RESUMO
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.
Assuntos
Adenocarcinoma/genética , Androgênios , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/fisiologia , Adenocarcinoma/metabolismo , Sítios de Ligação , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/metabolismo , Di-Hidrotestosterona/farmacologia , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Mucina-6 , Mucinas/biossíntese , Mucinas/genética , Proteínas de Neoplasias/biossíntese , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ornitina-Oxo-Ácido Transaminase/biossíntese , Ornitina-Oxo-Ácido Transaminase/genética , Neoplasias da Próstata/metabolismo , Proteína Quinase C-delta/biossíntese , Proteína Quinase C-delta/genética , Pirrolina Carboxilato Redutases/biossíntese , Pirrolina Carboxilato Redutases/genética , Receptores Androgênicos/genética , Canais de Cátion TRPV/biossíntese , Canais de Cátion TRPV/genética , Transcrição Gênica , delta-1-Pirrolina-5-Carboxilato RedutaseRESUMO
BACKGROUND: The androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) initiation and progression. To date, studies have focused disproportionately on androgen-stimulated genes such as prostate-specific antigen (PSA), while repressed genes have gained little attention, even though they too may be involved in regulating cell growth, differentiation, and apoptosis. METHODS: ChIP Display was used to identify putative AR target genes in the ablation-resistant human PCa cell line, C4-2B. Quantitative real-time reverse transcription-PCR analysis was used to measure gene expression in cells subjected to dihydrotestosterone (DHT) timecourse and dose-response, as well as AR knock-down and bicalutamide-treatments. RESULTS: We report on three genes, KIAA1217, CHRM1, and WBSCR28, which were newly identified in a screen for AR-occupied regions in C4-2B PCa cells, and which were repressed by treatment with DHT. AR knock-down resulted in increased KIAA1217, CHRM1, and WBSCR28 mRNA, indicating that, like PSA stimulation, AR represses these three genes even in the absence of added ligand. DHT decreased KIAA1217 and CHRM1 pre-mRNA levels, suggesting AR-mediated transcriptional inhibition. Cycloheximide attenuated DHT-mediated repression of CHRM1, suggesting the requirement of new protein synthesis. Furthermore, bicalutamide treatment did not mimic, but rather antagonized DHT-mediated KIAA1217 repression. Unlike the handful of androgen-repressed genes studied thus far, AR occupancy at KIAA1217, CHRM1, and WBSCR28 was mapped outside their respective 5'-promoter regions. CONCLUSIONS: Many more genes likely share AR-mediated gene repression through distal regulatory elements. Further study of such targets and their transcriptional regulation may help explain the receptor's tumorigenicity in PCa.