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. 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. 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. 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. 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. Murine kidney cells in culture undergo striking morphological changes during the transformation process. The cells become adherent by days 1-7, and after circumventing the crisis stage, become immortalized and later transformed (these stages can be robustly differentiated using gene expression profiling, suggesting that the morphologically defined stages have a genetic basis). In a first series of experiments we suggest using the murine in vitro transformation model of kidney cells to explore whether the conserved chromosome positioning in interphase nuclei changes during immortalization and transformation. Mouse chromosomes that were gene-rich (MMU7;19 genes/Mbp) and gene-poor (MMU18;9 genes/Mbp), and that contained sequences homologous to human chromosomes 19 and 18, respectively, were therefore selected. Primary kidney cells were processed for 3D-FISH on day 2-3, when they reached the pre-immortal stage around day 7, and at the subsequent stages of immortalization and transformation. Preliminary analyses revealed that MMU7 is predominantly central in the primary kidney cells, while MMU18 is predominantly peripheral, thereby reiterating the conservation of a gene density based non-random chromosome positioning pattern. Ongoing studies include observation of later stages and analysis of MMU2 (182 Mbp) &MMU19 (61Mbp) to address whether chromosome size has any impact on chromosome arrangement during cellular transformation. We are also currently performing SKY and extracting RNA from these same passages for karyotypic analysis and expression profiling to establish potential relationships between changes in the position of chromosome territories with genomic aberrations and chromosome-specific transcriptional activity. Aneuploidy, oncogene amplification, and epithelial/mesenchymal transition govern transformation of murine epithelial cells: models for human cancer Human carcinomas are defined by a recurrent distribution of genomic imbalances. The requirement for such specific genome mutations is less pronounced in murine cancer models induced by an oncogenic stimulus. To overcome these limitations, we established models for kidney and bladder cancer through spontaneous immortalization of murine epithelial cells. The sequence of genetic, genomic, and transcriptomic alterations was established at multiples stages, including senescence, immortalization, transformation, and after tumorigenesis in nude mice using SKY, arrayCGH, FISH, gene expression profiling, immunhistochemistry, and measurement of telomerase activity. The results show that specific chromosomal aneuploidies are necessary events that occur at the earliest stages;mouse chromosome 4 was consistently lost, whereas copy number increases were frequently mapped to chromosome 15. These aneuploidies resulted in expression changes of resident genes, and hence in a massive deregulation of the cellular transcriptome. In concert with such aneuploidy-induced transcriptional deregulation, we observed the consistent overexpression of Myc and Mdm2, which were at time of cellular transformation often present in double minute chromosomes. Pathway interrogation of gene expression changes during the sequential steps of transformation revealed a dominant role of changes consistent with epithelial/mesenchymal transition. We conclude that spontaneous transformation of murine epithelial cells requires the concomitant acquisition of specific chromosomal aneuploidies and deregulation of cellular oncogenes. Translocation induced oncogene activation was not observed. Therefore, our models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis and may therefore be useful for preclinical exploration of therapeutic interventions.
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