Consequences of Genomic Instability and Aneuploidy on the Colorectal Cancer Tran

基因组不稳定性和非整倍性对结直肠癌转移的影响

• 批准号: 8349195
• 负责人: Thomas Ried
• 金额: $ 56.34万
• 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/8349195
• 关键词: 13q; 18q; 20p; 20q; Address; Affect; Aneuploidy; Architecture; Biopsy; Bypass; C57BL/6 Mouse; Cancer cell line; Carcinoma; Cell Line; Cell Nucleus; Cell model; Cells; Chromosome Arm; Chromosome Mapping; Chromosome Positioning; Chromosome Territory; Chromosome Transfer; Chromosome abnormality; Chromosomes; Chromosomes, Human, Pair 18; Chromosomes, Human, Pair 19; Chromosomes, Human, Pair 20; Chromosomes, Human, Pair 7; Code; Colon; Colon Carcinoma; Colonic Neoplasms; Colorectal Cancer; Complement; Coupled; Cultured Cells; Cytogenetics; DNA copy number; Data; Diploidy; Disease; Down-Regulation; Epithelial; Epithelial Cells; Eukaryota; Event; Evolution; Exhibits; Gene Amplification; Gene Deletion; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic; Genome; Genomic Instability; Genomics; Goals; HMGA1 gene; Homologous Gene; Human; Human Chromosomes; Hybrids; Immune response; Immunologic Surveillance; Individual; International Union Against Cancer; Interphase; Laboratories; Large Intestine; Laser Scanning Confocal Microscopy; Lesion; Maintenance; Malignant Neoplasms; Maps; Mediating; Mesenchymal; Methods; Modeling; Modification; Molecular Cytogenetics; Molecular Profiling; Mucous Membrane; Mus; Mutation; Nature; Normal Cell; Nuclear; Nuclear Structure; Nude Mouse Assay; Oligonucleotide Microarrays; Oncogenes; PTGS2 gene; Pathway interactions; Pattern; Peripheral; Phenotype; Play; Positioning Attribute; Primary Carcinoma; Primary Neoplasm; Rectal Adenocarcinoma; Rectal Cancer; Recurrence; Relative (related person); Renal Cell Carcinoma; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Series; Serum; Signal Transduction; Solid Neoplasm; Staging; Structure-Activity Relationship; System; TGFB1 gene; Techniques; Time; Tissues; Trisomy; Tumor Biology; Tumor Cell Line; Tumor Suppressor Genes; Tumor-Derived; Tumorigenicity; Up-Regulation; Validation; Vascular Endothelial Growth Factors; WNT1 gene; base; cancer cell; carcinogenesis; cell type; colon cancer cell line; comparative genomic hybridization; density; design; established cell line; lymph nodes; mouse model; novel; pre-clinical; rectal; research study; therapeutic target; tumor; tumorigenesis

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 more than 100 locally advanced rectal adenocarcinomas and matched 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 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<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. Nuclear topography of aneuploid chromosomes and their consequences on transcriptional activity The 3D-position of chromosome territories in interphase nuclei is non-random and is conserved in evolution. Gene rich chromosomes, such as human chromosome 19, are located towards the center of the nucleus, whereas gene poor chromosomes, such as chromosome 18, are predominantly peripheral in many different cell types. We were eager to explore to which extent this remarkable conservation is maintained in cells containing chromosomal aneuploidies, in particular because we have previously established that these chromosomes are transcriptionally active. Therefore, a relationship could exist between nuclear position and transcriptional activity. We investigated, first, whether aneuploid chromosomes assume a nuclear position similar to that of their endogenous homologues. Using 3D-FISH and confocal laser scanning microscopy, we showed that human chromosomes 7, 18, or 19 introduced via microcell mediated chromosome transfer (MMCT) into the parental diploid colon cancer cell line DLD-1 maintain their conserved position. Our data is thus consistent with the model that each chromosome has an associated zip code that determines its nuclear localization. Whether the nuclear localization determines, or is determined by, the transcriptional activity of resident genes has yet to be ascertained. Similar analyses were extended to the murine donor cells used for MMCT which carried an individual human chromosome. Here we investigated (i) whether mouse nuclei recognize such a zip code of nuclear position on human chromosomes to facilitate their distinct partitioning and (ii) if chromosome positioning and transcriptional activity remain coupled under these trans-species conditions. We showed (i) that gene poor and gene rich human chromosomes do in fact maintain their divergent but conserved positions in mouse-human hybrid nuclei and (ii) that a foreign human chromosome is actively transcribed in mouse nuclei. The results suggest a species-independent conserved mechanism for the non-random positioning of chromosomes in the 3D interphase nucleus. Aneuploidy, oncogene amplification, and epithelial/mesenchymal transition govern transformation of murine epithelial cells: models for human cancer We extended these analyses and designed a novel mouse model for colon cancer. Normal primary murine epithelial colon cells were selectively isolated from the large intestine of eight different isogenic C57BL/6 mice. A portion of the primary colon cells were grown in culture under reduced serum conditions while another fraction of these normal cells were cryopreserved for further analysis. The cultured cells were sequentially recovered during different stages of transformation as morphological changes were observed: The cells were sequentially recovered from culture based on distinct morphological changes: first, when the cells were in the pre-immortal stage as cells were actively growing; second, as the cells bypassed crisis, formed colonies and became immortal; third, at early transformation, as the cells first formed foci; fourth, mid-transformed (higher proliferation rates, later passages); and finally, as cells became less adherent, and exhibiting multiple foci. The cells were analyzed at each stage using molecular cytogenetic methods and microarray techniques. SKY identified recurrent structural and numerical aberrations in all eight of the late transformed mouse colon cell lines. Array CGH revealed regions of gene amplifications and deletions. Global gene expression profiling identified deregulated gene specific to each stage of transformation. Three of the late transformed mouse colon cell lines produced tumors in nude mouse assays. This is a unique murine model that reproduces a stage-specific progression of alterations which is common in human colon tumorigenesis. The similarity of the cytogenetic and genetic aberration profiles suggest that this model can serve as a preclinical system for the validation of therapeutic targets.

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识别直肠癌发生背后的遗传改变，我们使用了一系列 100 多个局部晚期直肠腺癌的全局基因表达谱，并在寡核苷酸上匹配了正常直肠粘膜活检数组。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 < 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。匹配的样本。 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。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<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<0.001)，其功能注释揭示了在细胞免疫反应和监测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。 非整倍体染色体的核拓扑及其对转录活性的影响 间期核中染色体区域的 3D 位置是非随机的，并且在进化中是保守的。在许多不同的细胞类型中，基因丰富的染色体（例如人类 19 号染色体）位于细胞核的中心，而基因贫乏的染色体（例如 18 号染色体）主要位于细胞核的外围。我们渴望探索在含有染色体非整倍体的细胞中这种显着的保守性在多大程度上得以维持，特别是因为我们之前已经确定这些染色体具有转录活性。因此，核位置和转录活性之间可能存在某种关系。首先，我们研究了非整倍体染色体是否具有与其内源同源体相似的核位置。使用 3D-FISH 和共焦激光扫描显微镜，我们发现通过微细胞介导的染色体转移 (MMCT) 引入亲代二倍体结肠癌细胞系 DLD-1 中的人类 7、18 或 19 号染色体保持其保守位置。因此，我们的数据与每个染色体都有一个确定其核定位的相关邮政编码的模型一致。核定位是否决定常驻基因的转录活性，或者由常驻基因的转录活性决定，尚未确定。类似的分析扩展到用于 MMCT 的小鼠供体细胞，该细胞携带单个人类染色体。在这里，我们研究了（i）小鼠细胞核是否识别人类染色体上的核位置邮政编码，以促进其独特的划分；以及（ii）染色体定位和转录活性在这些跨物种条件下是否保持耦合。我们表明（i）基因贫乏和基因丰富的人类染色体实际上在小鼠-人类杂交细胞核中保持了它们不同但保守的位置，以及（ii）外源人类染色体在小鼠细胞核中活跃转录。结果表明，3D 间期核中染色体非随机定位存在一种独立于物种的保守机制。 非整倍性、癌基因扩增和上皮/间质转化控制小鼠上皮细胞的转化：人类癌症模型我们扩展了这些分析并设计了一种新型的结肠癌小鼠模型。从八只不同同基因 C57BL/6 小鼠的大肠中选择性分离正常原代小鼠上皮结肠细胞。一部分原代结肠细胞在低血清条件下培养，而另一部分正常细胞则冷冻保存以供进一步分析。在转化的不同阶段依次回收培养的细胞，观察形态变化：根据明显的形态变化，依次从培养物中回收细胞：首先，当细胞处于永生前阶段，细胞生长活跃；其次，当细胞处于永生前阶段时，细胞生长活跃；其次，细胞绕过了危机，形成了菌落并永生不朽；第三，在转化早期，细胞首先形成病灶；第四，中期转化（增殖率较高，传代较晚）；最后，细胞粘附性降低，并表现出多个病灶。使用分子细胞遗传学方法和微阵列技术对每个阶段的细胞进行分析。 SKY 在所有八种晚期转化的小鼠结肠细胞系中发现了反复出现的结构和数值畸变。阵列 CGH 显示了基因扩增和缺失的区域。全局基因表达谱鉴定了特定于转化每个阶段的失调基因。三种晚期转化的小鼠结肠细胞系在裸鼠试验中产生肿瘤。这是一种独特的小鼠模型，可重现人类结肠肿瘤发生中常见的特定阶段的改变进展。细胞遗传学和遗传畸变谱的相似性表明该模型可以作为验证治疗靶点的临床前系统。