Pyrosequencing analysis of multiple genes silenced by CSC treatment revealed progressive de novo DNA hypermethylation. ChIP analysis revealed that genes, which were hypermethylated and completely silenced by CSC treatment exhibited relatively high levels of H3K27Me3 prior to CSC exposure. In contrast, genes that were resistant to de-novo DNA methylation had very low or undetectable H3K27Me3 and high level H3K4Me3 prior to treatment. Interestingly, progressive increases in site-specific DNA methylation coincided with gradual reductions of H3K27Me3 of several epigenetically-silenced genes. These novel findings suggest that de-novo DNA methylation and gene silencing are not the result of random events during malignant transformation. Our data provide the first direct evidence for an instructive mechanism of de-novo DNA methylation during pulmonary carcinogenesis, and highlight the potential for our model to yield new insights regarding epigenetic mechanisms contributing to the DNA methylation paradox in lung cancer cells. Because it recapitulates critical epigenetic alterations observed in established lung cancers, this unique model may prove quite useful for evaluating novel chromatin remodeling agents for lung cancer prevention using surrogate epigenetic markers of malignant transformation as endpoints. Several studies suggest that smoking status at diagnosis or during treatment effects response to therapy and overall survival of lung cancer patients. As such, additional experiments have been performed to examine the effects of CSC on cultured lung cancer cells. Briefly, several lung cancer lines were treated with CSC using a variety of concentrations and exposure durations. Under conditions mimicking 1 pack per day (ppd) exposures for five days, CSC had no effects on in-vitro proliferation rates, yet dramatically enhanced tumorigenicity of A549 and Calu-6 cells in nude mice. Affymetrix arrays and quantitative RT-PCR experiments revealed that CSC markedly inhibited expression of Dkk-1, a secreted Wnt signaling antagonist and putative tumor suppressor. Subsequent ChIP, pyrosequencing, and methylation specific PCR experiments demonstrated that inhibition of Dkk-1 expression by CSC coincided with a dose-dependent increase in H3K27 trimethylation, and recruitment of polycomb repressor complexes without an appreciable increase in DNA methylation within the Dkk-1 promoter despite continuous CSC exposures ranging from 5-60 days. Cessation of CSC exposure resulted in diminution of Dkk-1 promoter-associated polycomb proteins, and restoration of Dkk-1 expression in these cancer cells. Western blot and focused qRT-PCR array experiments indicated that CSC mediated dose-dependent increases in Wnt signaling in A549 and Calu-6 cells. Similar findings were noted following siRNA mediated knock-down of Dkk-1 in these cells. CSC exposure also inhibited expression of Dkk-1 in normal respiratory epithelia. Interestingly, CSC exposure or knock-down of Dkk-1 in lung cancer cells dramatically up-regulated expression of Wnt5a, a non-canonical Wnt ligand implicated in cancer stem cell signaling. Knock-down of Dkk-1 recapitulated the pro-tumorigenic effects of CSC in lung cancer cells. Results of these studies were published in Cancer Research. Additional experiments have been conducted to specifically examine the effects of Wnt5a in lung cancer cells. Briefly, Calu-6 and H841 cells, which exhibit low endogenous levels of Wnt5a, and A549 cells, which express relatively high endogenous levels of this ligand, were transduced with lentiviral vectors expressing Wnt5a, shRNA targeting Wnt5a, or control sequences. Calu-6, and H841- but not A549 cells constitutively expressing Wnt5a exhibited significantly increased proliferation, migration, and invasion relative to vector controls. This phenomenon, which was also observed following exposure of parental Calu-6 cells to Wnt5a, was partially abrogated by Wnt5a-derived hexapeptide, Box-5. Knock-down of Wnt5a diminished proliferation, migration and invasion of A549 cells. Relative to vector controls, Calu-6 cells constitutively expressing Wnt5a exhibited significantly increased tumorigenicity in nude mice. Western blot, promoter-reporter, and focused quantitative RT-PCR array experiments demonstrated that Wnt5a increased expression of the nuclear orphan receptor, Ror2, and activated non-canonical Wnt signaling. Microarray array analysis revealed surprisingly little over-lap regarding either gene or micro-RNA expression between lung cancer lines following constitutive expression of Wnt5a, suggesting that the effects of Wnt5a in lung cancer cells are highly pleiotropic. Collectively, these data demonstrate that Wnt5a enhances the malignant phenotype of lung cancer cells, and support the development of therapies targeting Wnt5a-mediated signaling for lung cancer therapy. A manuscript pertaining to these experiments has been submitted for publication.