Modulating Cancer Stem Cell Signaling in Thoracic Malignancies

调节胸部恶性肿瘤中的癌症干细胞信号传导

• 批准号: 8349541
• 负责人: DAVID SCHRUMP
• 金额: $ 51.44万
• 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/8349541
• 关键词: 3' Untranslated Regions; A549; ABCG2 gene; Aryl Hydrocarbon Receptor; Attenuated; BMI1 gene; Binding; Biological Assay; Biological Models; CCAAT-Enhancer-Binding Proteins; Cell Cycle; Cells; Chest; Clinical; Clinical Protocols; Complex; Computer software; Critical Pathways; Cultured Cells; DNA; DNA Methylation; DNA Polymerase II; Dactinomycin; Decitabine; Dose; Embryo; Epigenetic Process; Epithelial Cells; Esophageal Squamous Cell; Flow Cytometry; Functional RNA; Gene Expression; Genes; Goals; Histology; Human; Immunohistochemistry; Immunoprecipitation; In Vitro; Ligands; Link; Lung; Maintenance; Malignant Neoplasms; Malignant neoplasm of esophagus; Malignant neoplasm of lung; Manuscripts; Mediating; Mediator of activation protein; Messenger RNA; MicroRNAs; Microarray Analysis; Molecular Profiling; Mutation; Nucleosomes; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Plicamycin; Population; Positioning Attribute; Precipitation; Prevention; Promoter Regions; Proto-Oncogene Proteins c-akt; Publications; Publishing; Pump; RNA; Relative (related person); Reporter; Repression; Response Elements; Reverse Transcription; SFRP4 gene; Seeds; Series; Side; Signal Transduction; Site; Smoker; Smoking Status; Specimen; Stem cells; Structure of parenchyma of lung; Structure of respiratory epithelium; Techniques; Tobacco; Transcript; Transfection; Transforming Growth Factor beta; Translational Repression; Tumor Suppressor Genes; Tumorigenicity; Up-Regulation; Western Blotting; Xenobiotics; cancer cell; cancer stem cell; cancer therapy; carcinogenesis; cell growth; chromatin immunoprecipitation; cigarette smoking; clinically relevant; crosslink; epigenomics; gene repression; histone modification; human TGFB1 protein; in vitro Model; in vivo; insight; knock-down; malignant phenotype; non-smoker; novel; pluripotency; promoter; research clinical testing; research study; response; sodium bisulfite; tumor; tumor progression

A series of experiments have been undertaken to comprehensively examine miRNAs that contribute to initiation and progression of tobacco-induced human lung cancers. Briefly, array techniques were used to examine miRNA expression profiles in lung cancer lines established from smokers and non-smokers as well as normal respiratory epithelia cultured in the presence or absence of CSC. This analysis revealed that miRNA signatures coincided with human lung cancer progression. Furthermore, miRNA profiles distinguished lung cancers derived from smokers relative to nonsmokers. Under relevant exposure conditions, CSC consistently up-regulated miR-31 in cultured normal respiratory epithelia and lung cancer cells. qRT-PCR and western blot experiments confirmed that CSC significantly increased miR-31 expression, and activated LOC554202 (the host gene for miR-31) in normal respiratory epithelia and lung cancer cells; miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure suggesting reprogramming in these cells. qRT-PCR experiments revealed that miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. RNA cross-link immunoprecipitation (CLIP) and 3' UTR reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over-expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC-mediated decreases in Dkk-1 and DACT-3 expression. Furthermore, over-expression of miR-31 diminished expression of several other Wnt antagonists including SFRP1, SFRP4, and WIF-1, and increased expression of Wnt-5a, a non-canonical Wnt ligand implicated in maintenance of cancer stem cells, which enhances the malignant phenotype of lung cancers in-vitro and in-vivo. Chromatin immunoprecipitation (ChIP) experiments demonstrated that CSC increased H3K4Me3, H3K9/14Ac and C/EBP-beta levels within the LOC554202 promoter. Knock-down of C/EBP-beta abrogated CSC-mediated activation of LOC554202. Over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells; knock-down of miR-31 inhibited growth of these cells. These experiments demonstrating that miR-31 functions as an oncomir during tobacco-induced human pulmonary carcinogenesis were published recently in PLoS One. More recent studies utilizing the same in-vitro model system revealed that CSC significantly repressed miR-487b in cultured normal respiratory epithelia and lung cancer cells. Interestingly, analogous to what was observed for miR-31, repression of miR-487b in cultured cells persisted following cessation of CSC exposure. qRT-PCR experiments demonstrated that miR-487b expression was significantly lower in primary lung cancers particularly those from smokers relative to adjacent normal lung parenchyma. Software-guided analysis revealed numerous potential targets for miR-487b including Wnt5a, SUZ12, BMI1, c-MYC and K-ras-mediators of stem cell pluripotency. Constitutive over-expression of miR-487b inhibited, whereas depletion of endogenous miR-487b enhanced expression of Wnt5a, BMI1, SUZ12, c-MYC and K-ras in SAEC and Calu-6 cells. ChIP analysis revealed that repression of miR-487b coincided with increased recruitment of SUZ12 and BMI1 to Dkk-1, SFRP1, SFRP4, and WIF-1 promoter regions, and down-regulation of these genes in normal respiratory epithelia and lung cancer cells. CLIP and 3' UTR experiments confirmed direct interference of miR-487b with Wnt5a, BMI1, SUZ12, c-MYC, and K-ras transcripts. Sodium bisulfite sequencing, methylated DNA precipitation (MeDIP) and ChIP, and nucleosome positioning experiments demonstrated that repression of miR-487b coincided with DNA methylation, de-novo nucleosome occupancy, and recruitment of SUZ12 and BMI1with decreased H2AZ and TCF1 levels within the miR-487b promoter region. Deoxyazacytidine induced miR-487b expression, and attenuated CSC-mediated repression of miR-487b. TGF-beta1 recapitulated CSC-mediated effects on miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling; inhibited lung cancer invasion mediated by CSC, or over-expression of c-MYC, k-ras, or TGF-beta1; and decreased proliferation, and tumorigenicity of lung cancer cells. These findings indicate that epigenetic silencing of miR-487b cooperates with up-regulation of miR-31to activate critical pathways mediating pluripotency during tobacco-induced human pulmonary carcinogenesis, and suggest that DNA demethylating agents may be useful for restoring miR-487b expression for lung cancer therapy. A manuscript pertaining to these studies, which are the first to directly implicate repression of miR-487b in the pathogenesis of human lung cancers, has been submitted for publication. Additional experiments have been performed to further examine mechanisms by which cigarette smoke increases the malignant phenotype of lung and esophageal cancer cells. Briefly, lung and esophageal cancer cells (A549, Calu-6, NCI-SB-ESC1 and NCI-SB-ESC2), were cultured in normal media (NM) with or without CSC under clinically relevant exposure conditions. Microarray analysis revealed that five day CSC exposure significantly up-regulated ABCG2, encoding a xenobiotic pump highly expressed in cancer stem cells. Quantitative reverse transcription-PCR (qRT-PCR), western blot, and immunohistochemistry experiments confirmed up-regulation of ABCG2 in cultured cancer lines, but not normal small airway epithelial cells (SAEC) or immortalized esophageal squamous cells (HET1A) exposed to CSC. Flow cytometry experiments demonstrated that CSC increased the side population (SP) of cultured cancer cells. Transient transfection experiments using ABCG2 promoter reporter constructs revealed that deletion of xenobiotic response elements as well as SP-1 sites markedly attenuated ABCG2 induction by CSC. ChIP experiments revealed that CSC-mediated induction of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor (AHR), SP-1, and Nrf2, as well as increased levels of RNA pol II and H3K9Ac within the ABCG2 promoter. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AHR, SP-1, and Nrf2 levels within the ABCG2 promoter, markedly down-regulated ABCG2 expression, decreased SP, and dramatically inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Micro-array analysis revealed that mithramycin treatment significantly repressed cell cycle and cancer-related genes. Approximately 1260 genes were commonly altered in Calu-6 and A549 cells at two mithramycin doses; the vast majority of these genes were repressed by drug treatment. For example, 37 of 38 cancer transformation genes were down-regulated by mithramycin. Additional analysis revealed significant inhibition of critical cancer pathways including AKT, TGF-beta, and Wnt following mithramycicn exposure. Collectively, these findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical evaluation of mithramycin for targeting cancer stem cell signaling in thoracic malignancies. A manuscript pertaining to these studies is nearing completion, and a novel clinical protocol utilizing mithramycin to eradicate cancer stem cells in patients with thoracic malignancies will be initiated in the next several months.

已经进行了一系列实验，以全面检查有助于烟草引起的人类肺癌的开始和进展的miRNA。简而言之，阵列技术用于检查由吸烟者和非吸烟者建立的肺癌线中的miRNA表达谱，以及在存在或不存在CSC的情况下培养的正常呼吸上皮。该分析表明，miRNA特征与人类肺癌进展一致。此外，miRNA特征区分开了源自吸烟者相对于非吸烟者的肺癌。在相关的暴露条件下，CSC在培养的正常呼吸性上皮和肺癌细胞中始终上调miR-31。 QRT-PCR和Western印迹实验证实，在正常呼吸性上皮和肺癌细胞中，CSC显着增加了miR-31的表达，并激活了LOC554202（miR-31的宿主基因）； MiR-31和LOC554202在停用CSC暴露的情况下持续存在表达这些细胞中的重新编程。 QRT-PCR实验表明，相对于相邻的正常肺组织，肺癌标本中的miR-31和LOC554202表达水平显着升高。 RNA交联免疫沉淀（夹）和3'UTR报告基因测定法证明了miR-31与Dickkopf-1（DKK-1）和DACT-3的直接相互作用。 miR-31的过表达显着降低了正常呼吸性上皮和肺癌细胞中DKK-1和DACT3表达水平。 miR-31的敲除增加了DKK-1和DACT3水平，并废除了DKK-1和DACT-3表达的CSC介导的降低。此外，miR-31的过表达降低了其他几种Wnt拮抗剂的表达，包括SFRP1，SFRP4和WIF-1，以及Wnt-5a的表达增加，Wnt-5a的表达是一种与维持癌症干细胞有关的非典型的Wnt配体，这与癌症干细胞有关，从而增强了肺癌症肺癌的恶性表型。染色质免疫沉淀（CHIP）实验表明，CSC在LOC554202启动子中增加了H3K4ME3，H3K9/14AC和C/EBP-BETA水平。 C/EBP-beta的敲除废除了CSC介导的LOC554202的激活。 miR-31的过表达显着增强了肺癌细胞的增殖和肿瘤性。 miR-31的敲除抑制了这些细胞的生长。这些实验表明，最近在PLOS ONE中发表了MiR-31在烟草诱导的人肺癌发生过程中起oncomir的作用。使用相同的维特罗模型系统的最新研究表明，在培养的正常呼吸性上皮和肺癌细胞中，CSC显着抑制了miR-487b。有趣的是，类似于miR-31观察到的，在停止CSC暴露后，培养细胞中miR-487b的抑制持续存在。 QRT-PCR实验表明，原发性肺癌中的miR-487b表达显着降低，尤其是吸烟者相对于邻近正常肺实质的表达。软件指导的分析揭示了miR-487b的许多潜在靶标，包括Wnt5a，Suz12，BMI1，C-Myc和干细胞多能的K-RAS-介体。 miR-487b的组成型过表达抑制了，而内源性miR-487b的耗竭增强了Wnt5a，BMI1，SUZ12，C-MYC和K-RAS在SAEC和CALU-6细胞中的表达。 CHIP分析表明，MiR-487b的抑制与SUZ12和BMI1的募集增加对DKK-1，SFRP1，SFRP4和WIF-1启动子区域以及正常呼吸呼吸性上皮细胞和肺癌细胞中这些基因的下调。夹子和3'UTR实验证实了miR-487b在Wnt5a，BMI1，SUZ12，C-MYC和K-RAS转录本上的直接干扰。亚硫酸钠测序，甲基化的DNA降水（MEDIP）和芯片以及核小体定位实验表明，miR-487b的抑制与DNA甲基化，DE-NOVO核小体占用率，Suz12和BMI1WITH降低了H2AZ和TCF1水平的SUZ12和BMI1WISH的募集。去氧齐瑞替丁诱导miR-487b表达，并减弱CSC介导的miR-487b的抑制。 TGF-BETA1概括了CSC介导的对miR-487b的影响。 miR-487b的组成型表达废除了Wnt信号传导；抑制由CSC介导的肺癌侵袭，或C-MYC，K-RAS或TGF-BETA1的过表达；并降低了肺癌细胞的增殖和肿瘤性。这些发现表明，miR-487b的表观遗传沉默与miR-31的上调合作，以激活关键途径，以介导烟草诱导的人类肺癌发生过程中介导多能性，并表明DNA脱甲基化剂可能有助于恢复miR-487b的表达对肺癌的恢复。与这些研究有关的手稿是第一个直接暗示对人类肺癌发病机理中对miR-487b的抑制。已经进行了其他实验，以进一步研究香烟烟雾增加肺和食管癌细胞的恶性表型的机制。简而言之，在临床相关的暴露条件下，在普通培养基（NM）中培养肺和食管癌细胞（A549，CALU-6，NCI-SB-ESC1和NCI-SB-ESC2）。微阵列分析表明，五天的CSC暴露显着上调了ABCG2，编码了在癌症干细胞中高度表达的异种生物泵。定量逆转录-PCR（QRT-PCR），Western印迹和免疫组织化学实验证实了在培养的癌细胞系中的ABCG2上调，但并非正常的小气道上皮细胞（SAEC）或永生化的食管食管细胞（HET1A）暴露于CSC。流式细胞仪实验表明，CSC增加了培养的癌细胞的侧群（SP）。使用ABCG2启动子报告构建体的瞬时转染实验表明，异种生物反应元件的缺失以及SP-1位点通过CSC显着减弱了ABCG2诱导。 CHIP实验表明，CSC介导的ABCG2的诱导与芳基烃受体（AHR），SP-1和NRF2的占用程度增加，以及ABCG2启动子中RNA POL II和H3K9AC的水平增加。在可能在临床环境中可以实现的条件下，毛霉素减少了ABCG2启动子内AHR，SP-1和NRF2水平的基础和CSC介导的增加，显着下调的ABCG2表达，SP降低，降低了SP，并极大地抑制了Lung and lung and lung and eclagealageal癌症的增殖和肿瘤性。微阵列分析表明，毛霉素治疗显着抑制了细胞周期和与癌症相关的基因。在两种毛霉素剂量下，在Calu-6和A549细胞中通常会改变约1260个基因。这些基因中的绝大多数都被药物治疗抑制了。例如，38个癌症转化基因中的37个被毛霉素下调。其他分析表明，在毛神经暴露后，包括AKT，TGF-β和WNT在内的关键癌症途径的显着抑制。总的来说，这些发现提供了吸烟状况与肺癌和食管癌患者结局之间的潜在机械联系，并支持密霉素的临床评估，用于靶向胸腔恶性肿瘤中的癌症干细胞信号。与这些研究有关的手稿即将完成，并在未来几个月内将启动一种利用毛霉素消除胸腔恶性肿瘤患者的癌症干细胞的新型临床方案。