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]

人类直肠癌和结肠癌中细胞转录组的非整倍性依赖性大规模失调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了证实，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，我们可以证明经常出现的非整倍体染色体臂 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。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。在另一系列结直肠癌中也观察到类似的失调，我们能够追踪正常上皮、早期低级别腺瘤、晚期高级别腺瘤、浸润性癌及其相关转移的转化顺序。为了确定基因组、转录组和蛋白质组的连续改变，这些改变定义了正常上皮的转化以及从腺瘤到侵袭性疾病的进展，我们分析了来自 20 个正常粘膜标本、12 个腺瘤、18 个原发性散发性结直肠癌、和12个肝转移。该集合中包括来自 17 名患者的粘膜-腺瘤-癌序列样本。比较基因组杂交 (CGH) 证实了之前的研究，揭示了阶段特异性、复发性基因组失衡的模式。在 9K cDNA 阵列上进行基因表达分析。 T检验组比较发现正常粘膜和腺瘤之间有58个差异表达基因，腺瘤和癌之间有118个基因，原发癌和肝转移之间有163个差异表达基因（p小于0.001）。从正常粘膜到腺瘤和癌，1750个基因的表达水平不断增加，而2099个基因的表达水平却下降。 差异表达的基因属于参与死亡受体信号传导、细胞凋亡抑制以及 p53-、TNF-α-、NF-K β-、TNFR1- 和整合素信号传导的细胞途径。使用 CGH 和表达谱对我们的样本进行平行分析，可以建立染色体拷贝数变化与 7、13、17、18 和 20 号染色体的染色体特异性平均基因表达水平的直接相关性（p 小于 0.01）。通过二维凝胶电泳和随后的质谱分析了一部分样品的蛋白质表达模式。虽然芯片上的 42 个差异表达蛋白和序列没有直接匹配，但大多数差异表达基因和蛋白属于相同的途径或网络。总之，基因组不稳定性的增加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。染色体非整倍体对非整倍体染色体上基因的平均表达水平产生直接影响，从而导致细胞转录组的大量失调。这种非整倍体依赖性转录失调并不是结肠癌所特有的，因为我们在原发性乳腺癌中也观察到了类似的现象。非整倍体染色体的核拓扑及其对转录活性的影响 染色体非整倍性是绝大多数癌症的一个定义特征。在非遗传性结肠癌中，7、13 和 20 号染色体的额外拷贝不仅在早期发育异常前病变中观察到，而且在整个转移过程中都忠实地维持着。已经确定染色体在间期核内呈现出高度保守的三维定位。 染色体拷贝数的增加也被证明与该染色体上基因表达的总体增加相关。因此，我们对含有人工衍生的三体性的细胞系进行三维荧光原位杂交（3D-FISH），以确定这种增加的表达是否需要非整倍体染色体采用与其内源同源物相同的核位置。我们的结果表明，内源性 7 号和 18 号染色体区域位于外围，而 19 号染色体区域则位于中心位置。值得注意的是，人工引入的三体染色体的位置与其在结肠癌细胞系 DLD-1 中的内源对应物相同。因此，我们的数据与每个染色体固有的邮政编码的想法是一致的[摘要截断为 7800 个字符]

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