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: 9343915
• 负责人: DAVID SCHRUMP
• 金额: $ 89.65万
• 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/9343915
• 关键词: A549; Accounting; Affect; Alkaline Phosphatase; Binding; Cell surface; Cells; Cessation of life; Chromatin; Chromosome abnormality; Complex; Cytokeratin; DNA Methylation; Data; Data Set; Databases; Development; Disease; Dose; Environmental Risk Factor; Epigenetic Process; Epithelial Cells; Esophageal carcinoma; Evaluation; Exhibits; Female; Gene Expression; Gene Expression Profiling; Genes; Genetic; Growth; Hispanics; Histone Code; Human; Immunoblotting; Immunohistochemistry; In Vitro; Lentivirus Vector; Lung; Maintenance; Malignant Neoplasms; Malignant Pleural Mesothelioma; Malignant neoplasm of esophagus; Malignant neoplasm of lung; Malignant neoplasm of thorax; Manuscripts; Mediating; Messenger RNA; Metabolism; Methylation; MicroRNAs; Microarray Analysis; Modeling; Modification; Morphology; Neoplasms; Non-Small-Cell Lung Carcinoma; Nucleosomes; Pathway interactions; Patients; Peer Review; Phenotype; Plicamycin; Polycomb; Population; Preparation; Primary Neoplasm; Proteins; Publications; Publishing; RNA; RNA-Binding Proteins; Regulation; Repression; Side; Signal Transduction; Smoker; Specificity; Spectral Karyotyping; Staging; Stem cells; Structure of parenchyma of lung; Techniques; Teratoma; Tetanus Helper Peptide; The Cancer Genome Atlas; Time; Tobacco; Tobacco smoke; Transgenes; Tumor Suppressor Genes; United States; Untranslated RNA; Up-Regulation; Xenograft procedure; cancer cell; cancer stem cell; cancer therapy; chromatin immunoprecipitation; cigarette smoke-induced; cigarette smoking; embryonic stem cell; epigenomics; in vivo; induced pluripotent stem cell; knock-down; lung small cell carcinoma; new therapeutic target; non-smoker; novel; novel strategies; novel therapeutics; overexpression; pluripotency; pressure; research study; respiratory; self-renewal; small hairpin RNA; stage-specific embryonic antigen 4; stem; stemness; symposium; transcriptome sequencing

To further examine epigenetic mechanisms regulating stemness in lung cancers and identify novel therapeutic targets in these neoplasms, we recently generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) by lentiviral transduction of OSKM (Yamanaka) factors. SAEC were derived from a 57 year old Hispanic female non-smoker. Numerous lung iPSC (Lu-iPSC) clones were generated, several of which were randomly selected for further analysis. These Lu-iPSC demonstrated hallmarks of pluripotency including morphology, cytoplasmic alkaline phosphatase expression, cell surface expression of SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, up-regulation of stem cell genes including Nanog, Oct4, and SALL4, with decreased expression of cytokeratins, as well as in-vitro proliferation and teratoma formation. Spectral karyotyping and qRT-PCR analyses demonstrated no chromosomal aberrations and complete silencing of reprogramming transgenes in Lu-iPSCs. Chromatin immunoprecipitation (ChIP)-seq, RNA-seq, qRT-PCR, and immunoblot experiments demonstrated complex alterations in DNA methylation with a shift toward a more hypermethylated state; this unexpected finding was also observed following analysis of A549 and Calu-6 lung cancer cells as well as publically available data sets for other iPSC as well as embryonic stem cells (ESC). RNAseq analysis demonstrated that 15,000 genes were commonly altered Lu-iPSC relative to SAEC. A marked up-regulation of PRC-2-related genes was observed. Notably, ASXL3, an epigenetic modifier not previously described in reprogrammed cells or thoracic malignancies, was up-regulated 400-fold in Lu-iPSC. No significant up-regulation of ASXL1 or ASXL2 was observed in Lu-iPSC relative to SAEC. Subsequent qRT-PCR and immunoblot experiments demonstrated over-expression of ASXL3 in SCLC lines relative to NSCLC lines or normal or immortalized respiratory epithelial cells. ASXL3 expression was particularly high in a subset of SCLC lines. Immunohistochemistry experiments confirmed significant up-regulation of ASXL3 in primary SCLC. Knock-down of ASXL3 inhibited proliferation and teratoma formation by Lu-iPSC, and significantly diminished growth of SCLC cells in-vitro and in-vivo. A comprehensive manuscript pertaining to these findings is in the final stage of preparation for peer review. Our Lu-iPSC model may be useful for identification of other novel epigenetic targets for lung cancer therapy. For example, RNA-seq analysis, qRT-PCR and immunoblot experiments demonstrated marked up-regulation of JARID2 in Lu-iPSC relative to parental SAEC. JARID2 is a PRC-2 associated Jumonji protein, which has been implicated in maintenance of pluripotency and regulation of differentiation in normal stem cells as well as EMT in cancer cells. JARID2 is highly enriched at PRC-2 targets on chromatin. Interaction with nucleosomes as well as noncoding RNAs confers specificity of binding of JARID2 to targets. Methylation of JARID2 (K116) by PRC-2 triggers an allosteric modification of PRC-2 which is critical for PRC-2 enzymatic activity. Recent observations that JARID2 interacts with G9a/GLP to mediate methylation of H3K9 indicate that JARID2 has additional repressive functions which are independent of PRC-2. qRT-PCR experiments were performed to examine JARID2 expression in lung cancer cells, which to date has not been examined in a comprehensive manner. This analysis demonstrated a spectrum of expression of JARID2 in NSCLC as well as SCLC lines, with about 50% of the lines exhibiting over-expression of JARID2. Immunoblot experiments confirmed over-expression of JARID2 in lung cancer lines relative to normal respiratory epithelial cells. Subsequent analysis of GENT, Oncomine and TCGA data bases confirmed that JARID2 is over-expressed in NSCLC, and suggested that JARID2 over-expression may be associated with decreased patient survival. Consistent with these observations, shRNA-mediated knock-down JARID2 inhibited proliferation of cultured lung cancer cells, and markedly decreased growth of lung cancer xenografts. Micro-array analysis demonstrated that knock-down of JARID2 affected numerous pathways mediating pluripotency and metabolism in lung cancer cells. Interestingly, mithramycin represses JARID2 in lung cancer cells and xenografts in a dose dependent manner. Collectively, these experiments demonstrate that JARID2 is a novel therapeutic target in lung cancers. We anticipate completing confirmatory experiments and submitting a manuscript for peer review within the next 3-6 months. In more recent studies, microarray analysis of cultured lung cancer cells and xenografts demonstrated that mithramycin decreased expression of musashi-2 (Msi-2), a novel RNA binding protein which mediates self-renewal in normal stem cells and aggressive phenotype of several human cancers. qRT-PCR and immunoblot experiments confirmed that mithramycin depletes Msi-2 in lung cancer cells in a time and dose-dependent manner. Expression levels of Msi-2 were significantly elevated in non-small cell as well as small-cell lung cancer lines relative to normal/immortalized human respiratory epithelial cells (p 0.001). Consistent with these findings, Msi-2 mRNA levels in primary lung cancers were significantly higher than those detected in adjacent paired normal lung parenchyma (p 0.0003). Msi-2 expression was enriched in side populations of cultured lung cancer cells, and was significantly increased in Lu-iPSC. si-RNA-mediated knock-down of Msi-2 decreased expression of Oct4, Nanog and Myc, and transiently inhibited proliferation of lung cancer cells. Attempts to permanently knockdown Msi-2 by shRNA techniques were unsuccessful, suggesting strong selection pressure to maintain Msi-2 expression in these cells. We have obtained tet-inducible lentiviral vectors targeting MSI-2, and experiments are in progress to definitively characterize effects of MSI-2 depletion in lung cancer cells in-vitro and in-vivo. Collectively, these data suggest that pharmacologic depletion of MSI-2 may be a novel strategy for lung cancer therapy. Results of these studies were presented at the World Lung Cancer Conference in September 2015, and a manuscript pertaining to these studies will be submitted for publication once the knock-down and gene expression profiling experiments have been completed.

为了进一步检查调节肺癌中干性干性的表观遗传机制并鉴定这些肿瘤中的新型治疗靶点，我们最近通过杀虫剂（Yamanaka）因素（Yamanaka）因胎病转导的正常小气道上皮细胞（SAEC）而产生了诱导的多能干细胞（IPSC）。 SAEC源自一名57岁的西班牙裔女性非吸烟者。生成了许多肺IPSC（LU-IPSC）克隆，其中一些被随机选择以进行进一步分析。这些Lu-ipsc表现出多能性的标志，包括形态，细胞质碱性磷酸酶表达，SSEA-3，SSEA-3，SSEA-4，SSEA-4，TRA-1-60和TRA-1-81的细胞表面表达，包括Nanog，Oct4和Sall4的干细胞基因的上调，以及与Cytoseramis的表达降低，以及cytosery的表达。光谱核分型和QRT-PCR分析显示没有染色体畸变和LU-IPSC中重编程转基因的完全沉默。染色质免疫沉淀（CHIP）-Seq，RNA-SEQ，QRT-PCR和免疫印迹实验表明，DNA甲基化的复杂改变，朝着更高甲基化的状态转移。在分析了A549和CALU-6肺癌细胞以及其他IPSC以及胚胎干细胞的公开数据集（ESC）之后，还观察到了这一意外发现。 RNASEQ分析表明，相对于SAEC，通常会改变15,000个基因。观察到PRC-2相关基因的明显上调。值得注意的是，在LU-IPSC中，ASXL3是先前未重编程的细胞或胸腔恶性肿瘤中未描述的表观遗传修饰符，在LU-IPSC中被上调了400倍。相对于SAEC，在LU-IPSC中未观察到ASXL1或ASXL2的明显上调。随后的QRT-PCR和免疫印迹实验表明，相对于NSCLC线或正常或永生化的呼吸上皮细胞，SCLC线中ASXL3的过表达。在SCLC线的子集中，ASXL3表达特别高。免疫组织化学实验证实了原代SCLC中ASXL3的显着上调。 ASXL3的敲除抑制了LU-IPSC的增殖和畸胎瘤形成，并显着减少了SCLC细胞在体外和体内的生长。与这些发现有关的全面手稿是准备同行评审的最后阶段。我们的LU-IPSC模型可能有助于鉴定其他新型的肺癌治疗表观遗传靶标。例如，RNA-seq分析，QRT-PCR和免疫印迹实验表明，相对于父母SAEC，LU-IPSC中JARID2的上调明显。 JARID2是一种PRC-2相关的Jumonji蛋白，它与维持多能性和调节正常干细胞的分化以及癌细胞中EMT的调节有关。 JARID2在染色质上的PRC-2靶标高度富集。与核小体以及非编码RNA的相互作用赋予了JARID2与靶标的结合的特异性。 PRC-2对JARID2（K116）的甲基化触发了PRC-2的变构修饰，这对于PRC-2酶促活性至关重要。最近观察到JARID2与G9A/GLP相互作用以介导H3K9的甲基化表明JARID2具有独立于PRC-2的其他抑制功能。进行了QRT-PCR实验，以检查肺癌细胞中的JARID2表达，迄今为止尚未以全面的方式进行检查。该分析表明，NSCLC和SCLC系中JARID2的表达谱，约有50％的线表现出JARID2的过表达。免疫印迹实验证实了相对于正常呼吸性上皮细胞，肺癌系中JARID2的过表达。随后对GENT，ONCOMINE和TCGA数据库的分析证实，JARID2在NSCLC中过表达，并建议JARID2过表达可能与患者生存率降低有关。与这些观察结果一致，SHRNA介导的敲除JARID2抑制了培养的肺癌细胞的增殖，并显着降低了肺癌异种移植的生长。微阵列分析表明，JARID2的敲除影响了许多途径，介导了肺癌细胞中的多能性和代谢。有趣的是，毛霉素以剂量依赖性方式抑制肺癌细胞和异种移植物中的JARID2。总的来说，这些实验表明JARID2是肺癌中新型的治疗靶标。我们预计将完成确认实验，并在接下来的3-6个月内提交手稿进行同行评审。在最近的研究中，对培养的肺癌细胞和异种移植的微阵列分析表明，毛霉素降低了肌肉2（MSI-2）的表达，这是一种新型的RNA结合蛋白，可介导正常干细胞中的自我更新，并在几种人类癌症的侵袭性表达中进行自我更新。 QRT-PCR和免疫印迹实验证实，在时间和剂量依赖性方式中，毛霉素在肺癌细胞中耗尽了MSI-2。相对于正常/永生的人类呼吸性上皮细胞，非小细胞以及小细胞肺癌线的MSI-2表达水平显着升高（P 0.001）。与这些发现一致，原发性肺癌中的MSI-2 mRNA水平明显高于相邻配对的正常肺实质中检测到的MRNA水平（P 0.0003）。 MSI-2表达在培养的肺癌细胞的侧面种群中富集，并在Lu-IPSC中显着增加。 SI-RNA介导的MSI-2敲低降低了Oct4，Nanog和Myc的表达，并瞬时抑制了肺癌细胞的增殖。通过SHRNA技术永久敲除MSI-2的尝试没有成功，这表明在这些细胞中保持MSI-2表达的强大选择压力。我们已经获得了针对MSI-2的TET诱导式慢病毒载体，并且正在进行实验，以确切地表征MSI-2耗竭对肺癌细胞中维特罗和体内的影响。总的来说，这些数据表明，MSI-2的药理耗竭可能是肺癌治疗的新型策略。这些研究的结果在2015年9月的世界肺癌会议上介绍，一旦完成敲低和基因表达谱实验，将提交与这些研究有关的手稿。