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Androgen-Induced Programs for Prostate Epithelial Growth and Invasion Arise in Embryogenesis and Are Reactivated in Cancer

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Androgen-Induced Programs for Prostate Epithelial Growth and Invasion Arise in Embryogenesis and Are Reactivated in Cancer Oncogene (2008) 27, 7180–7191 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Androgen-induced programs for prostate epithelial growth and invasion arise in embryogenesis and are reactivated in cancer EM Schaeffer1,2,3,5, L Marchionni3,5, Z Huang1,2, B Simons1, A Blackman3,WYu3, G Parmigiani1,3,4 and DM Berman1,2,3 1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 3Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA and 4Department of Biostatistics, Johns Hopkins University School of Medicine, Baltimore, MD, USA Cancer cells differentiate along specific lineages that most significant links to the development and cancer, we largelydetermine their clinical and biologic behavior. highlight coordinate induction of the transcription factor Distinct cancer phenotypes from different cells and organs Sox9 and suppression of the proapoptotic phospholipid- likelyresult from unique gene expression repertoires binding protein Annexin A1 that link earlyprostate established in the embryo and maintained after malignant development to earlyprostate carcinogenesis. These transformation. We used comprehensive gene expression results credential earlyprostate development as a reliable analysis to examine this concept in the prostate, an organ and valid model system for the investigation of genes and with a tractable developmental program and a high pathways that drive prostate cancer. propensityfor cancer. We focused on gene expression in Oncogene (2008) 27, 7180–7191; doi:10.1038/onc.2008.327; the murine prostate rudiment at three time points during published online 15 September 2008 the first 48 h of exposure to androgen, which initiates proliferation and invasion of prostate epithelial buds into Keywords: prostate organogenesis; androgen signaling; surrounding urogenital sinus mesenchyme. Here, we show prostate cancer; microarray that androgen exposure regulates genes previouslyim- plicated in prostate carcinogenesis comprising pathways for the phosphatase and tensin homolog (PTEN), fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), and Wnt signaling along with cellular Introduction programs regulating such ‘hallmarks’ of cancer as angiogenesis, apoptosis, migration and proliferation. We The discovery of proto-oncogenes in the 1970’s (Stehelin found statisticallysignificant evidence for novel androgen- et al., 1976) and tumor suppressor genes a decade later induced gene regulation events that establish and/or (Friend et al., 1986) launched an effort to identify a maintain prostate cell fate. These include modulation of common genetic basis for all cancers. This approach has gene expression through microRNAs, expression of yielded significant insights into common molecular specific transcription factors, and regulation of their machinery regulating tumor initiation and growth predicted targets. Byquerying public gene expression (Hanahan and Weinberg, 2000), but fails to account databases from other tissues, we found that rather than for dramatic differences in the behaviors of tumors generallycharacterizing androgen exposure or epithelial arising in different sites. As revealed by recent genomic budding, the earlyprostate development program more approaches, the site of origin is the dominant influence closelyresembles the program for human prostate cancer. on gene expression in cancers (Ramaswamy et al., 2001) Most importantly, early androgen-regulated genes and and specifies particular oncogenic mutations (Garraway functional themes associated with prostate development and Sellers, 2006). Tissue-specific behaviors of cancers were highlyenriched in contrasts between increasingly almost certainly reflect lineage-specific epigenetic pro- lethal forms of prostate cancer, confirming a ‘reactivation’ grams that operate during embryogenesis in cells and of embryonic pathways for proliferation and invasion in tissues from which cancers arise. Indeed, reawakening of prostate cancer progression. Among the genes with the embryonic programs has long been posited for cancer (Bailey and Cushing, 1925), and could underlie tissue Correspondence: Associate Professor DM Berman, Department of specific modes of regulating critical aspects of the Pathology, Johns Hopkins University School of Medicine and Sidney malignant phenotype such as survival, angiogenesis, Kimmel Cancer Center, CRB2 Room 545, 1550 Orleans street, invasion and migration. The advent of comprehensive Baltimore, MD, 21231 USA. genomic profiling techniques now permits this hypo- E-mail: [email protected] thesis to be tested, and to link cancer to embryogenesis 5These authors contributed equally to this work. Received 31 March 2008; revised 18 July 2008; accepted 1 August 2008; through cellular pathways that define specific lineages published online 15 September 2008 and organs (Garraway and Sellers, 2006). Identification Embryonic gene expression in prostate cancer EM Schaeffer et al 7181 of these pathways and their functions should identify urogenital sinus (UGS), an embryonic rudiment present targets for more specific and less toxic cancer therapies. in both sexes. These interactions lead to outgrowth of The prostate gland represents a prime target for such buds from the UGS epithelium (UGE) that proliferate an analysis as the genetic causes of prostate cancer are and invade surrounding urogenital sinus mesenchyme. poorly understood and because there are excellent AR expression has been described in both UGE and models of prostate development and homeostasis. While urogenital sinus mesenchyme (Drews et al., 2001; B other cancers have canonical cancer initiating mutations Simons, EM Schaeffer and DM Berman, unpublished (for example, K-ras for pancreatic carcinoma and observations), although epithelial AR is dispensable for Adenomatosis Polyposis Coli for colon carcinoma), induction of prostate development (Cunha et al., 2004). the driving force for prostate cancer initiation in humans remains uncertain. Answers may lie in cellular programs activated by androgen receptor (AR) signal- Results ing, which strictly controls prostate epithelial cell fate in embryogenesis and in cancer. Hormonal manipulation of mouse prostate development Upon binding androgen, AR signals through genomic In mouse, epithelial expression of the androgen-sensitive and nongenomic modes (Manolagas et al., 2002). homeobox family member Nkx3. 1 begins by embryonic Genomic responses entail nuclear translocation of day 16 (e16), and is the earliest reported marker of liganded AR and activation of transcription at regula- prostate development (Bhatia-Gaur et al., 1999). We tory regions containing AR-binding sites. Nongenomic comprehensively profiled gene expression in the UGS signaling, in contrast, comprises protein–protein inter- between e16 and e17.5, when prostate buds first emerge. actions in the cytoplasm and is exceedingly rapid, with The developmental fate of the UGS is bipotential in measurable responses within minutes of androgen both sexes and depends soley on androgens to drive exposure (Kousteni et al., 2001). AR signaling, through prostate formation in males (Jost, 1968). The absence of genomic and/or nongenomic routes, is necessary and androgens leads to vaginal/urethral formation in sufficient for prostate organogenesis. In response to females (Wilson et al., 1980). Taking advantage of this circulating testosterone and its local conversion to the dynamic, we induced prostate development in the more potent androgen, dihydrotestosterone, AR signal- androgen-naı¨ ve yet androgen-sensitive female UGS with ing induces epithelial-mesenchymal interactions in the precisely timed 6 and 12 h intrauterine exposure 15.5–16 d.p.c. 17.5 d.p.c. Mouse public domain Molecular Histologic Prostate degeneration/regeneration (GSE5901; Wang et al., 2007) Mesenchyme Epithelium Lung development (GSE1423; Lu et al., 2004) UGS Androgen-dependent gene expression in salivary gland (GSE3995: Treister et al., 2006) Androgen 48 h Endogenous (testis) Human public domain data Macrodissected prostate cancer profiling (Lapointe et al., 2004) Exogenous (injection) 12 h Androgen 6 h LCM prostate cancer profiling (GSE6099: Tomlins et al., 2007) Androgen start Differential gene expression analysis * Gene expression measurements: preprocessing, quality control evaluation * Statistical analysis of gene expression (Linear model analysis and empirical Bayes approach) * Gene cross-referencing with Entrez gene and homologene identifiers for cross platform and organismal analysis Analysis of functional annotation (AFA) * Enrichment evaluation using: Gene Ontology (GO), KEGG pathways and TFBS targets in prostate development and human cancer progression by Wilcoxon rank-sum test * Enrichment evaluation for mouse expression signatures in human prostate tumors by Wilcoxon rank-sum test Figure 1 Flowchart of data acquisition and analysis. (a) Schematic of early prostate development. The embryonic prostate rudiment, the urogenital sinus (UGS). Mesenchyme (light blue) surrounds epithelium (darker green). In the mouse, prostate-specific gene expression begins by embryonic day 16 (e16) followed by prostate epithelial budding at e17.5. Prostate development proceeds spontaneously in males in response to endogenous androgens or can be engineered in females in response to exogenous androgens. We comprehensively profiled androgen-induced gene
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