Androgen-induced programs for prostate epithelial growth and invasion arise in embryogenesis and are reactivated in cancer

Abstract

Cancer cells differentiate along specific lineages that largely determine their clinical and biologic behavior. Distinct cancer phenotypes from different cells and organs likely result from unique gene expression repertoires established in the embryo and maintained after malignant transformation. We used comprehensive gene expression analysis to examine this concept in the prostate, an organ with a tractable developmental program and a high propensity for cancer. We focused on gene expression in the murine prostate rudiment at three time points during the first 48 h of exposure to androgen, which initiates proliferation and invasion of prostate epithelial buds into surrounding urogenital sinus mesenchyme. Here, we show that androgen exposure regulates genes previously implicated 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 programs regulating such 'hallmarks' of cancer as angiogenesis, apoptosis, migration and proliferation. We found statistically significant evidence for novel androgen-induced gene regulation events that establish and/or maintain prostate cell fate. These include modulation of gene expression through microRNAs, expression of specific transcription factors, and regulation of their predicted targets. By querying public gene expression databases from other tissues, we found that rather than generally characterizing androgen exposure or epithelial budding, the early prostate development program more closely resembles the program for human prostate cancer. Most importantly, early androgen-regulated genes and functional themes associated with prostate development were highly enriched in contrasts between increasingly lethal forms of prostate cancer, confirming a 'reactivation' of embryonic pathways for proliferation and invasion in prostate cancer progression. Among the genes with the most significant links to the development and cancer, we highlight coordinate induction of the transcription factor Sox9 and suppression of the proapoptotic phospholipid-binding protein Annexin A1 that link early prostate development to early prostate carcinogenesis. These results credential early prostate development as a reliable and valid model system for the investigation of genes and pathways that drive prostate cancer.

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 expression changes in pharmacologically virilized female UGS at 6 and 12 h after injection with a potent androgen (dihidrotestosterone, 50 mg/kg) and in physiologic prostate development at e17.5, ∼48 h after the onset of androgen-induced transcriptional changes. (b) List of data sets from the public domain used in integrative analysis. (c) Linear models and Bayesian approaches were used to identify differentially expressed genes. (d) Significantly enriched themes were identified through functional annotation enrichment analysis (see Supplementary methods for analytic protocols). See online version for color figure. LCM, laser capture microdissection.

Figure 2

Androgen-induced gene expression in early prostate development is dynamic and organ specific. (a) Distinct and overlapping genomic responses to androgen at 6, 12 or 48 h of exposure (see text). Values represent differential expression at adj. P<0.05. (b) Nine genes showed concordant up- or downregulation at all time points. (c) Chart shows ratio of differentially expressed genes either suppressed (black) or induced (gray) by androgen at indicated time point. (d and e) Similarity of gene lists was determined by pairwise correspondence at the top plot analyses of statistically top ranking genes in (d) branching morphogenesis of prostate compared to lung (Lu et al., 2004) or (e) adult salivary gland (Treister et al., 2005).Y-axis represents the proportion of identical genes between two array sets, whereas X-axis represents the number of genes compared. Note there is a correspondence between all prostate comparisons (Pros vs Pros) with particularly high concordance (arrow) between pharmacologically regulated genes (12 h) and physiologically regulated genes (48 h).

Figure 3

Embryonic gene expression in human prostate cancer. (a) Genes differentially expressed at each time point (6, 12, or 48 h of androgen exposure; Y-axis) in early prostate development (top half of heat map) are identical to genes differentially expressed at different stages of prostate cancer progression (X-axis), whereas prostate regeneration (bottom half) shows little relationship to cancer. In this data set, based on macrodissected cancers (Lapointe et al., 2004), Gleason grade 6 tumors are labeled ‘low grade’ and ‘high grade’ are Gleason grades 8-10. Degree of shading indicates statistical significance in comparisons between two gene sets (b) Differentially expressed genes in early prostate development are also enriched in a similar prostate cancer progression study (Tomlins et al., 2007) using microdissected epithelial and cancer cells. Cancer comparisons include normal epithelium vs high-grade prostatic interaepithelial neoplasia (Nml vs PIN), PIN vs invasive cancer, cancer grade, (low vs high) and local vs metastatic tumors. Developmental genes enriched in cancer transitions (boxes labeled 1-6) are listed in Supplementary Tables 13-18.

Figure 4

Transitions to increasingly invasive cancers are characterized by the activation of distinct pathways, transcription factors and microRNA target genes. Analysis of functional annotation in development and cancer transition reveals distinct (a) Gene ontology (GO) categories, (b) predicted transcription factor binding sites in differentially expressed genes and (c) predicted targets of specific miRNAs at each cancer transition (for each category listed, there is enrichment in at least one of the three developmental time points and at least one of the four cancer transitions). Contrasts include benign vs prostatic intraepithelial neoplasia (Nml v PIN), PIN vs cancer, cancer grade (low vs high), and localized vs metastatic tumors.

Figure 5

Annexin and Sox9 in epithelial invasion. (a and b) Immunohistochemical localization of Annexin A1 (AnxA1) in UGS tissue from female (a) and male (b) e17.5 littermates. (c) Immunohistochemical localization of Annexin A1 in male e18 UGS showing decreased. Annexin A1 at tips of invading prostate epithelial buds (arrow). (d) Immunofluorsescent localization of Sox9 protein (green) at tips of invading prostate epithelial buds at e18. Antibodies against p63 (red) show nearly ubiquitous expression in UGS epithelium. Nuclei appear blue (DAPI stain). (e) Hematoxylin and eosin stain of PIN tissue microarray. Higher power inset demonstrates prominent nucleoli characteristic of PIN. (f) Adjacent tissue section showing immunofluorsescent localization of Sox9 protein (green) in predominantly luminal cells of the same PIN lesion shown in panel e. Antibodies against p63 (red) label basal cells. Nuclei appear blue (DAPI stain). Higher power inset demonstrates localization of Sox9 protein (green) in luminal epithelial cells in contrast with basal expression of p63 (red).