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: 10014669
• 负责人: DAVID SCHRUMP
• 金额: $ 146.38万
• 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/10014669
• 关键词: Aerodigestive Tract; Antibodies; Asbestos; Benign; Bile Acids; Biology; Carcinoma; Cell Cycle Regulation; Cell model; Cells; Cessation of life; Chromatin; Clinical; Clone Cells; Collaborations; Comb animal structure; Communication; Complex; DNA Methylation; DNase I hypersensitive sites sequencing; Data; Deoxyribonuclease I; Deoxyribonucleases; Development; Disease; Distant; Dose; Enhancers; Environmental Carcinogens; Environmental Risk Factor; Epigenetic Process; Epithelial Cells; Esophageal; Esophageal carcinoma; Evaluation; Exhibits; Expression Profiling; Family member; GABA Receptor; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genomics; Growth; Hepatic Stellate Cell; Histology; Histone Code; Human; Hypersensitivity; Immune; Immunologics; In Vitro; Intercistronic Region; Interleukin-15; Introns; Investigation; Link; Liver Fibrosis; Lung; Malignant - descriptor; Malignant Neoplasms; Malignant Pleural Mesothelioma; Malignant neoplasm of esophagus; Malignant neoplasm of lung; Malignant neoplasm of thorax; Manuscripts; Maps; Mediating; Mesothelial Cell; Metabolic Pathway; Metastatic to; Methods; MicroRNAs; Mitotic; Modeling; Molecular; NF1 gene; Neoplasms; Non-Small-Cell Lung Carcinoma; Oncogenic; PLK1 gene; PTEN gene; Pathway interactions; Peer Review; Pharmacology; Phenotype; Polycomb; Population; Preparation; Primary Neoplasm; Production; Property; Publishing; RNA analysis; Receptor Signaling; Regenerative Medicine; Regimen; Regulatory Element; Repression; Role; Sampling; Side; Signal Transduction; Site; Smoker; Solid Neoplasm; Stem cells; Study models; Teratoma; Tight Junctions; Time; Tobacco; Tobacco smoke; Transcript; Transplantation; Tumor Suppressor Genes; United States; Untranslated RNA; Up-Regulation; Work; anticancer research; cancer cell; cancer stem cell; cancer therapy; carcinogenesis; cigarette smoke; clinical development; cytokine; deep sequencing; differential expression; digital; epigenomics; experimental study; genome-wide; in vivo; induced pluripotent stem cell; knock-down; mouse model; neoplasm therapy; new therapeutic target; novel; novel strategies; novel therapeutics; oncology; overexpression; pluripotency; promoter; respiratory; sex; stem; stemness; transcriptome; transcriptome sequencing

In addition to having broad applicability as cellular therapies in regenerative medicine, transplantation, and oncology, induced pluripotent stem cells (iPSC) may be useful models for studying a variety of benign and malignant diseases. Limited information is available pertaining to the potential utility of iPSC for investigating molecular mechanisms mediating initiation and progression of thoracic cancers and identifying novel therapeutic targets in these malignancies. To examine these issues, we recently generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) by lentiviral transduction of Yamanaka factors. The lung iPSC (Lu-iPSC) exhibited complex alterations in DNA methylation, with marked up-regulation of PRC-2-related genes, and modulation of 15,000 additional genes. Additional Sex Combs Like-3 (ASXL3), an epigenetic modifier not previously described in reprogrammed cells or any human malignancy, was up-regulated 400-fold in Lu-iPSC and was markedly over-expressed in SCLC lines and primary tumors. Knock-down of ASXL3 inhibited proliferation and teratoma formation by Lu-iPSC, and significantly diminished growth of SCLC cells in-vitro and in-vivo. These initial studies were published in Cancer Research. Ongoing studies are focused on the mechanisms and clinical implications of ASXL3 up-regulation in SCLC. These studies have progressed a bit slower than anticipated due to the number of alternative transcripts, difficulty in constitutively over-expressing ASXL3 and lack of high-titer ASXL3-specific antibodies. Several additional genes which were noted to be upregulated in Lu-iPSC and subsequently found to be elevated in primary lung cancers are under investigation at this time. To further examine the potential of Lu-iPSC to model lung cancer stem cells, RNA- seq experiments were used to examine gene expression profiles in 2 Lu-iPSC clones, 10 SCLC lines, and 10 NSCLC lines relative to SAEC. 6318, 12051, and 6504 genes were differentially expressed in Lu-iPSC, SCLC, and NSCLC lines, respectively. 2804 differentially expressed genes (44%) in Lu-iPSC were unique to these cells. Approximately 1/3rd of genes differentially expressed in Lu-iPSC were modulated in a histology specific manner. 1515 genes representing 24%, 12%, and 23% of differentially expressed genes in Lu-iPSC, SCLC, and NSCLC lines, respectively, were commonly regulated across Lu-iPSC, SCLC and NSCLC. Top canonical pathways included cell cycle control of chromosomal replication, mitotic roles of Polo-like kinases, and role of cytokines in mediating communication between immune cells. 3499 genes were commonly regulated in SCLC and NSCLC; metabolic pathways were enriched in this gene subset. 1937 genes were commonly modulated in SCLC and Lu-iPSC; top canonical pathways included GABA receptor signaling, TREM1 signaling, and IL-15 production. In contrast, only 102 genes were commonly modulated in Lu-iPSC and NSCLC; top canonical pathways included tight junction signaling, hepatic fibrosis/hepatic stellate cell activation, and PTEN signaling. Pluripotency pathways modulated in Lu-iPSC were not enriched in SCLC or NSCLC, possibly due to limited numbers of pluripotent cells in these lines. RNA-seq is being performed on side population (SP) fractions of SCLC and NSCLC lines to identify novel genes/pathways mediating stemness that could be potential targets for lung cancer therapy. Collectively, these findings suggest that Lu-iPSC models may prove useful for examining mechanisms of pulmonary carcinogenesis, and developing novel regimens targeting pluripotency for lung cancer therapy. In collaboration with Dr. Gordon Hager's team, we have recently performed DNAse hypersensitivity sequencing (DHS) experiments to examine epigenomic perturbations in lung cancer cells in a genome-wide manner. 14 SCLC, 10 NSCLC and Lu-iPSC were compared to SAEC. Re-analysis of RNA-seq data confirmed that the Lu-iPSC transcriptome overlaps much more with SCLC than NSCLC. Infinium array analysis also indicated that DNA methylation signatures in iPSC more closely aligned with SCLC compared to NSCLC. 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 LuiPS 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. Further analysis demonstrated that the vast 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), a computational approach that combines genomic footprinting and genome-wide chromatin accessibility, identified NF1 family members as having the highest increase in digital footprinting and occupancy within open chromatin sites specifically in SCLC. Interestingly, NF1 has been recently linked to metastatic progression in S a mouse model of SCLC. A comprehensive manuscript pertaining to these studies is in final stages of preparation for peer review. In related efforts, we have developed reliable methods for isolation and propagation of cancer cells which exhibit stem-like properties from primary lung cancers. These putative cancer stem cells (CSC)/cancer progenitor cells (CPC) grow as spheroids and exhibit high level expression of pluripotency-associated genes when cultured under non-adherent conditions, and can be reproducibly isolated from subsequently established adherent lung cancer lines. As such, our unique models now enable us to systematically dissect epigenomic mechanisms contributing to pluripotency in pulmonary carcinomas and to evaluate novel pharmacologic and immunologic regimens targeting CSC in these malignancies. These efforts, which are a major focus of our investigative work, will facilitate clinical development of novel therapies for lung cancers with potentially broad applicability for the treatment of other solid tumors.

除了在再生医学，移植和肿瘤学的细胞疗法中具有广泛的适用性外，诱导的多能干细胞（IPSC）可能是研究各种良性和恶性疾病的有用模型。有限的信息与IPSC的潜在效用有关，用于研究介导胸腔癌的起始和进展并鉴定这些恶性肿瘤中新型治疗靶标的分子机制。为了检查这些问题，我们最近通过Yamanaka因子的慢病毒转导从正常人类小气道上皮细胞（SAEC）中产生了诱导的多能干细胞（IPSC）。肺IPSC（LU-IPSC）在DNA甲基化中表现出复杂的改变，并在PRC-2相关基因上明显上调，并调节了15,000个其他基因。额外的性梳子（例如3（ASXL3））是一种先前在重编程细胞或任何人类恶性肿瘤中未描述的表观遗传修饰剂，在Lu-IPSC中被上调了400倍，在SCLC系和原发性肿瘤中明显过表达。 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。这些最初的研究发表在癌症研究中。正在进行的研究集中在SCLC中ASXL3上调的机制和临床意义上。由于替代转录本的数量，组成性过度表达的ASXL3的难度以及缺乏高滴度ASXL3特异性抗体，这些研究的进展比预期的要慢一些。目前正在研究原发性肺癌中的其他几个基因，这些基因在原发性肺癌中被发现升高，此时正在研究中。为了进一步检查LU-IPSC对肺癌干细胞建模的潜力，使用RNA-SEQ实验来检查相对于SAEC的2个LU-IPSC克隆，10个SCLC线和10个NSCLC线中的基因表达谱。 6318、12051和6504基因分别在Lu-IPSC，SCLC和NSCLC系中差异表达。 LU-IPSC中2804个差异表达的基因（44％）是这些细胞独有的。以组织学特异性方式调节了在LU-IPSC中差异表达的基因的大约1/3。在LU-IPSC，SCLC和NSCLC系中，分别在LU-IPSC，SCLC和NSCLC中通常调节了1515个基因，分别为LU-IPSC，SCLC和NSCLC系中的差异表达基因的24％，12％和23％的基因。顶级规范的途径包括染色体复制的细胞周期控制，polo样激酶的有丝分裂作用以及细胞因子在介导免疫细胞之间通信中的作用。 3499个基因通常在SCLC和NSCLC中受到调节；代谢途径在该基因子集中富集。 1937年，基因通常在SCLC和LU-IPSC中进行调节。顶级规范途径包括GABA受体信号传导，TREM1信号传导和IL-15产生。相比之下，在Lu-IPSC和NSCLC中通常只调制102个基因。顶部规范的途径包括紧密的连接信号，肝纤维化/肝星状细胞激活和PTEN信号传导。在LU-IPSC中调节的多能途径未富集在SCLC或NSCLC中，这可能是由于这些线中的多能细胞数量有限。 RNA-seq正在SCLC和NSCLC系的侧种群（SP）部分进行，以鉴定可能是肺癌治疗的潜在靶标的新型基因/途径。总的来说，这些发现表明，LU-IPSC模型可能被证明可用于检查肺癌发生的机制，并开发针对多能性肺癌治疗的新型方案。与Gordon Hager博士的团队合作，我们最近进行了DNase超敏反应（DHS）实验，以全基因组的方式检查肺癌细胞中的表观基因组扰动。将14个SCLC，10个NSCLC和LU-IPSC与SAEC进行了比较。 RNA-seq数据的重新分析证实，LU-IPSC转录组与SCLC的重叠相比，与NSCLC更大。 Infinium阵列分析还表明，与NSCLC相比，IPSC中IPSC中的DNA甲基化特征更紧密地对齐。 IPA没有提供与与SCLC与Lu-IPSC相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对SCLC生物学有助于，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），以识别由LUIPS和SCLC之间染色质构型定义的调节元素，相对于SAEC。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。进一步的分析表明，绝大多数DHS都在启动子外部，包括内含子和基因间区域，表明增强子是LU-IPSC和SCLC中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）的双变量分析是一种结合基因组足迹和全基因组染色质的可及性的计算方法，确定NF1家族成员在SCLC中专门在开放的染色质位点内的数字足迹和占用率最高。有趣的是，NF1最近与S SCLC小鼠模型中的转移性进展相关。与这些研究有关的全面手稿是准备同行评审的最后阶段。在相关的工作中，我们开发了可靠的方法，用于分离和传播癌细胞，这些方法表现出来自原发性肺癌的茎样特性。这些假定的癌症干细胞（CSC）/癌症祖细胞（CPC）随着球体的生长，当在非粘附条件下培养时表现出多能相关基因的高水平表达，并且可以从随后确定的随后建立的辅助肺癌系中复制。因此，我们的独特模型现在使我们能够系统地剖析有助于肺癌多功能的表观基因组机制，并评估针对这些恶性肿瘤中CSC的新型药理和免疫学方案。这些努力是我们调查工作的主要重点，它将促进针对具有广泛适用于其他实体瘤的肺癌的新型疗法的临床开发。