Tamoxifen-elicited uterotrophy: cross-species and cross-ligand analysis of the gene expression program

Abstract

Background: Tamoxifen (TAM) is a well characterized breast cancer drug and selective estrogen receptor modulator (SERM) which also has been associated with a small increase in risk for uterine cancers. TAM's partial agonist activation of estrogen receptor has been characterized for specific gene promoters but not at the genomic level in vivo.Furthermore, reducing uncertainties associated with cross-species extrapolations of pharmaco- and toxicogenomic data remains a formidable challenge.

Results: A comparative ligand and species analysis approach was conducted to systematically assess the physiological, morphological and uterine gene expression alterations elicited across time by TAM and ethynylestradiol (EE) in immature ovariectomized Sprague-Dawley rats and C57BL/6 mice. Differential gene expression was evaluated using custom cDNA microarrays, and the data was compared to identify conserved and divergent responses. 902 genes were differentially regulated in all four studies, 398 of which exhibit identical temporal expression patterns.

Conclusion: Comparative analysis of EE and TAM differentially expressed gene lists suggest TAM regulates no unique uterine genes that are conserved in the rat and mouse. This demonstrates that the partial agonist activities of TAM extend to molecular targets in regulating only a subset of EE-responsive genes. Ligand-conserved, species-divergent expression of carbonic anhydrase 2 was observed in the microarray data and confirmed by real time PCR. The identification of comparable temporal phenotypic responses linked to related gene expression profiles demonstrates that systematic comparative genomic assessments can elucidate important conserved and divergent mechanisms in rodent estrogen signalling during uterine proliferation.

Figure 1

Dose Response Uterine Wet Weights (UWW). UWW was measured across several EE and TAM doses in the mouse and rat. A plot of the fold change increase in wet weight is plotted. A dose response curve was fit to the data (GraphPad 4.0) to estimate EC₅₀ values. 100 μg/kg b.w. approximates the maximum response in all four cases and was used in subsequent time course studies. ED₅₀ values were comparable between ligands in the rat while exhibiting only a two fold difference in the mouse, indicating conservation of sensitivity to EE and TAM.

Figure 2

Temporal Changes in Luminal Epithelial Height (LEH). LEH was quantified for each treatment group and compared to VEH controls. LEH induction was temporally delayed in response to TAM treatment in both species. Mouse induction was comparable between ligands while TAM-induced LEH was significantly (p < 0.05) greater than EE in rats.

Figure 3

Comparative Analysis of Species-Conserved, Ligand-Specific Gene Expression. (A) cDNA microarrays were used containing 13,361 mouse clones representing 8,734 unique genes and 8,507 rat clones representing 5,684 unique genes. (B) Differentially expressed genes regulated by each ligand were identified using relaxed criteria to minimize the likelihood of false-negatives that marginally failed to meet the selection criteria. Differentially expressed genes elicited by both EE and TAM were analyzed for similarity in their temporal profiles by comparative analysis. Genes were designated as either CoActive-Similar direction (CAS), CoActive-Divergent direction (CAD), Displaced Active Similar direction (DAS), or Displaced Active Divergent direction (DAD) based on the relationship between the time and direction of differential regulation, and the significance (P1(t)) of the expression profile relative to the VEH control. (C) The comparative results were plotted on a coordinate correlation graph. A majority of genes show positive correlation between ligands for both fold change and P1(t) value. (D) Cross species analysis of ligand-divergent (CAD) expression profiles indicate no conservation.

Figure 4

Comparative Analysis of Ligand-Conserved, Species-Specific Gene Expression. (A) cDNA microarrays were used containing 13,361 mouse clones representing 8,734 unique genes and 8,507 rat clones representing 5,684 unique genes. There were 3,417 orthologous genes represented on the mouse and rat cDNA microarrays as determined by HomoloGene. (B) Differentially expressed genes were assessed for similar expression patterns using relaxed criteria (|fold change| > 1.3; P1(t) > 0.99) to minimize the likelihood of false-negatives that marginally failed to meet the selection criteria. (C) Common differentially expressed orthologous genes were examined for comparable expression patterns, and designated according to the coactivity categories (CAS, CAD, DAS, DAD) as described in Figure 3. The results were plotted on a coordinate correlation graph. A high proportion of differentially expressed orthologs exhibited a positive correlation when considering both fold-change and P1(t). (D) Comparisons of species-divergent (CAD) expression profiles identified 35 genes that were differentially expressed in the mouse and the rat but exhibited putative divergent regulation elicited by EE and TAM.

Figure 5

Quantitative Real Time PCR Confirmation of Species-Specific ER Regulation of Ca2. The temporal gene expression of carbonic anhydrase 2 (designated Car2 in the mouse and Ca2 in the rat and human, hereafter referred to as Ca2 to represent all orthologs) was further validated by species-conserved and -unique primer sets. The QRT-PCR results confirm the microarray results indicating that Ca2 is differentially regulated in the mouse when compared to the rat. This differential regulation was elicited by both EE and TAM. Mouse Ca2 expression is significantly (p < 0.05) induced while rat expression is significantly (p < 0.05) repressed (n = 5, Tukey's HSD post hoc test), at multiple time points as indicated by the asterisk (*).

Figure 6

Four Way Venn Analysis of Active Genes. Each data set was converted into ortholog space and processed for activity using relaxed criteria prior to analysis using the 4-way Venn Diagram Generator . The Venn set was subsequently filtered for genes which met the initial, high-stringency criteria to ensure robust comparisons. Gene lists for each Venn set are available [see Additional File 7].