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: 7733266
• 负责人: Thomas Ried
• 金额: $ 69.47万
• 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/7733266
• 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; Class; Classification; Colon; Colon Carcinoma; Colonic Neoplasms; Colorectal; Colorectal Cancer; Complex; DNA copy number; Data; Development; Developmental Process; Diagnosis; Diploid Cells; Double-Stranded RNA; Down-Regulation; Endoribonucleases; Epithelium; 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; Localized; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Medical Surveillance; Messenger RNA; Modification; Molecular Profiling; Mucous Membrane; Mutation; Negative Lymph Node; Nuclear Pore Complex Proteins; Nucleic Acids; Nucleotides; Numbers; Oligonucleotide Microarrays; Oligonucleotides; Oncogenes; Operon; PTGS2 gene; Pathway interactions; Pattern; Phenotype; Play; Predisposition; Prevalence; Process; Purpose; 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; Transcriptional Activation; Transfection; Up-Regulation; Validation; Vascular Endothelial Growth Factors; Viral; WNT1 gene; base; cancer cell; carcinogenesis; chemotherapeutic agent; comparative genomic hybridization; cyclooxygenase 2; design; endoribonuclease; established cell line; functional genomics; gene function; human DICER1 protein; insight; neoplastic cell; novel; pathogen; promoter; rectal; small hairpin RNA; 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 less than 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 less than 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 less than 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. 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. Systematic knockdown of candidate colorectal cancer genes using RNA interference: The comprehensive and systematic analyses of gene expression profiles in primary colorectal carcinomas have suggested the involvement of several key regulators of malignant transformation of colorectal epithelium. We are now engaged in exploring the effects of these specific transcriptional changes on the transcriptome of colorectal cancer cells. In order to do so we have carefully generated a list of most significantly deregulated genes, validated their aberrant expression pattern in established cell lines from colorectal carcinomas, and designed siRNA oligonucleotides against them. We are now condu [summary truncated at 7800 characters]

原发性结肠癌的转录组：为了表征原发性结肠癌中整体转录失调的模式，我们使用寡核苷酸微阵列对 73 个肿瘤（UICC II 期，n = 33 和 UICC III 期，n = 40）进行了基因表达谱分析。对于 30 个肿瘤，将表达谱与匹配的正常粘膜样本的表达谱进行了比较。我们鉴定了一组 1,950 个基因，这些基因在肿瘤和粘膜样本之间具有高度显着的失调（P 小于 1e-7）。这些基因中有很大一部分映射到 20 号染色体（P=0.01）。正常结肠粘膜和癌症之间有 17 个基因的平均表达差异超过五倍，其中包括 MYC 和 HMGA1（一种假定的癌基因）的上调。此外，我们还鉴定了 68 个在淋巴结阴性和阳性肿瘤之间表达显着差异的基因（P 小于 0.001），其功能注释揭示了在细胞免疫反应和监视中发挥作用的基因的优势。使用定量实时 RT-PCR 在 40 多个肿瘤和正常粘膜样本中验证了 20 个失调基因的微阵列衍生基因表达水平，技术之间具有良好的一致性。 最后，我们通过比较基因组杂交对 32 个分析的结肠肿瘤绘制了特定基因组不平衡与整体转录活性改变之间的关系。此前，我们对原发性直肠癌进行了类似的分析。结肠癌和直肠癌的系统比较揭示了基因组失衡和转录失调的显着重叠，包括 Wnt/b-连环蛋白信号级联的激活，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背后的遗传改变，我们使用寡核苷酸阵列上的一系列 17 个局部晚期直肠腺癌和 20 个正常直肠粘膜活检的全局基因表达谱。正常直肠粘膜和直肠癌之间共有 351 个基因存在差异表达（p 小于 1.0e-7），其中 77 个基因具有大于 5 倍的差异，85 个基因在所有基因中始终具有至少两倍的变化。的匹配样本。 12 个基因满足所有这三个标准。 PTGS2 (COX2)、WNT1、TGFB1、VEGF 和 MYC 等基因的表达发生改变得到证实，而 PPARD 和 LEF1 等其他基因的数据与之前的报告不一致。此外，我们发现许多基因的表达失调，这些基因参与直肠癌的发生尚未有报道。通过使用比较基因组杂交绘制肿瘤中的基因组失衡图谱，我们可以证明经常性非整倍体染色体臂 7p、8q、13q、18q、20p 和 20q 的 DNA 拷贝数增益与其平均染色体臂表达谱显着相关。总而言之，我们的结果表明，特定基因的高水平、显着的转录失调和非整倍体染色体上基因的平均转录活性的一般修饰在直肠腺癌中共存。鉴定 13q 中与结直肠癌相关的假定癌基因：13 号染色体是结直肠癌中最常见的染色体之一。尽管这种非整倍性很普遍，但尚不清楚哪个或哪些基因是这种染色体失衡的目标。 因此，我们使用 Operon V2 寡核苷酸组（见上文），使用 NCI 寡核苷酸阵列 (22K) 分析了一组 73 个结肠癌样本。之前一项使用直肠癌的研究表明，大量差异表达（上调）基因映射到 13 号染色体。我们还在之前研究中包含的一组 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，我们能够分离出一组在特定染色体带上显示局部扩增的肿瘤。这使得能够识别该染色体上最小重叠的区域。使用 aCGH 和表达谱对大量样本进行一致分析，将潜在候选基因的数量减少到 13 号染色体上约 39 个基因的列表，这些基因在结直肠癌中的功能分析正在进行中。结直肠癌细胞系中的细胞凋亡筛选：恶性转化的途径之一包括癌细胞避免细胞凋亡的能力。我们目前正在探索调节结直肠癌细胞凋亡的基因的作用。使用一组商业化的 siRNA，我们对 418 个被认为在细胞凋亡中起作用的基因进行了筛选。对每个基因检查两个 siRNA，并使用结直肠癌细胞系 SW480 进行筛选。通过活力测定评估每种 siRNA 的效果。为了确定潜在的新颖的联合治疗干预措施，我们在有或没有 5-FU（一种用于治疗结肠癌的化疗剂）存在的情况下使用 RNAi。将细胞接种在 96 孔板中的 siRNA 和阳离子脂质复合物上进行反向转染。当适当时，48小时后添加5-FU（80M对应于EC50），并在进一步孵育72小时后测定细胞活力。添加 5-FU 使细胞活力均匀降低约 50%。我们鉴定了许多进一步增加 SW480 细胞对 5-FU 敏感性的基因。在没有 5-FU 处理的情况下，通过 siRNA 对细胞活力降低的严格过滤，使我们将注意力集中在两个基因上，作为降低细胞活力的潜在新靶标：一个属于核孔蛋白类的基因，另一个是半胱天冬酶相关基因。这两个基因的 siRNA 使 SW480 的活力降低了 40% 以上。 我们已经充分表征了这两个基因对应的 siRNA 介导的 mRNA 水平的降低，并一致观察到它们的下调会降低 SW480 细胞的活力。为了进一步验证其效果，我们沉默了其他细胞系中的 caspase 相关基因。与二倍体细胞系 SW48 相比，另一种非整倍体细胞系 SW837 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的下调具有潜在的敏感性。我们目前正在研究造成这种效应的功能机制。利用RNA干扰系统敲除候选结直肠癌基因：对原发性结直肠癌基因表达谱的全面系统分析表明，结直肠上皮恶性转化的几个关键调节因子参与其中。我们现在致力于探索这些特定转录变化对结直肠癌细胞转录组的影响。为此，我们仔细生成了一系列最显着失调的基因，验证了它们在已建立的结直肠癌细胞系中的异常表达模式，并设计了针对它们的 siRNA 寡核苷酸。我们现在正在 condu [摘要被截断为 7800 个字符]