Gene expression profiling identifies lobe-specific and common disruptions of multiple gene networks in testosterone-supported, 17beta-estradiol- or diethylstilbestrol-induced prostate dysplasia in Noble rats

Abstract

The xenoestrogen diethylstilbestrol (DES) is commonly believed to mimic the action of the natural estrogen 17beta-estradiol (E2). To determine if these two estrogens exert similar actions in prostate carcinogenesis, we elevated circulating levels of estrogen in Noble (NBL) rats with E(2/DES-filled implants, while maintaining physiological levels of testosterone (T) in the animals with T-filled implants. The two estrogens induced dysplasia in a lobe-specific manner, with E2 targeting only the lateral prostate (LP) and DES impacting only the ventral prostate (VP). Gene expression profiling identified distinct and common E2-disrupted versus DES-disrupted gene networks in each lobe. More importantly, hierarchical clustering analyses revealed that T + E2 treatment primarily affected the gene expression pattern in the LP, whereas T + DES treatment primarily affected the gene expression profile in the VP. Gene ontology analyses and pathway mapping suggest that the two hormone treatments disrupt unique and/or common cellular processes, including cell development, proliferation, motility, apoptosis, and estrogen signaling, which may be linked to dysplasia development in the rat prostate. These findings suggest that the effects of xenoestrogens and natural estrogens on the rat prostate are more divergent than previously suspected and that these differences may explain the lobe-specific carcinogenic actions of the hormones.

Figure 1

Hierarchical clustering analysis of T + E₂ and T + DES gene expression data set. (A) Dendrogram of T + E₂ expression data set in LP and VP. (B) Dendrogram of T + DES expression data set in LP and VP. (C) Venn diagram showing the number of genes differentially expressed in each treatment group compared with the respective untreated control. (D) Gene interaction network of a subset of differentially expressed genes that are common in both LP and VP dysplasia. Genes bordered with red were validated by quantitative real-time PCR.

Figure 2

Heat maps and gene interaction networks of differentially expressed genes found exclusively in the LP dysplasia following T + E₂ treatment. Red and green denote upregulated and downregulated expression, respectively, as compared with the overall gene's mean value normalized to the universal rat reference RNA. Columns represent data from a single prostate sample, and rows correspond to a single gene probe. (A) A single cluster of upregulated genes identified in the T + E₂ LP dysplasia, marked with pink (left panel); a selected region (blue box) of this cluster is enlarged (right panel). (B1 and B2) Two separate downregulated gene clusters observed only in T + E₂ LP dysplasia, marked with pink (left panels); selected clusters are magnified (right panels). (C and D) Two representative gene interaction networks (with the highest relevancy scores) generated by IPA analysis from the differentially expressed genes in the T + E₂ LP dysplasia panel. Green indicates downregulated; red, upregulated. Genes bordered in red were validated by real-time q-PCR. See Figure 1 for key to IPA network.

Figure 3

Post hoc real-time q-PCR analyses of selected genes in the T + E₂ LP dysplasia panel. Data were normalized to the levels of Rpl19. Bars indicate standard deviations (SD) of three to five animals in each treatment group. *P < .05, **P < .01 by one-way ANOVA with Tukey post hoc analysis.

Figure 4

Heat maps and gene interaction networks of differentially expressed genes found uniquely in the VP dysplasia following T + DES treatment. (A) A single cluster of upregulated genes identified only in the T + DES VP dysplasia, marked with pink (left panel), and the selected cluster enlarged (right panel). (B1 and B2) Clusters of downregulated genes in the VP but not in the LP following T + DES exposure. Interestingly, a single cluster (B2, left panel) corresponds to the downregulated genes in the T + DES-treated VP and is also underexpressed in both untreated and treated LPs. A selected region (blue box) of this cluster is enlarged (right panel). (C and D) Two representative gene interaction networks (with the highest relevancy scores) generated by IPA analysis from the differentially expressed genes in the T + DES VP dysplasia panel. Green indicates downregulated; red, upregulated. Genes bordered with red were validated by real-time q-PCR. See Figure 1 for key to IPA network.

Figure 5

Post hoc validation of selected genes in the T + DES VP dysplasia panel by real-time q-PCR. The data were normalized to the levels of Rpl19. Bars indicated standard deviations (SD) of three to five animals in each treatment group. *P < .05, **P < .01 by one-way ANOVA with Tukey post hoc analysis.

Figure 6

Heat maps of genes insensitive to (A) T + E₂ or (B) T + DES. (C) Venn diagram showing the number of genes insensitive to T + E₂ and/or T + DES treatment. Note that 41 genes are found in common in both hormone-insensitive panels and mapped to an IPA network (D) that includes the Ar, a key regulator of prostate function. Gray indicates no change in gene expression levels compared with those in untreated counterparts. See Figure 1 for key to IPA network.