Gene expression profiling reveals a massive, aneuploidy-dependent transcriptional deregulation and distinct differences between lymph node-negative and lymph node-positive colon carcinomas

Abstract

To characterize patterns of global transcriptional deregulation in primary colon carcinomas, we did gene expression profiling of 73 tumors [Unio Internationale Contra Cancrum stage II (n = 33) and stage III (n = 40)] using oligonucleotide microarrays. For 30 of the tumors, expression profiles were compared with 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 >5-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 lymph node-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 reverse transcription-PCR in >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/beta-catenin signaling cascade, suggesting similar pathogenic pathways.

Figures

Figure 1

Network annotation of genes with greater than 5-fold expression change in colon tumors relative to normal colonic epithelium using IPA. Red, genes up-regulated in colon tumors; green, genes down-regulated in colon tumors. Dark shade, genes with >5-fold differential expression; light shade, genes with lower difference in expression. PLF2 (yellow) was not spotted on the array, and TNF (blue) did not meet the filtering criteria. Genes whose names are printed in bold font were deregulated significantly (P < 0.0001). CTNNB1 was present twice on the arrays and revealed conflicting results. MYC assumes a central position in the network.

Figure 2

Chromosomal localization of genes with significant expression changes. A, 94% of the 16,037 genes that passed the filtering criteria had chromosome mapping locations. White columns, percentages of these genes that map to each chromosome; 94% of the 4,371 genes differentially expressed in the tumors with P < 0.0001 had known chromosome locations. Black columns, percentages of these genes, which map to each chromosome. B, percentage of genes indicated as black columns in (A) that were up-regulated (black) or down-regulated (white) in the tumors relative to the normal colon mucosa.

Figure 3

A, MDA of lymph node–negative (red) and lymph node–positive (green) colon cancers based on the set of 74 differentially expressed features. B, MDA of 73 colon cancers (red) and 30 matched normal mucosa samples (green). Note the stringent separation of the two groups.

Figure 4

Correlation of chromosomal copy numbers and resident gene expression levels. A, average CGH ratio value (x-axis) is plotted against the average gene expression value (y-axis) for each of 32 patients for whom we had done both analyses. The percentage correlation, its Ps, and R2 are indicated in each plot. The directionality of the copy number change is represented as a + (gain) or – (loss) preceding the chromosome number. B, the average expression of each gene along the length of the chromosome is plotted for those carcinomas without (left) and with (right) a copy number alteration. These plots correspond to the graphs in (A). Blue, genes with increased expression; red, genes with decreased expression relative to the reference RNA.