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: 10486839
• 负责人: DAVID SCHRUMP
• 金额: $ 170.38万
• 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/10486839
• 关键词: ASCL1 gene; Accounting; Barrett Esophagus; Binding; Bioinformatics; Cancer Biology; Carcinoma; Cell Culture Techniques; Cells; Cessation of life; Chromatin; Clinical; Clone Cells; Code; Comb animal structure; Complex; DNA Methylation; DNase I hypersensitive sites sequencing; Data; Deoxyribonuclease I; Deoxyribonucleases; Development; Diagnosis; Distant; Enhancers; Environmental Exposure; Epigenetic Process; Epithelial Cells; Esophageal Squamous Cell; Evaluation; Exhibits; Family member; Gametogenesis; Gene Amplification; Gene Expression; Gene Expression Regulation; Genes; Genomics; Growth; Histology; Human; Human Cell Line; Hypersensitivity; Immunodeficient Mouse; Immunologics; In Vitro; Intercistronic Region; Introns; Investigation; Knock-out; Link; Luciferases; Lung; Malignant Neoplasms; Malignant Pleural Mesothelioma; Malignant neoplasm of esophagus; Malignant neoplasm of lung; Malignant neoplasm of thorax; Manuscripts; Maps; Mediating; Mediator of activation protein; Metaplasia; Modeling; Molecular; NF1 gene; Neoplasm Metastasis; Neoplasms; Non-Small-Cell Lung Carcinoma; Normal Cell; Oncogenic; Operative Surgical Procedures; Organ; Pathway interactions; Patients; Peer Review; Pharmacology; Polycomb; Population; Preparation; Prevention; Public Health; Publishing; Regimen; Regulatory Element; Reporter; Reproducibility; Sampling; Signal Transduction; Site; Specimen; Structure of respiratory epithelium; Techniques; Teratoma; Testis; Time; Tissues; Transcript; Treatment Protocols; United States; Up-Regulation; Work; Xenograft procedure; airway epithelium; anticancer research; base; cancer cell; cancer stem cell; cancer subtypes; cancer therapy; clinical development; clinically relevant; deep sequencing; digital; epigenomics; experimental study; in vivo; induced pluripotent stem cell; knock-down; lung cancer cell; lung small cell carcinoma; neoplasm therapy; neoplastic cell; new therapeutic target; novel; novel strategies; novel therapeutics; overexpression; pluripotency; promoter; refractory cancer; sex; single cell analysis; single-cell RNA sequencing; stem; stem cell genes; stem cell model; stem cells; stem-like cell; stemness; transcriptome; transcriptome sequencing

Limited information is available pertaining to the potential utility of induced pluripotent stem cells (iPSC) for investigating molecular mechanisms mediating initiation and progression of thoracic malignancies and identifying novel therapeutic targets in these neoplasms. To examine this issue, we reprogrammed normal human small airway epithelial cells (SAEC) to pluripotency using lentiviral transduction of Yamanaka factors. The lung iPSC (Lu-iPSC) exhibited hallmarks of pluripotency in-vitro and in-vivo which coincided with complex alterations in DNA methylation, marked up-regulation of PRC-2-related genes, and modulation of 15,000 other transcripts. To explore the potential clinical relevance of the Lu-iPSC model, we compared transcriptome signatures in two Lu-iPSC clones, 14 small cell lung cancer (SCLC) lines, and 10 non-small cell lung cancer (NSCLC) lines relative to three SAEC cultures. RNA-seq analysis demonstrated that the Lu-iPSC transcriptomes overlap much more with SCLC than NSCLC. Infinium array analysis also indicated that DNA methylation signatures in the Lu-iPSC more closely aligned with SCLC compared to NSCLC. However, IPA did not provide useful information related to specific oncogenic pathways related to SCLC vs Lu-iPSC. To investigate how chromatin landscape contributes to SCLC biology, we expanded our study to perform DNase I hypersensitivity followed by deep sequencing (DNase-seq) to identify regulatory elements defined by the state of chromatin configuration among Lu-iPSC and SCLC relative to SAEC. More than 200,000 DNase 1 Hypersensitivity Sites (DHS) were identified in Lu-iPSC or SCLC but not in SAEC. Approximately 16% of these DHS were shared between Lu-iPSC and SCLC. The majority of DHS were outside promoters, encompassed by introns and intergenic regions, indicating that enhancers were the major contributors of genomic landscape in Lu-iPSC and SCLC. Integration of DHS and transcriptome data indicated that less than 5% of non-promoter differentially open regions (DOR) mapped to the nearest neighbor gene, indicating gene regulation by distant regulatory elements. A subset of DOR was unique to SCLC. Analysis of peak-to-gene links and gene-to-peak links across all samples showed that 95% of genes mapped to at least one open chromatin region, whereas each peak mapped to a mean number of 9 genes. Many of the predicted DOR-to-gene links occurred in clusters where multiple nearby peaks are predicted to be linked to the same gene, suggesting that these clusters function as part of a regulatory unit or enhancer. Bivariate analysis of Genomic Footprint (BaGFoot) identified NF1 family members as having the highest increase in digital footprinting and occupancy within open chromatin sites specifically in SCLC. A comprehensive manuscript pertaining to these studies is in final stages of preparation for peer review. Additional Sex Combs Like-3 (ASXL3), encoding a component of the polycomb deubiquitinase complex not previously described to be up-regulated in reprogrammed cells was identified as an essential mediator of pluripotency in human respiratory epithelia and a novel epigenetic target for small cell lung cancer (SCLC) therapy. ASXL3 over-expression correlated with gene amplification in SCLC. Knock-down of ASXL3 decreased the number and size of teratomas and inhibited growth of SCLC xenografts. Results of these studies were published in Cancer Research. Our recent analysis of single cell RNAs-seq data obtained from reprogramming of 5 human cell lines of different tissue origins revealed that ASXL3 up-regulation coincides with a transition state from normal to fully reprogrammed cells. Additional bioinformatics analysis indicates that ASXL3 is only up-regulated in the ASCL1 SCLC subtype; knock-out of ASCL1 or over-expression of myc which theoretically would drive SCLC from ASCL1 to either NeuroD1 or Yap subtypes does not appear to impact ASXL3 expression in SCLC. Studies are in progress using conditional ASXL3 knock-out as well as time course experiments using a FLAG-tagged sequence CRISPRed into the end of the ASXL3 coding region to examine if ASXL3 expression is essential for establishing and/or maintaining pluripotency during reprogramming in normal cells, elucidate the mechanisms by which ASXL3 expression is regulated in normal cells and cancer cells, identify ASXL3 binding partners, and define molecular and clinical implications of ASXL3 up-regulation in SCLC. Additional bioinformatics analysis indicates that ASXL3 is only up-regulated in the ASCL1 SCLC subtype; knock-out of ASCL1 or over-expression of myc which theoretically would drive SCLC from ASCL1 to either NeuroD1 or Yap subtypes does not appear to impact ASXL3 expression in SCLC. Several additional genes which were noted to be upregulated in a manner similar to ASXL3 in Lu-iPSC and subsequently found to be elevated in SCLC are also under investigation at this time. Based on our encouraging results pertaining to modeling of lung cancers, we have extended our reprogramming efforts to characterize and target epigenomic perturbations in esophageal cancer cells. Normal esophageal squamous epithelial cells (Heepic) as well as h-TERT immortalized metaplastic Barrett's esophageal cells (CP-A) were reprogrammed using established techniques. PCA demonstrated that the Eso-iPSC were fully reprogrammed; however, CP-A-iPSC were not. ASXL3 expression was much higher in the CP-A-iPSC compared to Eso-iPSC. Consistent with the notion that ASXL3 facilitates stemness, Eso-iPSC form teratomas in immunodeficient mice whereas CP-A-iPSC form high grade carcinomas. Also of note, whereas ASXL3 is not detected in 2D cultures of thoracic cancers (except SCLC), ASXL3 expression is upregulated when these cells are transitioned to non-adherent conditions. Lastly, ASXL3 is up-regulated in stem-like cells that we can now reproducibly isolate from primary surgical specimens irrespective of tissue histology. In related efforts, we isolated 24 separate stem-like clones from Lewis lung cancer cells stably transfected with a luciferase reporter construct. These clones exhibit distinct stem cell and cancer-testis gene expression profiles in-vitro, and unique, highly reproducible organ-specific metastases. ASXL3 expression is very high in many of the clones. As such, our unique models now enable us to systematically dissect epigenomic mechanisms contributing to pluripotency and metastatic potential in thoracic malignancies and to evaluate novel pharmacologic and immunologic regimens targeting CSC in these neoplasms. These efforts, which are a major focus of our current investigative work, will facilitate clinical development of novel therapies for thoracic cancers with potentially broad applicability for the treatment of other human malignancies.

有限的信息与诱导多能干细胞（IPSC）的潜在效用有关，以研究介导胸腔恶性肿瘤的起始和进展并鉴定这些肿瘤中新型治疗靶标的分子机制。为了检查这个问题，我们将正常的人类小气道上皮细胞（SAEC）重新编程为使用Yamanaka因子的慢病毒转导。肺IPSC（LU-IPSC）表现出多能体内和体内的多能性标志，与DNA甲基化的复杂变化相吻合，标志着PRC-2相关基因的上调，并调制了15,000个其他转录本。为了探索LU-IPSC模型的潜在临床相关性，我们比较了两个LU-IPSC克隆，14个小细胞肺癌（SCLC）系和10个非小细胞肺癌（NSCLC）的转录组特征。文化。 RNA-seq分析表明，LU-IPSC转录组与SCLC重叠率要比NSCLC多得多。 Infinium阵列分析还表明，与NSCLC相比，LU-IPSC中的DNA甲基化特征与SCLC更紧密地对齐。但是，IPA没有提供与与SCLC与LU-IPSC有关的特定致癌途径有关的有用信息。为了研究染色质景观如何有助于SCLC生物学，我们扩展了研究以执行DNase I超敏反应，然后进行深层测序（DNase-Seq），以鉴定由LU-IPSC和SCLC之间染色质构型所定义的调节元素。在LU-IPSC或SCLC中鉴定出超过200,000个DNase 1超敏反应位点（DHS），但在SAEC中未发现。这些DHS中约有16％在Lu-IPSC和SCLC之间共享。大多数DHS都是在启动子外部，包括内含子和基因间区域，表明增强子是LU-IPSC和SCLC基因组景观的主要贡献者。 DHS和转录组数据的整合表明，映射到最近的邻居基因的非启动者差异开放区域（DOR）的不到5％，表明通过远处调节元件调节基因。 DOR的子集是SCLC独有的。对所有样品的峰到基因链路和基因之间的峰值分析表明，95％的基因映射到至少一个开放的染色质区域，而每个峰映射到平均9个基因的峰值。许多预测的DOR到基因链接发生在预测附近多个峰与同一基因相关的集群中，这表明这些簇作为调节单元或增强子的一部分起作用。基因组足迹（Bagfoot）的双变量分析确定NF1家族成员在SCLC中专门在开放式染色质站点内数字足迹和占用率最高。与这些研究有关的全面手稿是准备同行评审的最后阶段。其他性梳子（例如-3（ASXL3））编码以前未被描述为在重编程细胞中上调的Polycomb去泛素酶配合物的成分，被鉴定为人类呼吸性上皮elia中多能性的必不可少的介体，用于小细胞肺肺肺肺部的新型表观遗传靶癌症（SCLC）疗法。 ASXL3过表达与SCLC中的基因扩增相关。 ASXL3的敲除减少了畸胎瘤的数量和大小，并抑制了SCLC异种移植物的生长。这些研究的结果发表在癌症研究中。我们最近对通过重编程不同组织起源的5个人类细胞系获得的单细胞RNAS-seq数据的分析表明，ASXL3上调与从正常到完全重编程的细胞的过渡态相吻合。其他生物信息学分析表明，ASXL3仅在ASCL1 SCLC亚型中上调。从理论上将SCLC从ASCL1驱动到NeuroD1或YAP亚型的ASCL1或MYC的过表达似乎不会影响SCLC中的ASXL3表达。使用有条件的ASXL3敲除以及使用标志标记的序列CRIS进行的时间课程实验正在进行研究，阐明在正常细胞和癌细胞中调节ASXL3表达的机制，鉴定ASXL3结合伴侣，并定义SCLC中ASXL3上调的分子和临床意义。其他生物信息学分析表明，ASXL3仅在ASCL1 SCLC亚型中上调。从理论上将SCLC从ASCL1驱动到NeuroD1或YAP亚型的ASCL1或MYC的过表达似乎不会影响SCLC中的ASXL3表达。此时，此时也正在研究其他几个基因，这些基因以类似于LU-IPSC的ASXL3的方式上调，此时也正在SCLC中发现升高。基于我们与肺癌建模有关的令人鼓舞的结果，我们扩大了重编程的努力，以表征和靶向食管癌细胞中的表观基因组扰动。使用已建立的技术对正常的食管鳞状上皮细胞（Heepic）以​​及H-TERT永生化生型巴雷特食管细胞（CP-A）进行了重编程。 PCA证明ESO-IPSC已完全重编程。但是，CP-A-IPSC不是。与ESO-IPSC相比，CP-A-IPSC的ASXL3表达要高得多。与ASXL3促进茎的概念一致，ESO-IPSC在免疫缺陷的小鼠中形成畸胎瘤，而CP-A-IPSC形成高级癌。同样值得注意的是，虽然在胸腔癌的2D培养物中未检测到ASXL3（SCLC除外），但当这些细胞转变为非粘附条件时，ASXL3表达会上调。最后，在干细胞中，ASXL3被上调，我们现在可以从原发性手术标本中可重复分离，而与组织学组织学无关。在相关的工作中，我们与用荧光素酶报告基因构建体稳定转染的刘易斯肺癌细胞分离了24个单独的茎状克隆。这些克隆表现出独特的干细胞和癌症基因表达谱，并具有独特的，高度可重现的器官特异性转移。在许多克隆中，ASXL3表达非常高。因此，我们的独特模型现在使我们能够系统地剖析胸腔恶性肿瘤中多能和转移潜力的表观基因组机制，并评估针对这些肿瘤中CSC的新型药理学和免疫学方案。这些努力是我们当前调查工作的重点的重点，它将促进针对胸腔癌的新疗法的临床开发，具有广泛适用于治疗其他人类恶性肿瘤的疗法。