Identification and functional characterization of colorectal cancer genes

结直肠癌基因的鉴定和功能表征

• 批准号: 8349193
• 负责人: Thomas Ried
• 金额: $ 70.42万
• 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/8349193
• 关键词: 13q; 18q; 20p; 20q; Affect; Aneuploidy; Biological; Biopsy; Cancer cell line; Candidate Disease Gene; Carcinoma; Cataloging; Catalogs; Cell Survival; Chromosome Arm; Chromosome Band; Chromosome Mapping; Chromosomes; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 20; Colon; Colon Carcinoma; Colonic Neoplasms; Colorectal Cancer; Complex; DNA copy number; Data; Development; Developmental Process; Diagnosis; Double-Stranded RNA; Down-Regulation; Endoribonucleases; Evidence based treatment; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Silencing; Gene Targeting; Genes; Genetic; Genomics; Goals; HMGA1 gene; Immune response; Immunologic Surveillance; International Union Against Cancer; Large Intestine Carcinoma; Link; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Modeling; Modification; Molecular Profiling; Mucous Membrane; Mutation; Nucleic Acids; Nucleotides; Oligonucleotide Microarrays; Oligonucleotides; Oncogenes; Operon; PLK1 gene; PTGS2 gene; Pathway interactions; Pattern; Phenotype; Play; Prevalence; Process; RNA Interference; RRM2 gene; Rectal Adenocarcinoma; Rectal Cancer; Reporting; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Screening procedure; Series; Signal Pathway; Signal Transduction; Small Interfering RNA; Specimen; Staging; Systems Biology; TACSTD2 gene; TGFB1 gene; Techniques; Technology; Time; Up-Regulation; Validation; Vascular Endothelial Growth Factors; Viral; WNT1 gene; base; cancer cell; carcinogenesis; comparative genomic hybridization; endoribonuclease; functional genomics; gene function; human DICER1 protein; insight; lymph nodes; neoplastic cell; novel; overexpression; pathogen; promoter; rectal; research study; small hairpin RNA; small molecule; therapeutic target; tool; tumor; tumorigenesis

The transcriptome of primary colon cancer: In order to characterize patterns of global transcriptional deregulation in primary colon carcinomas, we performed gene expression profiling of 73 tumors (UICC stage II, n=33 and UICC stage III, n=40) using oligonucleotide microarrays. For 30 of the tumors, expression profiles were compared to those from matched normal mucosa samples. We identified a set of 1,950 genes with highly significant deregulation between tumors and mucosa samples (P<1e-7). A significant proportion of these genes mapped to chromosome 20 (P=0.01). Seventeen genes had a greater than five-fold average expression difference between normal colon mucosa and carcinomas, including up-regulation of MYC and of HMGA1, a putative oncogene. Furthermore, we identified 68 genes that were significantly differentially expressed between lymph node negative and positive tumors (P<0.001), the functional annotation of which revealed a preponderance of genes that play a role in cellular immune response and surveillance. The microarray-derived gene expression levels of 20 deregulated genes were validated using quantitative real-time RT-PCR in more than 40 tumor and normal mucosa samples with good concordance between the techniques. Finally, we established a relationship between specific genomic imbalances, which were mapped for 32 of the analyzed colon tumors by comparative genomic hybridization, and alterations of global transcriptional activity. Previously, we had conducted a similar analysis of primary rectal carcinomas. The systematic comparison of colon and rectal carcinomas revealed a significant overlap of genomic imbalances and transcriptional deregulation, including activation of the Wnt/b-catenin signaling cascade, suggesting similar pathogenic pathways. The transcriptome of primary rectal adenocarcinomas: In order to identify genetic alterations underlying rectal carcinogenesis, we used global gene expression profiling of a series of 17 locally advanced rectal adenocarcinomas and 20 normal rectal mucosa biopsies on oligonucleotide arrays. A total of 351 genes were differentially expressed (p< 1.0e-7) between normal rectal mucosa and rectal carcinomas, 77 genes had a greater than five-fold difference, and 85 genes always had at least a two-fold change in all of the matched samples. 12 genes satisfied all three of these criteria. Altered expression of genes such as PTGS2 (COX2), WNT1, TGFB1, VEGF and MYC was confirmed, while our data for other genes like PPARD and LEF1 were inconsistent with previous reports. In addition, we found deregulated expression of many genes whose involvement in rectal carcinogenesis has not been reported. By mapping the genomic imbalances in the tumors using comparative genomic hybridization, we could show that DNA copy number gains of recurrently aneuploid chromosome arms 7p, 8q, 13q, 18q, 20p and 20q correlated significantly with their average chromosome arm expression profile. Taken together, our results demonstrate that both the high-level, significant transcriptional deregulation of specific genes and general modification of the average transcriptional activity of genes residing on aneuploid chromosomes coexist in rectal adenocarcinomas. A genomic strategy for the functional validation of colorectal cancer genes identifies potential therapeutic targets: Chromosome 13 is one of the most commonly gained chromosomes in colorectal carcinomas. Despite the prevalence of this aneuploidy, it is not clear which gene or genes are the targets of this chromosomal imbalance. We have therefore analyzed a set of 73 colon cancer samples using a NCI oligonucleotide arrays (22K) using the Operon V2 oligo set (see above). A previous study using rectal cancer showed that a high number of differentially expressed (upregulated) genes mapped to chromosome 13. We also performed array CGH in a set of 34 tumors included in the previous study. For this purpose, we used a high-resolution oligonucleotide-based microarray (185K) from Agilent. From this study, we were able to separate a set of tumors that show localized amplifications on specific chromosomal bands. This allowed identification of regions of minimal overlap on this chromosome. The concordant analysis of a significant number of samples using aCGH and expression profiling reduced the number of potential candidate genes to a list of some 39 genes residing on chromosome 13, whose functional analysis in colorectal cancers is now underway. These efforts resulted in the identification of some 11 genes whose down-regulation profoundly reduced the viability of colorectal cancer cell lines. We are now in the process of conducting time course experiments after knockdown to map the functional space in which these genes operate, and to identify small molecules that show similar gene expression changes as the siRNA mediated knockdown. Genes that are highly overexpressed in tumor cells can be required for tumor cell survival, and have the potential to be selective therapeutic targets. In an attempt to identify such targets, we combined a functional genomics and a systems biology approach to assess the consequences of RNAi-mediated silencing of overexpressed genes that were selected from 140 gene expression profiles from colorectal cancers (CRC) and matched normal mucosa. In order to identify credible models for in-depth functional analysis, we first confirmed the overexpression of these genes in 25 different CRC cell lines. We then identified five candidate genes that profoundly reduced the viability of CRC cell lines when silenced with either siRNAs or shRNAs, i.e., HMGA1, TACSTD2, RRM2, RPS2, and NOL5A. These genes were further studied by systematic analysis of comprehensive gene expression profiles generated following siRNA-mediated silencing. Exploration of these RNAi-specific gene expression signatures allowed the identification of the functional space in which the five genes operate, and showed enrichment for cancer specific signaling pathways, some known to be involved in CRC. By comparing the expression of the RNAi signature genes with their respective expression levels in an independent set of primary rectal carcinomas we could recapitulate these defined RNAi signatures, therefore establishing the biologically relevance of our observations. This strategy identified the signaling pathways that are affected by the prominent oncogenes HMGA1 and TACSTD2, established a yet unknown link between RRM2 and PLK1, and identified RPS2 and NOL5A as promising potential therapeutic targets in CRC.

原发性结肠癌的转录组：为了表征原发性结肠癌中全球转录放松管制的模式，我们使用寡核苷酸微阵列进行了73个肿瘤（UICC II期，N = 33和UICC III，N = 40）的基因表达分析（UICC II期，N = 33，N = 40）。在30个肿瘤中，将表达谱与匹配的正常粘膜样品的表达谱进行了比较。我们鉴定了一组1,950个基因，在肿瘤和粘膜样品之间具有高度显着的放松管制（P <1E-7）。这些基因中很大一部分映射到20号染色体（p = 0.01）。 17个基因在正常结肠粘膜和癌之间的平均表达差超过五倍以上，包括MYC和HMGA1的上调，这是一种推定的致癌基因。此外，我们确定了68个基因，这些基因在淋巴结阴性和阳性肿瘤之间得到了显着差异表达（P <0.001），其功能注释揭示了在细胞免疫反应和表面上发挥作用的基因优势。使用定量的实时RT-PCR在40多个肿瘤和正常粘膜样品中验证了20个失控基因的微阵列衍生基因表达水平，这些技术之间具有良好的一致性。最后，我们建立了特定的基因组失衡之间的关系，这些失衡是通过比较基因组杂交和全球转录活性改变的32个分析结肠肿瘤映射的。以前，我们已经对原发性直肠癌进行了类似的分析。结肠和直肠癌的系统比较表明，基因组失衡和转录放松管制的显着重叠，包括激活Wnt/B-catenin信号级联，表明相似的致病途径。原发性直肠腺癌的转录组：为了鉴定直肠癌发生的基础遗传改变，我们使用了一系列17种局部晚期直肠腺癌的全球基因表达分析和20个正常直肠直肠粘膜活检。正常直肠粘膜和直肠癌之间总共差异表达了351个基因（p <1.0e-7），77个基因的差异大于五倍以上，而85个基因的所有差异始终至少有两个变化。匹配的样品。 12个基因满足了所有这三个标准。确认了基因（例如PTGS2（COX2），Wnt1，TGFB1，VEGF和MYC）的基因表达改变，而我们对PPARD和LEF1（例如PPARD和LEF1）的数据与以前的报告不一致。此外，我们发现尚未报道许多基因的表达，这些基因的参与尚未报道。通过使用比较基因组杂交映射肿瘤中的基因组失衡，我们可以表明，复发性染色体染色体臂7p，8q，13q，13q，18q，20p和20q的DNA拷贝数增加与其平均染色体臂表达显着相关。综上所述，我们的结果表明，对特定基因的高级，显着的转录放松管制以及对直肠腺癌中非整倍体染色体共存的基因的平均转录活性的一般修饰。结直肠癌基因功能验证的基因组策略确定了潜在的治疗靶标：13染色体是结直肠癌中最常见的染色体之一。尽管这种非整倍性的流行率，但尚不清楚哪种基因或基因是该染色体失衡的靶标。因此，我们使用NCI寡核苷酸阵列（22K）使用操纵子V2寡核糖集（见上文）分析了一组73个结肠癌样品（22K）。先前使用直肠癌的研究表明，映射到13号染色体的大量差异表达（上调的）基因。我们还在先前研究中包括的34个肿瘤中进行了阵列CGH。为此，我们使用了Agilent的高分辨率寡核苷酸微阵列（185K）。从这项研究中，我们能够分离一组在特定染色体带上显示局部扩增的肿瘤。这允许鉴定该染色体上最小重叠区域。使用ACGH和表达分析对大量样品进行的一致分析将潜在候选基因的数量降低到位于13号染色体上的约39个基因的列表，该染色体13上的基因的功能分析正在进行中。这些努力导致了大约11个基因的鉴定，这些基因的下调大大降低了结直肠癌细胞系的生存能力。现在，我们正在进行敲低后进行时间课程实验以绘制这些基因工作的功能空间，并鉴定出与siRNA介导的敲低相似基因表达的小分子。在肿瘤细胞存活中可能需要高度表达在肿瘤细胞中的基因，并且具有选择性治疗靶标的潜力。为了识别此类靶标，我们结合了一种功能性基因组学和系统生物学方法，以评估RNAi介导的过表达基因的沉默的后果，这些基因是从结直肠癌（CRC）（CRC）中选择的140个基因表达谱并匹配正常粘膜的后果。为了识别用于深入功能分析的可靠模型，我们首先证实了这些基因在25种不同的CRC细胞系中的过表达。然后，我们确定了五个候选基因，这些基因用siRNA或shRNAS（即HMGA1，TACSTD2，RRM2，RPS2和NOL5A）沉默，从而大大降低了CRC细胞系的生存能力。通过系统分析siRNA介导的沉默后产生的综合基因表达谱进行进一步研究这些基因。对这些RNAi特异性基因表达特征的探索允许鉴定五个基因作战的功能空间，并显示出癌症特异性信号传导途径的富集，其中一些人已知参与CRC。通过比较RNAi特征基因的表达与它们在独立的原发性直肠癌中的表达水平各自的表达水平，我们可以概括这些定义的RNAi特征，因此建立了观察结果的生物学相关性。该策略确定了受突出的Oncogenes HMGA1和TACSTD2影响的信号传导途径，建立了RRM2和PLK1之间但未知的联系，并确定RPS2和NOL5A是CRC中有希望的潜在治疗靶标。