Systematic exploration of genomic alterations in colorectal cancer cell lines and their consequences on global gene expression levels: Specific genomic lesions have been identified in hereditary forms of colorectal cancer, however the mechanism responsible for the development of spontaneous colorectal cancer, which accounts for nearly 85% of all cases, remains unknown. These tumors are invariably aneuploid and contain both numerical as well as structural aberrations. While we have previously analyzed the extent to which gross genomic copy number imbalances affect gene expression in primary colon and rectal tumors, the inability to perform chromosome analyses on harvested tissue precluded our ability to look at the affect of structural alterations. We therefore combined Spectral Karyotyping, chromosome and array-based CGH to obtain high resolution mapping of colorectal cancer cell lines commonly utilized as model experimental systems. These results were combined with both centrosome analysis and gene expression profiling to devise a model for the effects of genomic alterations on gene expression leading to the development of colorectal carcinoma. As such, high-resolution aCGH confirmed the preponderance of chromosome breakpoints at sites of copy number variants in the human genome, a novel mechanism of DNA breakage in cancer that we recently described in primary colon carcinomas. Precision mapping of breakpoints and global gene expression profiling facilitated our ability to specifically address the consequences of chromosomal aberrations, both copy number alterations and structural rearrangements, with respect to their affect on gene expression. Although we did identify several instances where genes within the immediate proximity of breakpoints appeared to be preferentially altered in their expression, an event frequently observed in hematologic malignancies, the rearrangements by and large served to mediate a change in the copy number of genomic regions. The large coefficient of correlation (R= 0.69) between genomic copy number and the average expression of all genes mapping within those segments points to a more subtle and complex outcome. We did, however, identify several putative colorectal oncogenes whose high-level genomic amplification correlated with increased gene expression. These will be further explored as potential diagnostic and therapeutic targets. Chromosomal breakpoints in primary colon cancer cluster at sites of structural variants in the genome: Colorectal cancer is the second leading cause of cancer death in Europe and the United States, with about 300,000 new cases and 200,000 deaths each year. Cytogenetic and molecular cytogenetic studies clearly established that the colorectal cancer genome is defined by a specific distribution of genomic imbalances, most prominently gains of chromosomes and chromosome arms 7, 8q, 13, and 20q, as well as losses of chromosomes 4q, 8p, 17p, and 18q. Within the last decade, microarray technology has been extensively applied to survey the cellular transcriptome of common solid tumors, including colorectal cancer, and, for colon cancers, gene expression signatures were subsequently correlated with clinical outcome. However, high-resolution mapping of chromosomal copy number changes has only recently been achieved using BAC or cDNA clone-based arrays. Chromosome 8q is one of the most frequently gained chromosomal arms in colorectal cancers, and it is conceivable that it contains more oncogenes than just the MYC oncogene, which maps to chromosome band 8q24.21. A potential role of chromosome 8q for the development of lymph node metastases has been previously reported, and overexpression of a gene, PRL-3, that maps to chromosome 8q24.3 has been implied in the development of liver metastases. Moreover, the 8q24 locus contains SNPs that are associated with an increased risk for the development of colon cancer. Recently, a new class of genetic variation among human has become recognized as a major source of genetic diversity. Termed structural variations, these polymorphisms can present themselves as copy number variants (CNVs) and segmental duplications, which can be a CNV, but are not necessarily so. These polymorphisms can induce chromosomal rearrangements. One of our previous analyses of chromosomal aberrations in cell lines established from different carcinomas indicated that genomic copy number changes can be triggered by jumping translocations, many of which originated in the pericentromeric heterochromatin of several chromosomes. These regions frequently contain segmental duplications and other structural variants of the genome. Taken together, these data enticed us to systematically explore the genomic aberration profile of chromosome 8 and the potential involvement of structural variants of the human genome in the genesis of chromosomal aberrations in this common cancer. We therefore established a high-resolution map of genomic copy number changes in 51 primary colon carcinomas using comparative genomic hybridization on both a BAC-based genomic tiling array for chromosome 8 and, for a subset of those, using a 185K oligonucleotide platform for whole genome coverage. This analysis confirmed the dominant role of this chromosome. Unexpectedly, the position of breakpoints suggested co-localization with structural variants in the human genome. In order to map these sites with increased resolution and to extend the analysis to the entire genome, we analyzed a sub-set of these tumors (n=32) by comparative genomic hybridization on an 185K oligonucleotide array platform. Our comprehensive map of the colon cancer genome confirmed recurrent and specific low-level copy number changes of chromosomes 7, 8, 13, 18, and 20, and unveiled additional, novel sites of genomic imbalances including amplification of a histone gene cluster on chromosome 6p21.1-21.33 and deletions on chromosome 4q34-35. The systematic comparison of segments of copy number change with gene expression profiles showed that genomic imbalances directly affect average expression levels. Strikingly, we observed a significant association of chromosomal breakpoints with structural variants in the human genome: 41 per cent of all copy number changes occurred at sites of such copy number variants (p<2.2e-16). Such an association has not been described before and reveals a yet underappreciated plasticity of the colon cancer genome;it also points to potential mechanisms for the induction of chromosomal breakage in cancer cells. The systematic integration of genomic copy number changes with gene expression profiles in colorectal carcinomas revealed 44 highly overexpressed genes mapping to localized amplicons on chromosome 13, which is recurrently gained. RNA interference based silencing identified eight candidates whose loss of function resulted in viability reduction of at least 20% in colorectal cancer cell lines. Global expression profiles, established for each gene following RNAi defined their functional space. One candidate, LNX2, is involved in regulating notch signaling;silencing LNX2 resulted in reduced NOTCH levels, in downregulation of the transcription factor TCF7L2, and in markedly reduced WNT-signaling. Genomic amplification of chromosome 13 and overexpression of LNX2 therefore constitutively activates the WNT-pathway, supporting evidence for a NOTCH-WNT axis in colorectal cancer.
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