Transcription Factors Involved in Prostate Gland Adaptation to Androgen Deprivation

<div><p>Androgens regulate prostate physiology, and exert their effects through the androgen receptor. We hypothesized that androgen deprivation needs additional transcription factors to orchestrate the changes taking place in the gland after castration and for the adaptation of the epithelial cells to the androgen-deprived environment, ultimately contributing to the origin of castration-resistant prostate cancer. This study was undertaken to identify transcription factors that regulate gene expression after androgen deprivation by castration (Cas). For the sake of comparison, we extended the analysis to the effects of administration of a high dose of 17β-estradiol (E2) and a combination of both (Cas+E2). We approached this by (i) identifying gene expression profiles and enrichment terms, and by searching for transcription factors in the derived regulatory pathways; and (ii) by determining the density of putative transcription factor binding sites in the proximal promoter of the 10 most up- or down-regulated genes in each experimental group in comparison to the controls <i>Gapdh</i> and <i>Tbp7</i>. Filtering and validation confirmed the expression and localized EVI1 (Mecom), NFY, ELK1, GATA2, MYBL1, MYBL2, and NFkB family members (NFkB1, NFkB2, REL, RELA and RELB) in the epithelial and/or stromal cells. These transcription factors represent major regulators of epithelial cell survival and immaturity as well as an adaptation of the gland as an immune barrier in the absence of functional stimulation by androgens. <i>Elk1</i> was expressed in smooth muscle cells and was up-regulated after day 4. <i>Evi1</i> and <i>Nfy</i> genes are expressed in both epithelium and stroma, but were apparently not affected by androgen deprivation.</p></div

Kinetics of <i>Evi1</i>, <i>Elk1</i> and <i>Nfyb</i> expression.

<p>Quantitative RT-PCR determination of the mRNA content for the <i>Evi1</i> (A), <i>Elk1</i> (B) and <i>Nfyb</i> (C) genes in the prostate of non-castrated (NC) and castrated rats up to 7 days after surgery. The fold-change variation with respect to the controls is shown as the mean ± standard variation (n = 3 for each time point). The asterisks indicate p<0.05. The dotted lines in each figure correspond to the fourth-power exponential fitting curve, and are shown together for the sake of direct comparison in D.</p

TF with possible binding sites found in the 80 most-regulated genes.

<p>(A) TF binding sites not found in the internal control genes. (B) TF binding sites also found in the proximal promoter of internal control genes. Density values≤0.01 were omitted.</p

TF localization in epithelial and stroma cells.

<p>Immunohistochemical localization of selected TF (red) as indicated in the prostate of non-castrated controls (NC) and castrated rats 3 days after surgery (CAS). MYBL2 (A,B), GATA2 (C,D), EVI1 (E,F), ELK1 (G,H), NFYB (I,J), NFKB1 (K,L), NFKB2 (M,N), REL (O,P), RELA (Q,R), RELB (S,T). Nuclei were stained with DAPI (blue). White arrows indicate smooth-muscle cells. Yellow arrowheads indicate cells showing nuclear location of the TF. L  =  gland lumen; S  =  stroma. Scale bars = 50 µm.</p

Kinetics of <i>Mybl1</i>, <i>Mybl2</i> and <i>Gata2</i> expression.

<p>Quantitative RT-PCR determination of the mRNA content for the <i>Mybl1</i> (A), <i>Mybl2</i> (B) and <i>Gata2</i> (C) genes in the prostate of non-castrated (NC) and castrated rats up to 7 days after surgery. The fold-change variation with respect to the controls is shown as the mean ± the standard variation (n = 3 for each time point). The asterisks indicate p<0.05. The dotted lines in each figure correspond to the fourth-power exponential fitting curve, and are shown together for the sake of direct comparison in D.</p

Venn diagrams showing the number of genes that were differentially expressed.

<p>The diagrams show the genes that are exclusive to each treatment and those that are shared by two or all three experimental groups (Cas, E2 and Cas+E2) compared to the control group.</p

Number of enrichment terms exclusive or shared among groups.

<p>The three treatments shared 90 enrichment terms. The highest number of enrichment terms was found in the Cas group. The Cas+E2 group showed no exclusive enrichment term.</p

Summary of the sequential analyses performed in this study.

<p>Summary of the sequential analyses performed in this study.</p

The eighty most regulated genes.

<p>The ten most up- or down-regulated genes that were exclusive to each of the experimental groups or were shared by the three treatments. These genes are involved in different functions (see Appendix 1 for details on the annotated functions for these genes).</p

Kinetics of <i>Nfkb1</i>, <i>Nfkb2, Rel</i> and <i>RelA</i> expression.

<p>Quantitative RT-PCR determination of the mRNA content for the <i>Nfkb1</i> (A), <i>Nfkb2</i> (B), <i>Rel (c-Rel)</i> (C) and <i>RelA</i> (D) genes in the prostate of non-castrated (NC) and castrated rats up to 7 days after surgery. The fold-change variation with respect to the controls is shown as the mean ± standard variation (n = 3 for each time point). The asterisks indicate p<0.05. The dotted lines in each figure correspond to the fourth-power exponential fitting curve, and are shown together for the sake of direct comparison in E.</p