Aneuploidy-dependent massive deregulation of the cellular transcriptome and apparent divergence of the Wnt/beta-catenin signaling pathway in human rectal carcinomas

Abstract

To identify genetic alterations underlying rectal carcinogenesis, we used global gene expression profiling of a series of 17 locally advanced rectal adenocarcinomas and 20 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 >5-fold difference, and 85 genes always had at least a 2-fold change in all of the matched samples. Twelve genes satisfied all three of these criteria. Altered expression of genes such as PTGS2 (COX-2), WNT1, TGFB1, VEGF, and MYC was confirmed, whereas 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 show 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.

Figure 1

Network mapping of genes with >5-fold expression change using IPA. PTGS2 and DCN (arrows) connect the top two networks identified by IPA. Shades of red, genes with >5-fold higher expression in the tumors; shades of green, >5-fold decrease in expression in the tumors relative to the normal rectal mucosa. Gene names and the actual fold changes are indicated.

Figure 2

Chromosome localization of genes with significant expression changes. A, 93% of the 12,291 genes that passed the filtering criteria had chromosome mapping locations. White columns, percentages of these genes that map to each chromosome. Ninety-four percent of the 1,722 genes differentially expressed in the tumors with P < 0.0001 had known chromosome locations. Black columns, percentages of these genes that map to each chromosome. B, the percentage of genes indicated as black columns in (A), which were up-regulated (black) or down-regulated (white) in the tumors relative to the normal rectal mucosa.

Figure 3

CGH profiles of 21 rectal carcinomas. Red bars to the left of the chromosome, genomic copy number losses; green bars to the right of the chromosome, copy number gains. Thick bars, high-level copy number alterations (i.e., amplifications). The patient code corresponding to each bar is indicated.

Figure 4

Correlation between gene expression and alterations of chromosome copy number. A, the average gene expression value (Y axis) is plotted against the average CGH ratio value (X axis) for each of 12 patients for which we had done both analyses. The percentage correlation, its P value, and the R² are indicated on each plot. The directionality of the copy number change most commonly observed 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). Specifics about the number of samples in each category are included in Supplementary Table S8. Blue, genes with increased expression relative to the reference RNA; red, genes with decreased expression relative to the reference RNA.

Figure 5

Average chromosome arm expression patterns clearly discern tumors from rectal mucosa but also reveal two categories of normal mucosa. A, the gene expression values for every gene along the length of an individual chromosome arm were averaged for each of 20 normal mucosa biopsies (left) and 17 rectal carcinomas (right). There was a visible separation of the mucosa into two groups, each composed of 10 biopsies. The groups are differentially colored, representing Mucosa₀ (blue) and Mucosa₁ (red). B, unsupervised hierarchical cluster analysis of 20 normal mucosa (left) and 17 rectal carcinomas (right) using the average chromosome arm gene expression values plotted in (A). C, principle component analysis (left) based on the average chromosome arm gene expression values. Right, plot of the average chromosome arm gene expression values for the 20 normal mucosa from left to right, which are most useful in distinguishing Mucosa₀ (blue) from Mucosa₁ (red). D, unsupervised hierarchical cluster analysis of the 12 patients from whom we had normal rectal mucosa (left) and patient matched tumor biopsies (right). 0, Mucosa₀; 1, Mucosa₁.