Consequences of Genomic Instability and Aneuploidy on the Colorectal Cancer

基因组不稳定性和非整倍性对结直肠癌的影响

• 批准号: 7733268
• 负责人: Thomas Ried
• 金额: $ 55.58万
• 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/7733268
• 关键词: 13q; 18q; 20p; 20q; 3-Dimensional; Address; Adopted; Affect; Amino Acid Sequence; Aneuploidy; Architecture; Biopsy; Cancer cell line; Carcinoma; Cell Line; Cell Nucleus; Cessation of life; Chromosome Arm; Chromosome Mapping; Chromosome Positioning; Chromosome abnormality; Chromosomes; Chromosomes, Human, Pair 18; Chromosomes, Human, Pair 19; Chromosomes, Human, Pair 20; Chromosomes, Human, Pair 7; Classification; Collection; Colon; Colon Carcinoma; Colonic Neoplasms; Colorectal Cancer; Complement; DNA copy number; Data; Disease; Down-Regulation; Epithelial; Epithelium; Eukaryota; Eukaryotic Cell; Event; Evolution; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Proteins; Genes; Genome; Genomic Instability; Genomics; Genus Cola; Goals; HMGA1 gene; Homologous Gene; Human; Immune response; Individual; Inherited; Inhibition of Apoptosis; Integrins; International Union Against Cancer; Interphase; Invasive; Laboratories; Large Intestine Carcinoma; Lesion; Localized; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Medical Surveillance; Metastatic Neoplasm to the Liver; Modification; Molecular Profiling; Mucous Membrane; Mus; Mutation; Nature; Negative Lymph Node; Neoplasm Metastasis; Normal Cell; Nuclear; Nuclear Structure; Numbers; Oligonucleotide Microarrays; Oncogenes; PTGS2 gene; Pathway interactions; Patients; Pattern; Peptide Sequence Determination; Peripheral; Phenotype; Play; Positioning Attribute; Primary Carcinoma; Primary Neoplasm; Proteome; Receptor Signaling; Rectal Adenocarcinoma; Rectal Cancer; Recurrence; Relative (related person); Renal Cell Carcinoma; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Series; Signal Transduction; Solid Neoplasm; Specimen; Staging; Structure-Activity Relationship; System; TGFB1 gene; TNFRSF1A gene; TP53 gene; Techniques; Testing; Thinking; Time; Tissue Sample; Tissues; Transcriptional Activation; Trisomy; Tumor Biology; Tumor Cell Line; Tumor Necrosis Factor-alpha; Tumor Suppressor Genes; Tumor-Derived; Tumorigenicity; Two-Dimensional Gel Electrophoresis; Up-Regulation; Vascular Endothelial Growth Factors; WNT1 gene; Zip Code; adenoma; base; cDNA Arrays; cancer cell; carcinogenesis; cell type; colon cancer cell line; comparative genomic hybridization; comparison group; cyclooxygenase 2; density; established cell line; human TNF protein; malignant breast neoplasm; protein expression; rectal; research study; size; tumor; tumor progression

Aneuploidy-Dependant Massive Deregulation of the Cellular Transcriptome in Human Rectal and Colon Carcinomas 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 were 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. 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. A similar deregulation was also observed in another series of colorectal carcinomas, in which we were able to follow the sequence of transformation from normal epithelium, early low-grade adenoma, late high-grade adenoma, invasive carcinomas and their associated metastases. In order to identify sequential alterations of the genome, transcriptome, and proteome that define the transformation of normal epithelium and the progression from adenomas to invasive disease, we have analyzed tissue samples from 20 normal mucosa specimen, 12 adenomas, 18 primary sporadic colorectal carcinomas, and 12 liver metastases. Included in this collection were samples of the mucosa-adenoma-carcinoma sequence from 17 individual patients. Confirming previous studies, comparative genomic hybridization (CGH) revealed patterns of stage specific, recurrent genomic imbalances. Gene expression analysis was performed on 9K cDNA arrays. T-test group comparison identified 58 genes differentially expressed between normal mucosa and adenoma, 118 genes between adenoma and carcinoma, and 163 genes between primary carcinoma and liver metastasis (p less than 0.001). Expression levels of 1750 genes increased constantly from normal mucosa to adenoma and carcinoma, whereas 2099 genes showed decreased expression levels. The differentially expressed genes belong to cellular pathways involved in death receptor signaling, inhibition of apoptosis, and p53-, TNF-alpha-, NF-K beta-, TNFR1-, and integrin-signaling. The parallel analysis of our samples using both CGH and expression profiling allowed establishing a direct correlation of chromosomal copy number changes and chromosome specific average gene expression levels for chromosomes 7, 13, 17, 18, and 20 (p less than 0.01). Protein expression patterns of a subset of the samples were analyzed by 2-dimensional gel electrophoresis and subsequent mass spectrometry. While there was no direct match of the 42 differentially expressed proteins and sequences on the array, most of the differentially expressed genes and proteins belonged to identical pathways or networks. In conclusion, increasing genomic instability and a recurrent pattern of chromosomal aberrations as well as distinct gene- and protein expression patterns correlate with distinct stages of colorectal cancer progression. Chromosomal aneuploidies exert a direct effect on average expression levels of the genes residing on the aneuploid chromosomes thereby contributing to a massive deregulation of the cellular transcriptome. This aneuploidy-dependent transcriptional deregulation is not specific for colon cancer, as we have observed a similar phenomenon in primary breast cancer as well. The nuclear topography of aneuploid chromosomes and their consequences on transcriptional activity Chromosomal aneuploidy is a defining feature of the vast majority of carcinomas. In non-hereditary forms of colon cancer, additional copies of chromosomes 7, 13 and 20 are not only observed in early pre-dysplastic lesions, but are faithfully maintained throughout progression to metastasis. It has been established that chromosomes assume a well conserved 3-dimensional positioning within the interphase nucleus. An increase in chromosome copy number has also been shown to correlate with an overall increased expression of genes residing on that chromosome. We are therefore performing 3-dimensional fluorescence in situ hybridization (3D-FISH) on cell lines containing artificially derived trisomies to determine if this increased expression required the aneuploid chromosome to adopt the same nuclear position, as its endogenous homologues. Our results demonstrate that endogenous chromosome 7 and 18 territories are peripheral in position, while chromosome 19 territories are centrally localized. Remarkably, the artificially introduced trisomic chromosomes are positioned identically to their endogenous counterparts in the colon cancer cell line DLD-1. Our data is therefore consistent with the idea that inherent to each chromosome is a zip code th [summary truncated at 7800 characters]

为了识别直肠癌的基础遗传变化，我们使用了直肠癌的遗传变化，我们使用了17个局部局部高级直肠腺癌的一系列全球基因表达分析，对人的直肠和结肠癌中细胞转录组的大规模消耗管制进行了大规模消耗管制。正常直肠粘膜和直肠癌之间总共差异表达了351个基因（P小于1.0E-7），77个基因的差异大于五倍，而85个基因在所有匹配的样品中始终具有至少两倍的变化。 12个基因满足了所有这三个标准。确认了基因（COX2），WNT1，TGFB1，VEGF和MYC等基因的表达改变，而我们的其他基因（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。为了表征原代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。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。我们确定了一组1,950个基因，在肿瘤和粘膜样品之间具有高度显着的管制（P小于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小于0.001），其功能注释揭示了在细胞免疫反应和表现中起作用的大量基因。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。在另一系列的结直肠癌中也观察到了类似的失调，其中我们能够遵循正常上皮，早期低级腺瘤，晚期高级腺瘤，浸润性癌及其相关转移的转化序列。为了确定基因组，转录组和蛋白质组的顺序改变，这些改变定义了正常上皮的转化以及从腺瘤到浸润性疾病的进展，我们分析了来自20个正常粘膜标本，12个腺瘤，18个原发性孢子虫的孢子虫型结肠癌癌的组织样品。该系列中包括17例患者的粘膜 - 腺瘤 - c癌序列的样品。确认先前的研究，比较基因组杂交（CGH）揭示了阶段特异性，复发性基因组失衡的模式。基因表达分析在9K cDNA阵列上进行。 t检验组比较鉴定了正常粘膜和腺瘤之间差异表达的58个基因，腺瘤和癌之间的118个基因以及原代癌和肝转移之间的163个基因（P小于0.001）。 1750个基因的表达水平从正常粘膜到腺瘤和癌不断增加，而2099年基因的表达水平降低了表达水平。 差异表达的基因属于与死亡受体信号传导，凋亡抑制以及p53-，TNF-Alpha-，NF-Kβ-，TNFR1-和整合素信号有关的细胞途径。使用CGH和表达分析对我们的样品的平行分析允许建立染色体染色体拷贝数变化和染色体特异性基因表达水平7、13、17、18和20的直接相关性（P小于0.01）。通过二维凝胶电泳和随后的质谱法分析样品子集的蛋白质表达模式。虽然在数组上没有42个差异表达的蛋白质和序列的直接匹配，但大多数差异表达的基因和蛋白质都属于相同的途径或网络。总之，增加基因组不稳定性和染色体畸变的复发模式以及不同的基因和蛋白质表达模式与结直肠癌进展的不同阶段相关。染色体非整倍体对位于非整倍体染色体上的基因的平均表达水平产生直接影响，从而有助于大规模的细胞转录组进行大规模放松管制。这种非整倍性依赖性转录放松管制并非针对结肠癌，因为我们也观察到原发性乳腺癌的现象类似。非整倍型染色体的核形象及其对转录活性染色体型非整倍性的后果是绝大多数癌的定义特征。在非遗传形式的结肠癌形式中，染色体7、13和20的其他副本不仅在早期发育前病变中观察到，而且在整个过程中都忠实地保持了转移。已经确定染色体假定相间核中保守的3维定位。 还显示染色​​体拷贝数的增加与该染色体上的基因表达的总体表达相关。因此，我们正在对包含人为衍生的三体的细胞系上进行3维荧光原位杂交（3D-FISH），以确定这种增加的表达是否需要非整倍型染色体以采用与内源同源物相同的核位置。我们的结果表明，内源性染色体第7和18个领土位于外围，而19号染色体则位于中心位置。值得注意的是，人为引入的三体染色体位于结肠癌细胞系DLD-1中的内源性对应物。因此，我们的数据与每个染色体固有的想法是一致的，即邮政编码th [摘要以7800个字符截断]

项目成果

Artificially introduced aneuploid chromosomes assume a conserved position in colon cancer cells.

• DOI: 10.1371/journal.pone.0000199
• 发表时间: 2007-02-07
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Sengupta K;Upender MB;Barenboim-Stapleton L;Nguyen QT;Wincovitch SM Sr;Garfield SH;Difilippantonio MJ;Ried T
• 通讯作者: Ried T