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Colorectal Cancer Transcriptome: Effects of Genomic Instability and Aneuploidy – Rockville

Thomas Ried

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National Cancer Institute (NIH)
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.

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