Identification of novel androgen receptor target genes in prostate cancer

Background: The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression. Results: Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells – D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1) – most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. Conclusion: AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general, response is stronger in C4-2B compared to LNCaP cells. Some of the genes near AR-occupied regions appear to be regulated by the AR in vivo as evidenced by their expression levels in prostate cancer tumors of various stages. Several AR target genes discovered in the present study, for example PRKCD and PYCR1, may open avenues in PCa research and aid the development of new approaches for disease management.Unnati Jariwala, Jennifer Prescott, Li Jia, Artem Barski, Steve Pregizer, Jon P Cogan, Armin Arasheben, Wayne D Tilley, Howard I Scher, William L Gerald, Grant Buchanan, Gerhard A Coetzee and Baruch Frenke

Identification of novel androgen receptor target genes in prostate cancer-0

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>ation with target loci. Two independent AR ChIPs, and IgG control ChIPs were subjected to the CD procedure as described in Methods. In the example shown here, PCRs were performed with the 'AC' and the 'TG' PCR primers (see Methods and Additional file ) with the annealing temperature set at either 70°C or 71°C as indicated. Amplified products were resolved using 8% PAGE and visualized by EtBr staining. The arrowheads point at bands amplified more prominently in the AR compared to the Control (IgG) lanes. , marker DNA; numbers above bands indicate size in bps. . The two bands indicated in panel A by arrowheads were excised, purified and re-amplified with the same 'AC' and 'TG' primers used for CD. The products were subjected to secondary digestion with the indicated enzymes, followed by agarose gel electrophoresis. Arrowheads point at similar III sub-fragments obtained from the two AR ChIPs. – , no template control, , uncut, , marker DNA. . The III subfragments from B were excised, purified and sequenced. By blasting against the human genome using Ensembl [65], both sequences mapped to chromosome 17q25.3, ~1.5-kb upstream of the MAFG gene and ~2.5-kb downstream of the PYCR1 gene as shown in the diagram. The two genes are transcribed in the same direction as indicated by the horizontal arrows. , polyadenylation signal. The AR binding region discovered through CD ('''') abuts a CpG island (), but does not overlap with any repetitive elements (). Several AREs () were identified in this region using Consite [66]. . AR occupancy at the PYCR1/MAFG locus was tested by conventional ChIP assay. The PSA enhancer serves as positive control. A non-target locus serves as the negative control. Genomic DNA was used to demonstrate that the ChIP amplification was performed within a dynamic range. – , no template control. , marker DNA

Identification of novel androgen receptor target genes in prostate cancer-4

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>roarray sets (21) and results are mined for all probesets (rows) interrogating each of the 32 CD-disclosed genes (Table 1). Heat map shows relative expression for each of the indicated probesets, where darker shades represent higher mRNA levels. Tumors included 23 prostate cancers from patients not receiving therapy (), 17 primary prostate cancers following 3-month neoadjuvant androgen ablation therapy (), and 7 AR-positive metastatic lesions (). All Grade A probesets interrogating each gene are shown, except for probesets 59776_at (WBSCR28) and 36904_at (KIF1A), which did not detect significant expression in any sample. Samples are grouped and ranked as follows. – probesets for the known AR-stimulated genes KLK3/PSA and TMPRSS2. – probesets exhibiting statistically greater mean expression in untreated compared to AAT-treated primary PCa samples (< 0.05), thereby representing putative AR-stimulated genes. – probesets exhibiting statistically lower mean expression in untreated compared to AAT-treated PCa samples (p < 0.05), thereby representing putative AR-repressed genes. – probesets exhibiting no statistical difference between samples without or with AAT. Probesets in Groups II-IV are ranked by -value in descending order

Identification of novel androgen receptor target genes in prostate cancer-3

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>wo days in CSS-supplemented medium. Gene expression was analyzed side-by-side by RT-qPCR and corrected for 18S rRNA. Bars represent the comparative ratio between the expression in C4-2B and LNCaP cells, where the expression level in LNCaP cells is defined as 1. Included in this Figure are only genes for which the expression levels were significantly different between the two cell lines in two independent experiments (n = 3; Mean ± SD). TRPV3 mRNA was detected in LNCaP cells in only one of the three measurements, and this value was used as the upper limit for TRPV3 expression in these cells. See additional file for details

Identification of novel androgen receptor target genes in prostate cancer-1

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>SS-containing medium for three days, and then re-fed (time 0) with the same medium supplemented with either 10 nM DHT or ethanol vehicle. RNA was extracted at the indicated time points during the time course and expression of the specified genes was measured by RT-qPCR. Expression levels relative to 18S rRNA (which itself stayed stable throughout the time course) are shown with the 0 time values defined as 1 for each cell line. Representative data is shown from one of two independent experiments with n = 3, except for panels 2L, O, U, Y and ZC, where the C4-2B data is derived from 6 measurements (see Additional file for the complete set of raw data). Error bars are SEM. Genes are roughly ordered based on the DHT-responsiveness in C4-2B cells, with stimulated genes first (panels A-J) to repressed genes last (panels ZC-ZF). TRVP3 mRNA was barely detectable in LNCaP cells

Identification of novel androgen receptor target genes in prostate cancer-2

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>A (black bars), followed by administration of either DHT (10 nM) or Ethanol vehicle for 16 hours. Expression levels of the indicated genes were analyzed in triplicate by RT-qPCR and corrected for 18S rRNA levels. Values measured with the non-specific siRNA and ethanol were defined as 1. Results (Mean ± SD) are representative of three independent experiments