Fig 1.
Venn diagram of genes whose transcript levels differ between colonic adenoma and normal epithelium for three genera.
The sets of genes with differential transcript levels between colonic adenomas and normal colonic epithelium are summarized for the untreated Apc Min/+ mouse, Apc Pirc/+ rat, and human. Up in adenoma presents genes with increased levels in adenomas compared to the normal colonic epithelium. Down in adenoma presents genes with decreased levels in adenomas. The mouse and rat data were collected from experiments described in Methods and accessible in GEO datasets GSE107139 and GSE54036. Human data were retrieved from the published GEO dataset GDS2947 comparing adenomatous tumors with normal colonic tissue from 32 patients with spontaneous, non-familial colorectal cancer. For each genus, the comparison of transcript level was performed using a stringency of a change in transcript level by at least a factor of 2 with a false discovery rate of 0.05. The pairwise and three-way intersections for these sets were determined from this Venn diagram. The Venn diagram in Panel A displays the pairwise and three-way intersections of these gene lists between genera. For each differential gene list, the corresponding set of enriched GO categories was determined using a multi-set approach that accounts for category size variation and category overlaps. The Venn diagram in Panel B displays the pairwise and three-way intersections between genera of these lists of enriched GO categories. The higher agreement between genera at the GO level, suggested by these Venn diagrams, was tested by permutation analysis in Fig 2.
Fig 2.
Permutation test of significance of agreement between differential transcriptome signals shared by all three genera.
Each panel compares the observed mean pairwise overlap fraction (red) of the three species lists with the distribution of sharing rates for randomized data (boxplot, from 1000 gene-permutations). The extent of sharing among gene lists (Panel A) is lower than the GO level (19% and 13%, for up and down, respectively), but still higher than expected by chance. On a GO basis (Panel B), the observed intersection between genera for enhanced signals is 43% (p = 0.001) and for diminished signals 33% (p = 0.002). Though relatively few adenoma-associated transcripts are shared among all three genera, the agreement is statistically significant and is accentuated at the level of GO functional categories.
Fig 3.
Dominant functional categories of the genes whose transcript levels are enhanced in adenomas of any of the three genera—Mouse, rat, and human.
Dominant functional categories (rows) enriched in the large list of 5602 genes (columns) having increased transcript levels in tumors in any one of the three genera–mouse, rat, and human from among 19169 annotated genes. 274 populated GO sets showed enrichment in the union of gene lists at p < 10−5; a subset of these covering the union with low redundancy is shown in the Figure. Shaded area in each row counts the number of tumor-associated genes having that functional property; categories are ordered from the top by decreasing numbers of tumor-associated genes not contained in a prior category. Colors indicate the genus in which the transcript level is enhanced in adenomas.
Fig 4.
Dominant functional categories of the genes whose transcript levels are reduced in adenomas of any of the three genera—Mouse, rat, and human.
Functional categories (rows) enriched in the large list of 5477 genes (columns) having decreased transcript levels in tumors in any one of the three genera–mouse, rat, and human from among 19169 annotated genes. 218 populated GO sets showed enrichment at p < 10−5; a subset of these covering the union with low redundancy is shown in the figure. The shaded area in each row counts the number of tumor-associated genes having that functional property; categories are ordered from the top by decreasing numbers of tumor-associated genes not contained in a prior category. Colors indicate the genus in which the transcript level is diminished in adenomas.
Fig 5.
Heatmap summarizing the transcript levels from 89 genes (75 up; 14 down) that show altered expression in all three genera.
Both the mouse and the rat models are genetically homogeneous, whereas the human is not. Thus, genetic heterogeneity can explain the dispersion of the human data. Color indicates deviation from gene- and genus-specific average expression (log2 scale). The arrow notes the position of the Pde4b/PDE4B transcript.
Fig 6.
Dominant functional categories (GO terms) enriched in the 75 genes with increased transcript level in all three genera.
Functional categories (rows) enriched in the75 genes (columns) having increased transcript levels in tumors in all three genera–mouse, rat, and human from among 19169 annotated genes. Shaded area in each row counts the number of tumor-associated genes having that functional property; categories are ordered from the top by decreasing numbers of tumor-associated genes not contained in a prior category.
Table 1.
Test of effect of the Pde4b genotype on the number of adenomas in the colon of Apc Min/+ mice over a series of backcross-intercross generations.
Gross tumor numbers were measured in the colons of ApcMin/+ mice from the series of backcross-intercross generations, carrying the Pde4b+/+, Pde4b+/- and Pde4b-/- genotypes. Pde4b+/+ animals (110) show the lowest average colon tumor count 2.4 +/- 2.2. Pde4b+/- animals (192) show higher average colon tumor counts than Pde4b+/+ animals 3.3 ± 2.9. Finally Pde4b-/- mice (66) show the highest average colon tumor number 4.3 ± 4.6. p = 0.0004 by the Kruskal-Wallis Test of association between tumor number and number of mutant alleles of Pde4b. We reject the null hypothesis that there is no difference in gross colonic tumor number as a function of Pde4b genotype.
Fig 7.
Heatmap summarizing the transcripts from the normal colonic epithelium of ApcMin/+ mice whose levels differ between tumor-bearing and tumor-free mice.
Expression analysis was carried out for transcripts of the normal colonic epithelium of ApcMin/+ mice. The levels of these transcript populations were compared between mice bearing 1 or 2 colonic adenomas versus mice free of colonic adenomas. Analysis of differences between groups in hybridization signal was performed using EBarrays. The stringency cutoff was set at a difference of at least a factor of 2 fold and FDR of 20%. The Pde4b signal was confirmed by RT PCR analysis (Fig 9).
Fig 8.
Real time PCR analysis of transcripts that differ in level between the ApcMin/+ adenoma and the normal colonic epithelium of tumor-bearing versus tumor-free ApcMin/+ mice.
Real time PCR results for transcripts of the normal colonic epithelium (NCE) whose level differ between ApcMin/+ mice bearing colonic adenomas (T+) and ApcMin/+ mice free of colonic tumors (T-). The cycle number (ΔCT), normalized to GAPDH, is shown on the Y axis. The relevant significant pairwise comparisons are marked: * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001; NS for Not Significant. WT, wildtype.
Fig 9.
Test of the effect of Pde4b genotype on colonic adenoma numbers in Apc Min/+ mice treated with DSS.
Apc Min/+ animals of Pde4b+/+, Pde4b+/- and Pde4b-/- genotypes treated with 2% and 4% DSS. Gross colonic tumor counts were measured in the colons. Two treated heterozygotes were sacrificed at 87 days; the treated homozygous mutants were sacrificed between 43 and 100 days. The other animals were all sacrificed at 100 days. The colonic tumor count of each animal is presented in this scatter plot. For the 2% DSS treatment, the numbers of mice and average colonic tumor multiplicity of each Pde4b genotype were: Pde4b+/+ animals (17) tumor count of 3.9 ± 1.4; Pde4b+/- animals (13) tumor count of 6.5 ± 4.6; Pde4b -/- mice (5) tumor count of (3.6 ± 1.8). We cannot rule out the null hypothesis that there is no difference between gross colonic tumor number based on Pde4b genotype with 2% DSS treatment (p = 0.38 by the Kruskal-Wallis test). For animals treated with 4% DSS, 30 Pde4b+/- animals show a higher average colon tumor count than 21 Pde4b+/+ animals (12.2 ± 9.9 versus 5.4 ± 3.9); p = 0.009 by the Wilcoxon rank sum test. Only 9 Pde4b-/- animals entered the study; those colon tumor counts (9.7 ± 9.9) are not significantly different from counts for the other two genotypes. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; two-sided Wilcoxon rank sum test. The median for each group is indicated by a thin horizontal line.
Table 2.
Effect of the Pde4b genotype on the survival of Apc Min/+ and Apc +/+ mice treated with DSS.
For each cohort of mice, the number surviving to 100 days of age is given in the numerator and the total number in the denominator.
Fig 10.
Kaplan-Meier survival curve of 4% DSS-treated Apc Min/+ mice versus Pde4b genotype.
The distribution of ages at which 4% DSS treated Apc Min/+ mice of Pde4b+/+, Pde4b+/- and Pde4b+/- genotypes became moribund is presented as Kaplan-Meier survival curves. Pde4b-/- mice demonstrate significantly accelerated morbidity compared to either Pde4b+/- or Pde4b+/+ mice.
Fig 11.
Feedback model for the interaction of Wnt and cyclic AMP signaling.
The APC complex mediates the destruction of β-catenin. Its activity in turn is neutralized by WNT signaling allowing for β-catenin accumulation and nuclear translocation. This double-negative chain makes WNT a positive-regulator of β-catenin activity. When APC is truncated, increased β-catenin-mediated transcription ensues, causing tumorigenesis in the colon. Promoter-binding assays [51] are interpreted to show that nuclear β-catenin activates the transcription of PDE4B, whose hydrolytic activity inactivates cyclic AMP (cAMP). Since cAMP is a positive effector of protein kinase A, which in turn activates β-catenin, the hydrolysis of cAMP blunts the activation of β-catenin. The observed silencing of PDE4B in advanced colonic cancer, which would be predicted to enhance β-catenin activity, may involve the repressive histone deacetylase HDAC4, that is also β-catenin target gene.