Colorectal Cancer Transcriptome: Effects of Genomic Instability and Aneuploidy

结直肠癌转录组：基因组不稳定性和非整倍性的影响

• 批准号: 8937866
• 负责人: Thomas Ried
• 金额: $ 109.05万
• 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/8937866
• 关键词: 13q; 17p; 18q; 20q; Address; Affect; Aneuploidy; Architecture; Biological; Biological Assay; Bladder; C57BL/6 Mouse; Cancer Biology; Cancer Model; Carcinoma; Cell Adhesion; Cell Cycle Checkpoint; Cell Line; Cell Nucleus; Cell fusion; Cells; Centrosome; Cervix Uteri; Cervix carcinoma; Characteristics; Chemicals; Chromosomal Gain; Chromosomal Instability; Chromosomal Loss; Chromosomal translocation; Chromosome Arm; Chromosome Mapping; Chromosome Transfer; Chromosome abnormality; Chromosomes; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 16; Chromosomes, Human, Pair 20; Chromosomes, Human, Pair 3; Chronic Myeloid Leukemia; Classification; Clear Cell; Clonal Expansion; Colon; Colon Carcinoma; Colonic Neoplasms; Colorectal Adenocarcinoma; Colorectal Cancer; Colorectal Neoplasms; Constitutional; Cytogenetic Analysis; Cytokinesis; DNA Methylation; DNA Repair; DNA copy number; Diploidy; Disease; Distant Metastasis; Down-Regulation; Dysplasia; Epithelial; Epithelial Cells; Epithelium; Equilibrium; Evaluation; Event; Failure; Fibrous capsule of kidney; Fructose; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic; Genome; Genomic Instability; Genomics; Glycolysis; Goals; Hematologic Neoplasms; Hour; Human; Immunocompromised Host; Impairment; In Vitro; Injection of therapeutic agent; Kidney; Lamins; Large Intestine Carcinoma; Lesion; Life; Lung; Maintenance; Malignant Epithelial Cell; Malignant Neoplasms; Mammary gland; Maps; Mediating; Metabolic Pathway; Metabolism; Mitosis; Modeling; Modification; Molecular Abnormality; Molecular Cytogenetics; Molecular Profiling; Mucous Membrane; Mus; Mutation; Normal tissue morphology; Nuclear; Nude Mice; Oligonucleotide Microarrays; Oncogenes; Oncogenic; Ontology; Organ; Patau' s syndrome; Pathway interactions; Pattern; Phenotype; Philadelphia Chromosome; Play; Premalignant; Primary Neoplasm; Process; Pyruvate Metabolism Pathway; Radiation; Rectal Cancer; Rectum; Recurrence; Regulation; Resources; Role; Sampling; Signal Transduction; Solid Neoplasm; Specificity; Staging; Stem cells; Stimulus; Telomerase; Tetraploidy; Tissues; Transformed Cell Line; Trisomy; Tumor Suppressor Genes; Up-Regulation; Viral; arm; base; cancer cell; cellular imaging; chromatin remodeling; chromosome 3q gain; chromosome 4 loss; comparative genomic hybridization; epithelial to mesenchymal transition; established cell line; fatty acid metabolism; genome-wide; interest; kidney cell; microdeletion; mouse model; overexpression; pressure; segregation; theories; transcription factor; tumor; tumorigenesis; tumorigenic

By systematically integrating gene expression profiles and 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 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 colorectal adenocarcinomas. In order to characterize patterns of global transcriptional deregulation in primary colon carcinomas, we performed gene expression profiling of some 300 tumors, including colon and rectum using oligonucleotide microarrays. For most of these 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 (P1e-7). A significant proportion of these genes mapped to chromosome 20 (P=0.01), which is frequently gained in colorectal tumors. Finally, we established a relationship between specific genomic imbalances, which were mapped for many 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. Nuclear topography of aneuploid chromosomes and their consequences on transcriptional activity. We and others previously showed that chromosomal aneuploidies affect the chromosome wide expression levels of resident genes and therefore result in a massive alteration of the transcriptome of cancer cells. This is true for constitutional chromosomal aneuploidies, for cancer cells, both murine and human, and for cells in which an artificial trisomy was generated experimentally via microcell mediated chromosome transfer (MMCT). We demonstrated that (i) genes mapping to the induced aneuploid chromosome are expressed and that (ii) genes on other chromosomes are affected as well. In theory, this effect could be stochastic or deterministic. In order to address this question we generated seven independent clones, i.e., biological replicates of the diploid CRC cell line DLD1 containing an artificially induced trisomy of chromosome 13. First, we confirmed our previous findings that average chromosome wide gene expression levels follow genomic copy number. Second, we demonstrated that the expression changes in the biological replicates were in fact extremely similar, hence deterministic. The consequence of a chromosome 13 copy number increase, confirmed by aCGH, resulted in a significant average upregulation of most genes on chromosome 13 (P=0.001). Some genes were consistently highly upregulated, including MAB21L1. We also observed an overall, albeit slight, downregulation of genes on chromosome 16 compared to other chromosomes (P=0.02). In addition to expression levels on chromosome 13, we were interested in detecting whether genes on other chromosomes were consistently differentially regulated as a consequence of the artificial trisomy. A total of 409 up-regulated and 372 down-regulated genes were specifically and consistently affected throughout the genome (1.5 fold difference, FDR 0.05). Gene ontology analysis revealed that these genes were enriched in metabolic pathways, including genes associated with fatty acid metabolism, glycolysis, pyruvate metabolism and fructose metabolism. We conclude that aneuploidies result in a non-random massive deregulation of the transcriptional equilibrium. We are now investigating whether specific overexpressed transcription factors on chromosome 13 are responsible for the genome-wide effects. Modeling human tumors in the mouse helps to dissect specific aspects of human tumorigenesis. Most murine cancer models are induced by the overexpression of known or putative oncogenes, or by deletion of tumor suppressor genes. Comprehensive molecular cytogenetic analyses, however, have revealed that in particular those models induced by a strong oncogenic stimulus have a lower degree of chromosomal instability compared to human tumors, and to models induced by deletion of tumor suppressor genes. Consequently, they are not suited as models to study the most common and recurrent genetic aberrations in human carcinomas, i.e., chromosomal gains and losses. We hypothesized that during the process of spontaneous transformation of murine cells aneuploidy would play a more dominant role compared to tumors induced by the overexpression of oncogenes. Therefore, we established 45 spontaneously transformed cell lines from the epithelium of six organs of wild-type C57B1/6 mice (mammary gland, colon, lung, cervix, bladder and kidney), without radiation, chemical or viral induction. Phenotypic changes, chromosomal aberrations, centrosome number and telomerase activity were assayed in control uncultured epithelial cells and in three subsequent stages of transformation, i.e., pre-immortal, immortal and transformed. Supernumerary centrosomes, binucleate cells and tetraploidy were observed as early as 48 hours after initiation of in vitro cultures. In addition, telomerase activity increased throughout progression. Live-cell imaging revealed that failure of cytokinesis, not cell fusion, generated the binucleate cells and thereby promoted genome duplication. SKY demonstrated that aneuploidy commenced early and preceded cellular immortalization. The chromosomal imbalances consisted predominantly of whole chromosome losses (4, 9, 12, 13, 16, and Y) and gains (1, 5 10, 11, 15, and 19). After transformation, focal amplifications of several oncogenes, such as Myc and Mdm2 on commonly gained chromosomes and microdeletions of Cdkn2a/p16 were frequently detected. Seventy-four percent of the transformed bladder, kidney and colon lines resulted in tumors following injection into immunocompromised nude mice. Subsequently, these tumorigenic kidney cell lines were injected both subcutaneously and into the renal capsule of isogenic mice (C57BL/6). All three cell lines developed tumors subcutaneously, and one formed tumors in the renal capsule. Histological evaluation revealed phenotypic features resembling human clear-cell carcinomas; therefore, our models are a valuable resource for further study of this cancer. In order to map chromosomal aberrations and genomic imbalances and their consequences on the transcriptome, we proceeded with a detailed analysis using aCGH, FISH and gene expression profiling of the kidney, bladder and colon lines. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and mitosis. Genes that modulate the epithelial to mesenchymal transition (EMT), as well as genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes observed in human bladder, kidney and colon tumorigenesis revealed remarkable overlap with changes detected in the spontaneously transformed murine cultures.

通过使用比较基因组杂交在肿瘤中系统地整合肿瘤中的基因表达谱和基因组失衡，我们可以表明，DNA拷贝数的复制数量相关的非整倍体染色体臂臂7p，8q，13Q，13Q，18Q，18Q和20Q与其平均染色体臂表达谱显着相关。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驻留在结直肠腺癌中的非整倍体染色体上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大约300个肿瘤的基因表达分析，包括结肠和直肠。对于大多数这些肿瘤，将表达谱与匹配的正常粘膜样品的表达谱进行比较。我们鉴定了一组1,950个基因，在肿瘤和粘膜样品之间进行了高度显着的管制（P1E-7）。这些基因中很大一部分映射到20号染色体（p = 0.01），通常在结直肠肿瘤中获得。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许多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。非整倍型染色体的核超图及其对转录活性的后果。我们和其他人先前表明，染色体肾上腺素会影响居民基因的染色体广泛表达水平，因此导致癌细胞转录组发生了巨大变化。对于鼠类和人类的癌细胞，以及通过Microcell介导的染色体转移（MMCT）实验生成人造三体菌的细胞，对于鼠类和人类的癌细胞以及人工三体的细胞都是正确的。我们证明了（i）映射到诱导的非整倍体染色体的基因表达，并且（ii）其他染色体上的基因也受到影响。从理论上讲，这种效果可能是随机或确定性的。为了解决这个问题，我们生成了七个独立的克隆，即二倍体CRC细胞系DLD1的生物学重复，其中包含人为诱导的染色体13的三体术。首先，我们证实了以前的发现，我们的发现表明，平均染色体广泛的基因表达水平遵循基因组拷贝数。其次，我们证明了生物学重复的表达变化实际上非常相似，因此是确定性的。 ACGH证实的13拷贝数增加的后果导致大多数基因在13染色体上的平均上调（p = 0.001）。一些基因始终高度上调，包括MAB21L1。我们还观察到与其他染色体相比，尽管染色体16染色体上的基因的总体下调，但仍略有下调（p = 0.02）。除了在13染色体上的表达水平外，我们对检测其他染色体上的基因是否始终受到差异性调节，这是人造三体切开术。在整个基因组中，总共有409个上调和372个下调的基因受到特异性和始终如一的影响（1.5倍差异，FDR 0.05）。基因本体分析表明，这些基因富含代谢途径，包括与脂肪酸代谢，糖酵解，丙酮酸代谢和果糖代谢相关的基因。我们得出的结论是，非随机的转录平衡导致非随机大规模放松管制。我们现在正在研究13染色体上的特定过表达转录因子是否负责全基因组效应。对小鼠中的人类肿瘤进行建模有助于剖析人类肿瘤发生的特定方面。大多数鼠类癌模型是由已知或推定的癌基因的过表达或通过缺失肿瘤抑制基因引起的。然而，综合分子细胞遗传学分析表明，与人类肿瘤相比，强烈的致癌刺激诱导的那些模型具有较低程度的染色体不稳定性，而与肿瘤抑制基因的缺失引起的模型有关。因此，它们不适合研究人癌中最常见和反复的遗传像差，即染色体的增益和损失。我们假设在鼠细胞自发转化的过程中，与癌基因过表达诱导的肿瘤相比，非整倍性的作用更为主导。因此，我们从野生型C57B1/6小鼠的六个器官的上皮（乳腺，结肠，肺，子宫颈，膀胱，膀胱和肾脏）中自发地转化了45个细胞系，没有辐射，化学或病毒诱导。在对照未培养的上皮细胞中测定了表型变化，染色体畸变，中心体数和端粒酶活性，并在转化的三个后期阶段，即预皮前，不朽和转化。在开始体外培养物后48小时，就观察到了超生物中心体，双核细胞和四倍体。此外，端粒酶活性在整个进展过程中都增加。活细胞成像表明，细胞因子而不是细胞融合的失败产生了双核细胞，从而促进了基因组重复。天空表明，非整倍性早早开始，并且先于细胞永生化。染色体失衡主要由整个染色体损失（4、9、12、13、16和Y）和增益（1、5 10、11、15和19）组成。转化后，经常检测到几种癌基因（例如MYC和MDM2）对通常获得的染色体和CDKN2A/P16的微缺失的局灶性扩增。转化后的膀胱，肾脏和结肠系中有74％导致肿瘤注射到免疫功能低下的裸鼠中。随后，将这些肿瘤性肾细胞系皮下注射并置于等生小鼠的肾囊中（C57BL/6）。这三个细胞系皮下出现肿瘤，并在肾囊中形成肿瘤。组织学评估显示表型特征类似于人类透明细胞癌。因此，我们的模型是进一步研究该癌症的宝贵资源。为了映射染色体畸变和基因组失衡及其对转录组的后果，我们使用ACGH，FISH，FISH，基因和基因表达分析进行了详细的分析，膀胱和结肠线的基因表达分析。结果表明，与人类癌症一样，基因组失衡的复发性和组织特异性分布也是广泛的。染色体非整倍性导致居民基因的表达变化，因此导致细胞转录组的大规模放松管制。在转化的顺序步骤中，对表达变化的途径询问揭示了与DNA修复，中心体调节，干细胞特征和有丝分裂相关的基因的富集。调节上皮到间充质转变（EMT）的基因以及定义染色体不稳定性表型的基因起主要作用，并且在与细胞粘附，侵入性和增殖丧失一致的方向上发生了变化。与在人膀胱，肾脏和结肠肿瘤发生中观察到的基因表达变化的比较表明，与自发转化的鼠培养物中发现的变化显着重叠。