Chromosome transfer induced aneuploidy results in complex dysregulation of the cellular transcriptome in immortalized and cancer cells.

Chromosomal aneuploidies are observed in essentially all sporadic carcinomas. These aneuploidies result in tumor-specific patterns of genomic imbalances that are acquired early during tumorigenesis, continuously selected for and faithfully maintained in cancer cells. Although the paradigm of translocation induced oncogene activation in hematologic malignancies is firmly established, it is not known how genomic imbalances affect chromosome-specific gene expression patterns in particular and how chromosomal aneuploidy dysregulates the genetic equilibrium of cells in general. To model specific chromosomal aneuploidies in cancer cells and dissect the immediate consequences of genomic imbalances on the transcriptome, we generated artificial trisomies in a karyotypically stable diploid yet mismatch repair-deficient, colorectal cancer cell line and in telomerase immortalized, cytogenetically normal human breast epithelial cells using microcell-mediated chromosome transfer. The global consequences on gene expression levels were analyzed using cDNA arrays. Our results show that regardless of chromosome or cell type, chromosomal trisomies result in a significant increase in the average transcriptional activity of the trisomic chromosome. This increase affects the expression of numerous genes on other chromosomes as well. We therefore postulate that the genomic imbalances observed in cancer cells exert their effect through a complex pattern of transcriptional dysregulation.

Silence of chromosomal amplifications in colon cancer.

Oncogene activation by gene amplification is a major pathogenetic mechanism in human cancer. Using comparative genomic hybridization, we determined that metastatic human colon cancers commonly acquire numerous extra copies of chromosome arms 7p, 8q, 13q, and 20q. We then examined the consequence of these amplifications on gene expression using DNA microarrays. Of 55,000 transcripts profiled, 2,146 were determined to map to one of the four common colon cancer amplicons and to also be expressed in normal or malignant colon tissues. Of these, only 81 transcripts (3.8%) demonstrated a 2-fold increase over normal expression among cancers bearing the corresponding chromosomal amplification. Chromosomal amplifications are common in colon cancer metastasis, but increased expression of genes within these amplicons is rare.

Carcinogen-induced colon tumors in mice are chromosomally stable and are characterized by low-level microsatellite instability.

The azoxymethane (AOM)-induced mouse colon tumor model recapitulates many of the histopathological features associated with the multistage progression of human sporadic colorectal cancers (CRCs). To better define the genetic events associated with tumorigenesis in this murine model, we analysed tumors from A/J mice for chromosomal (CIN) and microsatellite (MSI) instabilities, two fundamental pathways of genomic instability that play a critical role in the pathogenesis of human CRCs. Male A/J mice, 6-week old, were injected with either AOM (n=5) (10 mg/kg b.w., i.p.) or vehicle (n=5) (0.9% NaCl solution) once a week for 6 weeks. At 32 weeks after the last dose, comparative genomic hybridization (CGH) was performed on 16 tumors harvested from five animals. Although 25% of the tumors displayed either a gain of chromosome 2 or loss of Y, the majority (75%) showed no genomic imbalances. Further analysis of chromosomal aberrations, using CGH and spectral karyotyping (SKY) was performed in our recently established A/J colon tumor-derived cell line, AJ02-NM0. Results showed a pseudotetraploid karyotype with loss of only the Y chromosome in these cultured cells, thereby providing additional evidence for the minimal role of CIN in the primary AOM-induced tumors. Interestingly, the majority (81%) of A/J tumors displayed low-level microsatellite instability (MSI-L) when analysed using mono- and dinucleotide repeat markers, and showed a significant expansion to high-level instability (MSI-H) in the AJ02-NM0 cells. This finding in cultured cells additionally provides evidence that a mild mutator pathway may contribute to the development of behaviorally benign carcinomas in situ in A/J mice. To better understand the tumorigenic process in the A/J colons, we screened for mutational alterations in key regions of the K-ras and Apc genes. Results showed a very low frequency (6%) of K-ras activating mutations, together with the absence of Apc truncation mutations in primary tumors and AJ02-NM0 cells. However, these tumors displayed intense nuclear accumulation of beta-catenin protein, indicating activation of the Wnt signaling pathway. Based on our molecular and cytogenetic findings, we propose that carcinogen-induced tumors may develop via mechanisms independent of the 'classical' CIN or MSI pathways.

Objective method of comparing DNA microarray image analysis systems.

Many image analysis systems are available for processing the images produced by laser scanning of DNA microarrays. The image processing system takes pixel-level intensity data and converts it to a set of gene-level expression or copy number summaries that will be used in further analyses. Image analysis systems currently in use differ with regard to the specific algorithms they implement, ease of use, and cost. Thus, it would be desirable to have an objective means of comparing systems. Here we describe a systematic method of comparing image processing results produced by different image analysis systems using a series of replicate microarray experiments. We demonstrate the method with a comparison of cDNA microarray data generated by the UCSF Spot and the GenePix image processing systems.

Pronounced chromosomal instability and multiple gene amplifications characterize ulcerative colitis-associated colorectal carcinomas.

Patients with ulcerative colitis have a significantly increased lifetime risk for the development of colorectal carcinomas. While genetic and genomic changes during carcinogenesis have been thoroughly studied in sporadic colorectal cancers, less is known about ulcerative colitis-associated colorectal carcinomas. The aim of this study was to extend the identification of specific genomic imbalances to ulcerative colitis-associated colorectal carcinomas and to establish a comprehensive map of DNA gains and losses by investigating 23 tumor specimens from 23 patients. The molecular cytogenetic characterization was performed using comparative genomic hybridization; immunohistochemistry was used to measure proliferative activity and laminin-5 expression as a marker for invasiveness. The results indicate that these tumors are invariably aneuploid, with a high proliferative activity and increased invasive potential. The average number of copy alterations correlates with increased cyclin A levels (P=0.044), which is an independent predictor of risk of carcinoma development in ulcerative colitis. Despite severe genetic instability, the general pattern of specific chromosomal aberrations that defines sporadic colorectal carcinomas is maintained in ulcerative colitis-associated malignancies. High-level copy number increases (amplifications) are dispersed throughout the genome. Strikingly, these amplifications are much more frequent than in sporadic carcinomas and map to chromosomal regions that have not been described before.

Chromosomal aneuploidy affects the global proteome equilibrium of colorectal cancer cells.

BACKGROUND: Chromosomal aneuploidy has been identified as a prognostic factor in the majority of sporadic carcinomas. However, it is not known how chromosomal aneuploidy affects chromosome-specific protein expression in particular, and the cellular proteome equilibrium in general. OBJECTIVE: The aim was to detect chromosomal aneuploidy-associated expression changes in cell clones carrying trisomies found in colorectal cancer. METHODS: We used microcell-mediated chromosomal transfer to generate three artificial trisomic cell clones of the karyotypically stable, diploid, yet mismatch-deficient, colorectal cancer cell line DLD1--each of them harboring one extra copy of either chromosome 3, 7 or 13. Protein expression differences were assessed by two-dimensional gel electrophoresis and mass spectrometry, compared to whole-genome gene expression data, and evaluated by PANTHER classification system and Ingenuity Pathway Analysis (IPA). RESULTS: In total, 79 differentially expressed proteins were identified between the trisomic clones and the parental cell line. Up-regulation of PCNA and HMGB1 as well as down-regulation of IDH3A and PSMB3 were revealed as trisomy-associated alterations involved in regulating genome stability. CONCLUSIONS: These results show that trisomies affect the expression of genes and proteins that are not necessarily located on the trisomic chromosome, but reflect a pathway-related alteration of the cellular equilibrium.

Cancer drug resistance: the central role of the karyotype.

Current genetic and epigenetic theories of cancer-specific drug resistance do not adequately explain: (i) the karyotypic changes that coincide with resistance, (ii) the high rates at which cancer cells acquire and enhance resistance compared to the rates of conventional mutation, (iii) the wide ranges of resistance such as multidrug resistance, (iv) the frequent occurrence of intrinsic drug resistance. We have recently proposed, that specific karyotypic alterations are sufficient for drug resistance via new transcriptomes of cooperative genes, independent of gene mutation. This mechanism generates new phenotypes just like trisomy 21 generates Down syndrome. These karyotypic changes are generated by cancer-specific aneuploidy autocatalytically, because aneuploidy destabilizes the karyotype by misbalancing teams of proteins that synthesize, repair and segregate chromosomes. Evidence for this chromosomal mechanism is as follows: (i) resistance is proportional to the number of clonal chromosomal alterations compared to drug-sensitive precursors. (ii) The high rates at which cancer cells acquire drug resistance are comparable with the rates, as high as 10(-2) per cell generation, at which their karyotypes change-dimming hopes for gene-specific therapies. (iii) Multidrug resistance probably reflects un-selected transcriptomes of karyotypes selected for resistance against specific drugs. (iv) Intrinsic drug resistance probably reflects unselected transcriptomes of karyotypes selected for oncogenicity. We also adduce evidence that resistance of chronic myeloid leukemia against the drug imatinib is chromosomal, although it is widely believed to be due to mutation of a kinase.

Artificially introduced aneuploid chromosomes assume a conserved position in colon cancer cells.

BACKGROUND: Chromosomal aneuploidy is a defining feature of carcinomas. For instance, in colon cancer, an additional copy of Chromosome 7 is not only observed in early pre-malignant polyps, but is faithfully maintained throughout progression to metastasis. These copy number changes show a positive correlation with average transcript levels of resident genes. An independent line of research has also established that specific chromosomes occupy a well conserved 3D position within the interphase nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated whether cancer-specific aneuploid chromosomes assume a 3D-position similar to that of its endogenous homologues, which would suggest a possible correlation with transcriptional activity. Using 3D-FISH and confocal laser scanning microscopy, we show that Chromosomes 7, 18, or 19 introduced via microcell-mediated chromosome transfer into the parental diploid colon cancer cell line DLD-1 maintain their conserved position in the interphase nucleus. CONCLUSIONS: Our data is therefore consistent with the model that each chromosome has an associated zip code (possibly gene density) 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.

Stage-specific alterations of the genome, transcriptome, and proteome during colorectal carcinogenesis.

To identify sequential alterations of the genome, transcriptome, and proteome during colorectal cancer progression, we have analyzed tissue samples from 36 patients, including the complete mucosa-adenoma-carcinoma sequence from 8 patients. Comparative genomic hybridization (CGH) revealed patterns of stage specific, recurrent genomic imbalances. Gene expression analysis on 9K cDNA arrays identified 58 genes differentially expressed between normal mucosa and adenoma, 116 genes between adenoma and carcinoma, and 158 genes between primary carcinoma and liver metastasis (P < 0.001). Parallel analysis of our samples by CGH and expression profiling revealed a direct correlation of chromosomal copy number changes with chromosome-specific average gene expression levels. Protein expression was analyzed by two-dimensional gel electrophoresis and subsequent mass spectrometry. Although there was no direct match of differentially expressed proteins and genes, the majority of them belonged to identical pathways or networks. In conclusion, increasing genomic instability and a recurrent pattern of chromosomal imbalances as well as specific gene and protein expression changes correlate with distinct stages of colorectal cancer progression. Chromosomal aneuploidies directly affect average resident gene expression levels, thereby contributing to a massive deregulation of the cellular transcriptome. The identification of novel genes and proteins might deliver molecular targets for diagnostic and therapeutic interventions.