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: 7965738
• 负责人: Thomas Ried
• 金额: $ 75.82万
• 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/7965738
• 关键词: 13q; 18q; 20p; 20q; Aneuploid Cells; Aneuploidy; Apoptosis; Biological; Biological Assay; Biopsy; Cancer cell line; Cancerous; Candidate Disease Gene; Carcinoma; Caspase; Cataloging; Catalogs; Cell Line; Cell Survival; Cells; Chromosome Arm; Chromosome Band; Chromosome Mapping; Chromosomes; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 20; Classification; Colon; Colon Carcinoma; Colonic Neoplasms; Colorectal Cancer; Complex; DNA copy number; Data; Development; Developmental Process; Diagnosis; Diploid Cells; Double-Stranded RNA; Down-Regulation; Endoribonucleases; Evidence based treatment; Fluorouracil; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Silencing; Gene Targeting; Genes; Genetic; Genomics; Genus Cola; Goals; HMGA1 gene; Hour; Immune response; International Union Against Cancer; Large Intestine Carcinoma; Lipids; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Messenger RNA; Modification; Molecular Profiling; Mucous Membrane; Mutation; Nuclear Pore Complex Proteins; Nucleic Acids; Nucleotides; Oligonucleotide Microarrays; Oligonucleotides; Oncogenes; Operon; PTGS2 gene; Pathway interactions; Pattern; Phenotype; Play; Predisposition; Prevalence; Process; RNA Interference; 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; TGFB1 gene; Techniques; Technology; Therapeutic Intervention; Time; Transfection; Up-Regulation; Validation; Vascular Endothelial Growth Factors; Viral; WNT1 gene; base; cancer cell; carcinogenesis; chemotherapeutic agent; comparative genomic hybridization; endoribonuclease; functional genomics; gene function; human DICER1 protein; insight; lymph nodes; neoplastic cell; novel; pathogen; promoter; rectal; research study; small hairpin RNA; small molecule; 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. Identification of a putative oncogene in 13q involved in colorectal cancer: 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. Apoptosis screen in colorectal cancer cell lines: One of the pathways of malignant transformation includes the ability of cancerous cells to avoid apoptosis. We are currently exploring the role of genes that regulate apoptosis in colorectal cancer cells. Using a commercial set of siRNAs, we have conducted screens of siRNAs targeting 418 genes considered to have a role in apoptosis. Two siRNAs were examined for each gene and the screen was performed using the colorectal cancer cell line SW480. The effect of each siRNA was evaluated with a viability assay. In order to identify potentially novel as well as combination therapeutic interventions, we used RNAi with and without the presence of 5-FU, a chemotherapeutic agent used for the treatment of colon cancer. Cells were seeded on a complex of siRNA and cationic lipid in 96-well plates for reverse transfection. When appropriate, 5-FU (80M corresponding to the EC50) was added after 48 hours, and cell viability was assayed after a further 72 hours of incubation. The addition of 5-FU uniformly decreased the viability of the cells by approximately 50%. We identified numerous genes that further increased the sensitivity of SW480 cells to 5-FU. Stringent filtering of decreased viability by siRNAs in the absence of 5-FU treatment led us to focus on two genes as potential novel targets that reduce cell viability: a gene that belongs to the class of nucleoporins, and a caspase-associated gene. The siRNAs to both genes reduced SW480 viability by greater than 40%. We have fully characterized the reduction in mRNA levels mediated by the siRNAs corresponding to these two genes and consistently observe that their down-regulation reduces the viability of SW480 cells. As a further validation of their effect, we have silenced the caspase-associated gene in additional cell lines. The reduction in cell viability was more pronounced in another aneuploid cell line, SW837, than in a diploid cell line, SW48, indicating a potential susceptibility of aneuploid cell lines to the down-regulation of this target gene. We are currently investigating the functional mechanism responsible for this effect.

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。匹配的样本。 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。 在结直肠癌的13Q中鉴定了假定的致癌基因：13染色体是大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。结直肠癌细胞系中的凋亡筛查：恶性转化的途径之一包括癌细胞避免凋亡的能力。我们目前正在探索调节大肠癌细胞中凋亡的基因的作用。使用一组商业的siRNA，我们进行了靶向418个基因的siRNA筛选，该基因被认为在凋亡中起作用。对每个基因检查了两个siRNA，并使用结直肠癌细胞系SW480进行筛选。通过使用生存力测定法对每个siRNA的效果进行评估。为了鉴定潜在的新颖和组合治疗干预措施，我们使用有或不存在5-FU的RNAi，这是一种用于治疗结肠癌的化学治疗剂。将细胞在96孔板中的siRNA和阳离子脂质的络合物中播种，进行反转染。在适当的时候，在48小时后添加5-FU（80m与EC50相对应），并在孵育72小时后测定细胞活力。 5-FU的添加均匀地降低了细胞的生存能力约50％。我们发现了许多基因，这些基因进一步提高了SW480细胞对5-FU的敏感性。在没有5-FU治疗的情况下，严格过滤siRNA的生存能力降低，导致我们专注于两个基因，作为降低细胞生存力的潜在新靶标：属于核孔蛋白类和caspase相关基因的基因。这两个基因的siRNA都使SW480的生存能力降低了40％。 我们已经充分表征了与这两个基因相对应的siRNA介导的mRNA水平的降低，并始终观察到它们的下调降低了SW480细胞的生存能力。为了进一步验证它们的作用，我们已经在其他细胞系中沉默了与caspase相关的基因。与二倍体细胞系SW48相比，在另一个非整倍性细胞系SW48中，细胞活力的降低更为明显，表明非整倍体细胞系对该靶基因下调的潜在敏感性。我们目前正在研究负责此效果的功能机制。