Epigenetic Mechanisms of Gene Expression in Lung Cancer Cells

肺癌细胞基因表达的表观遗传机制

• 批准号: 8552990
• 负责人: DAVID SCHRUMP
• 金额: $ 48.65万
• 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/8552990
• 关键词: 3' Untranslated Regions; A549; Agar; Alleles; Allogenic; Apoptosis; Attenuated; CTAG1 gene; Cell Cycle Progression; Cell Differentiation process; Cells; Chest; Chromatin; Chromatin Structure; Clinical; Coculture Techniques; Complex; Cytolysis; DNA; DNA Sequence; Dermal; Development; Disease; Dose; EZH2 gene; Effector Cell; Epigenetic Process; Epithelial Cells; Evaluation; Excision; Exhibits; Extended Family; Fibroblasts; Financial compensation; Gene Activation; Gene Expression; Gene Family; Gene Silencing; Genes; Germ Cells; Global Change; Goals; Growth; H1299; HLA A*0201 antigen; Heterochromatin; Histone Deacetylase; Histone-Lysine N-Methyltransferase; Histones; Human; Hypermethylation; Immunoblot Analysis; Immunoblotting; Immunohistochemistry; KDM5B gene; Lung; Lysine; Malignant Neoplasms; Malignant Pleural Mesothelioma; Malignant neoplasm of lung; Malignant neoplasm of testis; Maps; Mediating; Mesothelial Cell; Mesothelium; Messenger RNA; Methylation; Molecular Profiling; Mono-S; Nude Mice; Oncogenes; Ovary; Patients; Pattern; Pharmaceutical Preparations; Pleura; Pleural Mesothelioma; Polycomb; Proteins; Pseudogenes; Publishing; RNA Splicing; Recombinants; Regulator Genes; Relative (related person); Repression; Research Project Grants; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Secondary to; Somatic Cell; Specimen; Spermatocytes; Spermatogonia; Staging; Structure; T-Cell Receptor; Techniques; Testis; Time; Toxic effect; Trichostatin A; Tumor Suppressor Genes; Tumor Suppressor Proteins; Up-Regulation; Variant; X Chromosome; Xenograft procedure; anticancer research; bronchial epithelium; cancer cell; cancer immunotherapy; carcinogenesis; cell mediated lymphocytolysis test; chromatin immunoprecipitation; cytokine; cytotoxic; demethylation; epigenomics; genome-wide; histone methyltransferase; imprint; inhibitor/antagonist; insight; intraperitoneal; knock-down; malignant phenotype; melanoma-associated antigen-A1; migration; novel; pluripotency; promoter; research study; small hairpin RNA; tumor

Preliminary qRT-PCR experiments allowed us to select for further study four lung cancer lines (H1299, H841, A549, Calu-6) exhibiting heterogeneous patterns of NY-ESO-1, MAGE-A1, and MAGE-A3 CT-X gene expression, as well as normal human bronchial epithelia (NHBE) and human small airway epithelial cells (SAEC), which do not express any CT-X genes. Immunoblot analysis confirmed qRT-PCR results. Extensive pyrosequencing and chromatin immunoprecipitation (ChIP) experiments demonstrated that lack of NY-ESO-1, MAGE-A1 or MAGE-A3 expression in lung cancer cells was attributable to persistence of normal heterochromatin structure, rather than global activation of these genes via promoter hypomethylation, followed by selective silencing associated with bivalent chromatin.Additional experiments were performed to ascertain if modulation of histone lysine methylation altered NY-ESO-1, MAGE-A1, and MAGE-A3 expression in lung cancer cells. Briefly, lentiviral shRNA techniques were used to knock-down LSD-1(KDM1) and JARID1B (KDM5B) that mediate demethylation of mono, di-, and trimethylated H3K4 (histone activation marks, or the histone lysine methyltransferase EZH2 (KMT6) that mediates trimethylation of H3K27 (histone repressive mark) in H841 cells that do not express NY-ESO-1, MAGE-A1, or MAGE-A3. ChIP experiments revealed that global changes in activation and repression marks coincided with similar alterations, and decreased occupancy of the respective histone modifiers within the NY-ESO-1, MAGE-A1 and MAGE-A3 promoters in knock-downs relative to control cells. Whereas knock-down of EZH2, LSD1, or JARID1B alone was insufficient to activate NY-ESO-1, MAGE-A1, or MAGE-A3, inhibition of EZH2, LSD1, or JARID1B expression significantly enhanced DAC-mediated induction of these CT genes in lung cancer cells. The effects of targeted modulation of histone lysine methylation were more pronounced than those observed following knock-down of the class III histone deacetylase, SirT1. Additional qRT-PCR and immunoblot experiments demonstrated that 3-deazaneplanocin A (DZNep), a novel pharmacologic inhibitor of EZH2 expression, at a concentration one log lower than the cytotoxic dose of this agent in cancer cells, significantly enhanced DAC-mediated activation of NY-ESO-1, MAGE-A1 and MAGE-A3 in H841 cells (Figure 2). This phenomenon extended to other CT-X genes such as MAGE-A6 and MAGE-A12, and was observed across numerous other lung cancer lines. The magnitude of enhancement of DAC-mediated de-repression of CT-X genes in lung cancer cells by DZNep was markedly higher than that observed in SAEC or NHBE, and approximated or exceeded that observed following sequential DAC-DP or DAC-trichostatin A (TSA) treatment. Pyrosequencing and ChIP experiments demonstrated that this phenomenon was not attributable to enhanced demethylation, but instead coincided with decreased EZH2 and H3K27Me3 levels within the NY-ESO-1, MAGE-A1 and MAGE-A3 promoters. Consistent with these observations, constitutive expression of EZH2 significantly attenuated the enhancement effect of DZNep on DAC-mediated induction of NY-ESO-1, MAGE-A1 or MAGE-A3 in lung cancer cells. Subsequent cytokine and chromium release assays demonstrated that DZNep significantly enhanced DAC-mediated recognition and lysis of lung cancer cells by allogeneic PBL expressing recombinant T cell receptors specific for NY-ESO-1 or MAGE-A3 in the context of HLA-A*0201. No cytokine release or lysis was observed following co-culture of effector cells with drug treated HLA-A*0201-transduced normal airway epithelial cells or dermal fibroblasts. Full details of these experiments, which were the first to demonstrate that modulation of histone lysine methylation may be a novel epigenetic strategy for cancer immunotherapy, have been published in Cancer Research.In additional studies, Affymetrix microarrays were used to examine global gene expression profiles, and specifically identify genes encoding PcG proteins in a panel of malignant pleural mesothelioma (MPM) lines relative to cultured normal mesothelia. This analysis demonstrated over-expression of EZH2 (KMT6) and to a lesser extent, EED and SUZ12, which encode core components of polycomb repressor complex-2 (PRC-2), in MPM lines. Quantitative RT-PCR (qRT-PCR) experiments using primers recognizing both EZH2 splice variants, and immunoblot analysis confirmed over-expression of EZH2, but not EED or SUZ12 in MPM lines relative to cultured normal mesothelial cells; up-regulation of EZH2 coincided with a global increase in the PRC-2 mediated repressive chromatin mark, H3K27Me3 in MPM cells. Subsequent qRT-PCR and immunohistochemistry experiments confirmed over- expression of EZH2 in 80%-85% of primary MPM specimens relative to normal pleura. In general, EZH2 levels tended to coincide with mRNA copy numbers, although some variations were noted, suggesting that post-transcriptional mechanisms also contribute to EZH2 over-expression in MPM. Consistent with these findings, levels of miR-101 or miR-26, which normally target the 3' UTR of EZH2, were significantly decreased in MPM specimens compared to normal pleura. Subsequent studies demonstrated that EZH2 protein levels did not correlate with stage of disease; however, intratumoral expression of either of the two EZH2 variants assessed by Illumina array techniques correlated with survival in patients with locally advanced MPM undergoing potentially curative resections. Additional experiments were performed to examine if aberrant PRC-2 activity directly contributes to the malignant phenotype of pleural mesothelioma cells. Briefly, shRNA techniques were used to knock-down EZH2 in cultured MPM cells; the shRNA used for these experiments targeted both splice variants of EZH2. Similar experiments were undertaken to knock-down EED, which although not over-expressed in primary MPM, is critical for maintaining stability of PRC-2, and histone methyltransferase activity of EZH2. Specific knock-down of these PRC-2 components significantly inhibited proliferation, migration, soft agar clonogenicity, as well as tumorgenicity of MPM cells. The effects of EED knock-down on global H3K27Me3 levels, as well as proliferation, migration, clonogenicity and tumorgenicity were more pronounced than EZH2 knock-down in MPM cells; these results may have been due to relative knock-down efficacies of the shRNAs, compensation of EZH2 knock-down by EZH1, or more profound destabilization of PRC-2 by depletion of EED. Subsequent immunoblot and pyrosequencing experiments demonstrated that DZNep mediated time and dose-dependent depletion of EZH2 and EED, and decreased H3K27Me3 levels without inducing global DNA demethylation in cultured MPM cells; these effects coincided with significantly decreased proliferation, migration and soft agar clonogenicity of these cells. In addition, intraperitoneal (IP) administration of DZNep significantly inhibited growth of established MPM xenografts in athymic nude mice without obvious systemic toxicities. Micro-array, qRT-PCR, and ChIP experiments demonstrated that the growth inhibitory effects of targeted disruption of PRC-2 or DZNep treatment in MPM cells coincided with decreased promoter occupancy of H3K27Me3, and up-regulation of numerous tumor suppressors modulating pluripotency, cell cycle progression and apoptosis in cancer cells. Detailed results of these experiments have been published recently in Clinical Cancer Research.

Preliminary qRT-PCR experiments allowed us to select for further study four lung cancer lines (H1299, H841, A549, Calu-6) exhibiting heterogeneous patterns of NY-ESO-1, MAGE-A1, and MAGE-A3 CT-X gene expression, as well as normal human bronchial epithelia (NHBE) and human small airway epithelial cells (SAEC), which do没有表达任何CT-X基因。 免疫印迹分析证实了QRT-PCR结果。肺癌细胞中广泛的焦糖测测和染色质免疫沉淀（CHIP）实验表明，缺乏NY-ESO-1，MAGE-A1或MAGE-A1或MAGE-A3表达，这是由于正常异质素结构的持久性，而不是通过启动者下甲基化的实验，而不是正常的异质素结构，而不是通过这些基因的全球性激活，而不是全球性地激活，均与选择性相关。确定肺癌细胞中组蛋白赖氨酸甲基化的调节是否改变了NY-ESO-1，MAGE-A1和MAGE-A3的表达。 简而言之，使用慢病毒SHRNA技术来敲击LSD-1（KDM1）和JARID1B（KDM5B），这些技术介导了单，二甲基和三甲基化的H3K4的脱甲基化（组蛋白激活或组蛋白蛋白蛋白赖氨酸甲基转移酶EZH2（KMT6）的脱甲基化H3K4（kmt6）的hymine rymetiate hylytriptiate hermantiatey hylytription kmt6）在不表达NY-ESO-1，MAGE-A1或MAGE-A3的H841中，芯片实验的全球激活和抑制作用都与相似的变化相吻合，并且在NY-ESO-1，MAGE-A1和MAGE-A3蛋白蛋白蛋白蛋白蛋白蛋白蛋白酶中的optery ockingers sepers sepers sepers sepers sephss sepers sepers sepers sepers sepers sepers sepers seperss coptions chip-a3。仅LSD1或JARID1b不足以激活NY-ESO-1，MAGE-A1或MAGE-A3，抑制EZH2，LSD1或JARID1b表达显着增强了DAC介导的这些CT对这些CT基因的诱导，而不是肺癌的靶向型甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基的效果。脱乙酰基酶，SIRT1。 Additional qRT-PCR and immunoblot experiments demonstrated that 3-deazaneplanocin A (DZNep), a novel pharmacologic inhibitor of EZH2 expression, at a concentration one log lower than the cytotoxic dose of this agent in cancer cells, significantly enhanced DAC-mediated activation of NY-ESO-1, MAGE-A1 and MAGE-A3 in H841 cells (Figure 2). 这种现象扩展到其他CT-X基因，例如MAGE-A6和MAGE-A12，并在许多其他肺癌系中观察到。 DZNEP在肺癌细胞中DAC介导的CT-X基因抑制抑制的抑制的大小明显高于在SAEC或NHBE中观察到的，并且在顺序DAC-DAC-DP或DAC-DAC-DAC-DAC-TRICHOSTATATITATIN A（TSA）治疗后观察到的近似或超过。 焦磷酸测序和芯片实验表明，这种现象不是归因于增强的脱甲基化，而是与NY-ESO-1，MAGE-A1和MAGE-A3启动子内的EZH2和H3K27me3水平降低相吻合。 与这些观察结果一致，EZH2的组成型表达显着减弱了DZNEP对肺癌细胞中DAC介导的NY-ESO-1，MAGE-A1或MAGE-A3的DAC介导的诱导的增强作用。随后的细胞因子和铬释放分析表明，DZNEP通过表达对NY-ESO-1或MAGE-A3的重组T细胞受体的同种异体PBL显着增强了DAC介导的识别和对肺癌细胞的识别，并在HLA-A*0201的背景下显着增强了重组T细胞受体。 在与药物处理的HLA-A*0201转导的正常气道上皮细胞或皮肤成纤维细胞共同培养后，未观察到细胞因子释放或裂解。 这些实验的全部细节已在癌症研究中发表了，这些实验首先证明了组蛋白赖氨酸甲基化的调节可能是一种新型的表观遗传学策略。间皮。 该分析表明，在MPM线中，ED和SUZ12的EZH2（KMT6）和SUZ12的过度表达在较小程度上，它们在MPM线上编码了Polycomb Repressor Complex-2（PRC-2）的核心成分。 定量RT-PCR（QRT-PCR）实验使用识别EZH2剪接变体的引物和免疫印迹分析证实了EZH2的过表达，但在MPM线中没有EED或SUZ12相对于培养的正常中皮细胞。 EZH2的上调与PRC-2介导的抑制性染色质标记，H3K27me3在MPM细胞中的全球增加相吻合。 随后的QRT-PCR和免疫组织化学实验证实了EZH2在80％-85％的初级MPM标本中相对于正常胸膜的表达。 通常，尽管注意到一些变化，但EZH2水平倾向于与mRNA拷贝数一致，这表明转录后机制也有助于MPM中的EZH2过表达。 与这些发现相比，与正常的胸膜相比，MiR-101或miR-26的水平通常靶向EZH2的3'UTR。 随后的研究表明，EZH2蛋白水平与疾病阶段无关。然而，通过Illumina阵列技术评估的两个EZH2变体中的任何一个与局部晚期MPM患者的生存有关的两种EZH2变体的表达。进行了其他实验，以检查异常的PRC-2活性是否直接有助于胸膜间皮瘤细胞的恶性表型。 简而言之，SHRNA技术用于在培养的MPM细胞中敲低EZH2。用于这些实验的shRNA针对EZH2的两个剪接变体。 进行了类似的实验来敲破EED，尽管在初级MPM中并未表达过表达，但对于维持PRC-2的稳定性和EZH2的组蛋白甲基转移酶活性至关重要。 这些PRC-2成分的特异性敲除可以显着抑制增殖，迁移，软琼脂克隆性以及MPM细胞的肿瘤性。 EED敲低对全局H3K27me3水平以及增殖，迁移，克隆性和肿瘤的影响比MPM细胞中的EZH2敲低更为明显。这些结果可能是由于shRNA的相对敲低效力，EZH1敲击EZH2的补偿，或通过EED耗尽而对PRC-2的更深刻稳定。 随后的免疫印迹和焦磷酸测序实验表明，DZNEP介导的时间和EZH2和EED的剂量依赖性耗竭，并降低了H3K27me3水平，而没有诱导培养的MPM细胞中的全局DNA脱甲基化；这些影响与这些细胞的增殖，迁移和软琼脂克隆性显着降低。 此外，腹膜内（IP）DZNEP的给药可显着抑制无胸腺裸鼠中已建立的MPM异种移植物的生长，而没有明显的全身毒性。 Micro-Array，QRT-PCR和CHIP实验表明，在MPM细胞中有针对性破坏PRC-2或DZNEP治疗的生长抑制作用与H3K27me3的启动子占用率降低，并上调了众多肿瘤抑制型肿瘤的启动子占用，并调节多能型和细胞循环型进度和APOPTOSS综合癌症。 这些实验的详细结果最近在临床癌症研究中发表了。