Epigenetic Alterations Induced by Tobacco Smoke

培养的肺癌细胞用不同浓度和暴露时间的烟草烟雾凝聚物（TSC）处理。在模拟每天1包（ppd）暴露5天的条件下，TSC介导的体外增殖率没有明显变化，但却显著增强了裸小鼠A549和Calu-6细胞的致瘤性。affmetrix长寡核苷酸阵列和定量RT-PCR实验显示，TSC显著抑制DKK1的表达，DKK1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 （MSP）实验表明，尽管连续暴露于TSC 5-60天，TSC对DKK1表达的抑制与H3K27三甲基化的剂量依赖性增加和多梳抑制子复合物的募集一致，但DKK1启动子内的DNA甲基化没有明显增加。停止TSC暴露导致DKK1启动子相关多梳蛋白的减少，并恢复DKK1在这些癌细胞中的表达。Western blot和聚焦qRT-PCR阵列实验表明，TSC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DKK1敲低后也发现了类似的结果。有趣的是，TSC暴露或DKK-1的敲除显著上调了Wnt5A的表达，Wnt5A是一种激活与肿瘤转移有关的非规范平面细胞极性网络的配体。这些研究的结果在几个月前发表了。最近的研究表明，Wnt5A的组成性过表达激活了PCP通路的下游靶点，并显著增强了Calu-6细胞的侵袭性和致瘤性。有趣的是，吸烟的肺癌患者的预后明显差于前吸烟者或从不吸烟者，Wnt5A表达是肺癌患者治疗失败和预后不良的独立决定因素。其他实验正在进行中，以表征Wnt5A增强肺癌细胞恶性表型的机制，并确定Wnt5A的表达是否以器官特异性的方式增强肺癌细胞的转移潜力。这些实验结果可能为靶向Wnt5A作为减少肺癌患者癌症干细胞信号传导和转移的策略提供了理论依据。尽管绝大多数肺癌可直接归因于吸烟，但关于介导这些肿瘤发生和发展的早期表观遗传事件的信息有限。因此，我们试图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，将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9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的高甲基化-&amp；#946；它们在人类肺癌中通常是沉默的。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。综上所述，这些数据表明，在培养的呼吸上皮细胞中，吸烟诱导癌症相关的表观基因组改变。这种体外模型可能有助于描述肺癌发生过程中基因调控的早期表观遗传机制。与这些研究有关的手稿已提交出版。研究人员还开展了其他实验，以检测未经治疗和tsc暴露的HBEC和SAEC中与肺癌患者的癌细胞系或原发肿瘤标本相关的微rna表达谱。选择四组细胞系进行分析，包括短期NHBE和SAEC，永生化HBECs，来自非吸烟者的肺癌系（H1650和H1975）以及来自吸烟者的肺癌系（H1299, H358， Calu-6和A549）。简单地说，从未经处理的NHBE、SAEC、HBEC、A549和Calu-6细胞中分离出小rna， TSC处理72小时，A549和Calu-6细胞暴露于DAC/DP，使用我们的标准治疗方案。使用NCode Multi-Species miRNA Microarray V2芯片评估miRNA表达谱，该芯片包含针对Sanger mirBase 9.0中所有miRNA物种的探针，适用于人类、小鼠、大鼠、果蝇、秀丽隐杆线虫和斑马鱼。对芯片数据进行处理和归一化，使用genesspring软件进行统计和聚类分析。采用双向方差分析（ANOVA）技术鉴定未处理和tsc暴露细胞中具有统计学差异的miRNA表达水平。类似的技术被用于比较miRNA在原发性呼吸上皮、HBEC、吸烟者和非吸烟者衍生的肺癌系中的表达，以建立癌症进展模型。这个分析产生了几个有趣的结果，包括：miRNA标记可以对人类肺癌进展进行分类。基于全球miRNA表达聚类，可以很容易地区分原代呼吸上皮、永活支气管上皮细胞和吸烟者/非吸烟者衍生的癌细胞系。相对于原代细胞，每一组都有自己的一组mirna过表达或过表达。2. MiRNA谱区分来自吸烟者和非吸烟者的肺癌。42个mirna在烟草相关肺癌细胞系A549、Calu-6、H1299和H358中相对于来自非吸烟者的H1650和H1975细胞有差异表达。值得注意的是，mir-31、mir-21和几个let-7家族成员在烟草相关肺癌中显著过表达。3. 烟草烟雾调节正常呼吸道上皮细胞和肺癌细胞中miRNA的表达。比较tsc处理和未处理的SAEC、NHBE、HBEC、A549和Calu-6细胞之间的miRNA表达谱，发现烟草烟雾暴露显著改变了mir-21、mir-31和mir-487b的表达。特别有趣的是，我们最近的实验显示mir-31靶向DKK-1和其他几种编码Wnt拮抗剂的转录本。编码mir-31的宿主基因已经被确定，并且正在进行实验以确定TSC诱导该基因的机制。这些研究提供了香烟致癌物与Wnt信号激活之间的直接联系，这对维持癌症干细胞至关重要；与这些研究有关的手稿已接近完成，不久将提交出版。正在进行的翻译工作集中在确认这些发现[摘要被截断为7800个字符]

Cultured lung cancer cells were treated with tobacco smoke condensates (TSC) using a variety of concentrations and exposure durations. Under conditions mimicking 1 pack per day (ppd) exposures for five days, TSC mediated no appreciable changes regarding in-vitro proliferation rates, but instead dramatically enhanced tumorigenicity of A549 and Calu-6 cells in nude mice. Affimetrix long-oligo array and quantitative RT-PCR experiments revealed that TSC markedly inhibited expression of DKK1, a secreted Wnt signaling antagonist and putative tumor suppressor. Subsequent ChIP, pyrosequencing, and methylation specific PCR (MSP) experiments demonstrated that inhibition of DKK1 expression by TSC coincided with a dose-dependent increase in H3K27 trimethylation, and recruitment of polycomb repressor complexes without an appreciable increase in DNA methylation within the DKK1 promoter despite continuous TSC exposures ranging from 5-60 days. Cessation of TSC exposure resulted in diminution of DKK1 promoter-associated polycomb proteins, and restoration of DKK1 expression in these cancer cells. Western blot and focused qRT-PCR array experiments indicated that TSC mediated dose-dependent increases in Wnt signaling in A549 and Calu-6 cells. Similar findings were noted following siRNA mediated knock-down of DKK1 in these cells. Interestingly, TSC exposure or knock-down of DKK-1 dramatically upregulated expression of Wnt5A, a ligand which activates a non-canonical planar-cell-polarity network implicated in tumor metastasis. Results of these studies were published several months ago. More recent studies have demonstrated that constitutive over-expression of Wnt5A activates downstream targets of the PCP pathway, and markedly enhances invasion and tumorigenicity of Calu-6 cells. Interestingly, lung cancer patient who smoke have significantly worse outcomes than former or never smokers, and Wnt5A expression is an independent determinant of treatment failure and poor outcome in lung cancer patients. Additional experiments are in progress to characterize the mechanisms by which Wnt5A enhances the malignant phenotype of lung cancer cells, and, to ascertain if expression of Wnt5A enhances metastatic potential of lung cancer cells in an organ specific manner. Results of these experiments may provide the rationale for targeting Wnt5A as a strategy to diminish cancer stem cell signaling and metastases in lung cancer patients. Whereas the vast majority of lung cancers are directly attributable to cigarette smoking, limited information is available regarding early epigenetic events mediating initiation and progression of these neoplasms. As such, we sought 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) were cultured in normal media (NM) with or without cigarette smoke condensate (CSC) for up to nine 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-β 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. Collectively these data indicate that cigarette smoke induces cancer-associated epigenomic alterations in cultured respiratory epithelia. This in-vitro model may prove useful for delineating early epigenetic mechanisms of gene regulation during pulmonary carcinogenesis. A manuscript pertaining to these studies has been submitted for publication. Additional experiments have been undertaken to examine micro-RNA expression profiles in untreated and TSC-exposed HBEC and SAEC relative to cancer lines or primary tumor specimens from lung cancer patient. Four sets of cell lines were chosen for this analysis including short term NHBE and SAEC, immortalized HBECs, lung cancer lines from non-smokers (H1650 and H1975), as well as lung cancer lines from smokers (H1299, H358, Calu-6, and A549). Briefly, small RNAs were isolated from untreated NHBE, SAEC, HBEC, A549, and Calu-6 cells treated with TSC for 72h, and A549 and Calu-6 cells exposed to DAC/DP using our standard treatment regimen. miRNA expression profiles were evaluated using NCode Multi-Species miRNA Microarray V2 chips containing probes that target all of the miRNA species in the Sanger mirBase 9.0 for human, mouse, rat, Drosophila, C. elegans, and zebrafish. Microarray data were processed and normalized, with statistic and clustering analyses performed using Genespring software. Two-way analysis of variance (ANOVA) techniques were used to identify statistically different miRNA expression levels in untreated and TSC-exposed cells. Similar techniques were used to compare miRNA expression in primary respiratory epithelia, HBEC, smoker-, and nonsmoker- derived lung cancer lines for cancer progression modeling. This analysis yeilded several interesting results including: 1. miRNA signature can classify human lung cancer progression. Based on the global miRNA expression clustering, primary respiratory epithelia, immortalized bronchial epithelial cells, and smoker/ non-smoker derived cancer lines could be readily distinguished from one another. Each group exhibited its own set of miRNAs that were over- or under-expressed relative to the primary cells. 2. MiRNA profiles distinguish lung cancers derived from smoker and non-smokers. Forty-two miRNAs were differentially expressed in tobacco- associated lung cancer lines A549, Calu-6, H1299, and H358 relative to H1650 and H1975 cells that were derived from non-smokers. Notably, mir-31, mir-21 and several let-7 family members were significantly over-expressed in tobacco- associated lung cancers. 3. tobacco smoke modulates miRNA expression in normal respiratory epithelia and lung cancer cells. Comparison of the miRNA expression profiles between TSC-treated and untreated SAEC, NHBE, HBEC, A549 and Calu-6 cells, revealed that tobacco smoke exposure significantly altered expression of mir-21, mir-31 and mir-487b. Of particular interest, our recent experiments revealed that mir-31 targets DKK-1 and several other transcripts encoding Wnt antagonists. The host gene encoding mir-31 has been identified, and experiments are in progress to ascertain the mechanisms by which TSC induces this gene. These studies provide a direct link between cigarette carcinogens and activation of Wnt signaling, which is critical for maintenance of cancer stem cells; manuscripts pertaining to these studies are nearing completion, and will be submitted for publication soon. Ongoing translational efforts are focused on confirming these findings in [summary truncated at 7800 characters]