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.

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