Roles for Nkx3.1 in prostate development and cancer

Abstract

In aging men, the prostate gland becomes hyperproliferative and displays a propensity toward carcinoma. Although this hyperproliferative process has been proposed to represent an inappropriate reactivation of an embryonic differentiation program, the regulatory genes responsible for normal prostate development and function are largely undefined. Here we show that the murine Nkx3.1 homeobox gene is the earliest known marker of prostate epithelium during embryogenesis and is subsequently expressed at all stages of prostate differentiation in vivo as well as in tissue recombinants. A null mutation for Nkx3.1 obtained by targeted gene disruption results in defects in prostate ductal morphogenesis and secretory protein production. Notably, Nkx3.1 mutant mice display prostatic epithelial hyperplasia and dysplasia that increases in severity with age. This epithelial hyperplasia and dysplasia also occurs in heterozygous mice, indicating haploinsufficiency for this phenotype. Because human NKX3.1 is known to map to a prostate cancer hot spot, we propose that NKX3.1 is a prostate-specific tumor suppressor gene and that loss of a single allele may predispose to prostate carcinogenesis. The Nkx3.1 mutant mice provide a unique animal model for examining the relationship between normal prostate differentiation and early stages of prostate carcinogenesis.

Figure 1

Prostate-specific expression of Nkx3.1 in adult male mice. (A) Diagram of the male urogenital system in adult mice, showing the embryological relationships of the tissues (adapted from Cunha et al. 1987; Podlasek et al. 1997). The anterior, dorsolateral, and ventral prostatic lobes, as well as the BUGs (dark gray) are ductal derivatives of the urogenital sinus; the bladder and urethra (medium gray) are its nonductal derivatives. The seminal vesicles, ductus deferens, epididymides, and ampullary glands (light gray) are derived from the Wolffian duct, and the testes (white) from the genital ridge. In the ventral view, only the base of the bladder is shown for clarity. (B) Diagram of the male urogenital system in a newborn mouse [postnatal day 0 (P0; adapted from Cunha et al. 1987)]. By 17.5 dpc, the prostatic buds (dark gray) arise as outbuddings of the urogenital sinus epithelium (white) into the surrounding mesenchyme (medium gray). Also shown are the Wolffianduct-derived seminal vesicles and ductus deferens (light gray). (C) Ribonuclease protection analysis using total RNA (20 μg) from the indicated tissues of adult (8-week) male mice, using a Nkx3.1 antisense riboprobe. The rpL32 riboprobe serves as an internal control for RNA loading.

Figure 2

Expression of Nkx3.1 in embryonic and neonatal prostate. (A) Diagram showing transverse planes of section through the urogenital sinus, shown in panels B–M. The rostral region (R) corresponds to the location of the prospective prostatic buds; the caudal region (C) corresponds to the prospective bulbourethral glands. (B–I) In situ hybridization analysis of Nkx3.1 expression in transverse sections through the rostral male urogenital sinus, shown at low (B–E) and high (F–I) power. (B,F) No expression is detected at 14.5 dpc. (C,G) At 15.5 dpc, Nkx3.1 expression is restricted to the lateral urogenital sinus epithelium (UGE) and is excluded from the dorsal and ventral sides (forming the parentheses pattern). (D,H) Nkx3.1 expression continues in the lateral UGE, with elevated expression in the emerging anterior prostatic buds. (E,I) At 17.5 dpc, expression is restricted to the newly formed dorsolateral and ventral prostatic buds and is not found in the prospective urethral epithelium. (J–M) Nkx3.1 expression in transverse sections through the caudal male urogenital sinus. (J) Expression at 14.5 dpc is found in bilateral outpouchings (arrow) from the UGE. (K–M) At 15.5–17.5 dpc, expression is found in the nascent BUGs and the ducts (arrow in M) that join them to the prospective urethra. (N–W) Nkx3.1 expression in isolated tissues from male mice at P0 and P8; staining is more intense at the ends of the outgrowing prostatic ducts (arrows in O, P, and S). (AP) Anterior prostate; (BUG) bulbourethral gland; (C) caudal; (DD) ductus deferens; (DLP) dorsolateral prostate; (Int) large intestine; (R) rostral; (UGE) urogenital sinus epithelium; (UGM) urogenital sinus mesenchyme; (UGS) urogenital sinus; (Ure) urethra; (VP) ventral prostate. Scale bar, 50 μm.

Figure 3

Nkx3.1 marks prostate differentiation in tissue recombinants. (A) Design of the tissue recombination assay. Recombinants of urogenital sinus mesenchyme (UGM) with either urogenital sinus epithelium (UGE) or bladder epithelium (BLE) form prostate; recombinants of bladder mesenchyme (BLM) with either epithelium form bladder. (B–E) In situ hybridization analysis of Nkx3.1 expression in tissue recombinants harvested at 1 week. Expression is found in recombinants that form prostate (UGM + UGE and UGM + BLE) but not in those that form bladder (BLM + UGE and BLM + BLE). Arrows in C and E indicate bladder-like structures that do not express Nkx3.1. (F–I) Nkx3.1 expression in tissue recombinants of UGM with wild-type BLE (WT BLE) vs. UGM with BLE from Tfm mice (Tfm BLE), at 2 and 4 weeks of growth. (B–I) Scale bars, 50 μm.

Figure 4

Analysis of Nkx3.1 mutant mice. (A–E) Targeted disruption of Nkx3.1. (A) Strategy for gene disruption. The Nkx3.1 locus comprises two exons (gray boxes), with the coding region (medium gray) contained in both exons and the homeobox in the second exon (dark gray). Homologous recombination with the targeting vector deletes most of the coding region, including the homeobox. The positions of the 5′- and 3′-flanking probes used for Southern blot analysis are shown. (E) EcoRI; (H) HindIII; (N) NotI; (X) XbaI. (B) Southern blot analysis of genomic DNA using the 5′- flanking probe, showing recovery of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) adult mice. This probe detects a 9-kb HindIII wild-type fragment and a 6-kb fragment from the targeted allele (arrows). (C) Southern blot analysis using an internal probe containing the homeobox, confirming its deletion in Nkx3.1 homozygotes. This probe detects a 9-kb HindIII wild-type fragment (arrow) and does not hybridize to the targeted allele. Dashes in B and C indicate positions of markers at 10 and 5 kb. (D) PCR analysis of genomic DNA from wild-type, heterozygous, and homozygous adult mice. Primers (described in Materials and Methods) amplify a 707-bp fragment from wild-type genomic DNA and a 232-bp fragment from the targeted allele (arrows). Dashes indicate positions of markers at 1018, 506, and 220 bp. (E) Ribonuclease protection analysis of total RNA from the APs of 8-week-old mice, using an Nkx3.1 antisense riboprobe corresponding to the homeobox. Dashes indicate positions of markers at 220, 201, and 154 nucleotides. (F–H) Morphology of male urogenital tissues from wild-type and Nkx3.1 mutant littermates. (F) Urogenital systems from wild-type (left) and Nkx3.1 homozygote (right) at 8 weeks of age, showing positions of prostatic lobes (AP, DLP, VP), bladder (Bl), ductus deferens (DD), urethra (Ure), and seminal vesicles (SV). (G) Higher-power view of the mutant anterior prostate shown in E, with semitransparent ducts (arrow). (H) BUGs from wild-type (left) and Nkx3.1 homozygote (right) at 6 weeks of age. Scale bars in F–H, 0.5 mm. (I) Microdissected prostatic lobes from wild-type and Nkx3.1 homozygous mice at 12 weeks of age. Scale bar, 1.0 mm. (J) Quantitation of ductal tips, analyzed as in H. The mean number of ductal tips was significantly smaller in each of the mutant prostatic lobes, at P < 0.1 (*) or P < 0.05 (**). (K) Quantitation of the histological composition of the wild-type and Nkx3.1 mutant BUGs. The total area analyzed was 6.1 × 10⁷ μm² for wild-type glands and 2.5 × 10⁷ μm² for mutant glands; significant differences from the wild-type (P < 0.05) are indicated (*). In J and K, error bars represent standard error of the mean (s.e.m.). (L) Analysis of secretory proteins from VP and AP prostatic lobes, BUG, and SV. Protein secretions were collected from tissues of 8-week-old male mice and resolved on a 10%–20% SDS–polyacrylamide gradient gel. (Equal volume) Lanes contain 4 μl of secretory material; (equal amount) lanes contain 10 μg of total protein. Asterisks (*) indicate proteins that are decreased in −/− mice; (^†) a protein increased in homozygotes. Arrowheads indicate the protein bands analyzed by microsequencing. Dashes at right mark the positions of molecular mass standards at 102, 81, 46.9, 32.7, 30.2, and 24 kD.

Figure 5

Histology of Nkx3.1 mutant mice. (A–U) H&E staining of paraffin sections of BUG, AP, and DLP in wild-type (Nkx3.1^+/+), heterozygous (Nkx3.1^+/−), and homozygous (Nkx3.1^−/−) mice at 4, 12, and 45 weeks of age. (A–D) At 12 weeks of age, the wild-type BUG (A,B) contains differentiated mucin-producing cells; while the homozygous gland (C,D) largely contains cells with ductal morphology. (E–H) At 4 weeks of age, the wild-type anterior prostate (E,F) contains immature columnar epithelial cells arranged in characteristic papillary tufts (arrow); the homozygous anterior prostate (G,H) contains a multilayered hyperplastic epithelium, with little lumenal space. (I–L) At 12 weeks of age, the wild-type anterior prostate (I,J) contains differentiated columnar epithelial cells with lumenal spaces filled with secretions (lightly staining eosinophilic material). The homozygous AP (K,L) contains hyperplastic epithelium with mildly dysplastic regions (arrows), and little secretory material. (M–R) At 45 weeks of age, the wild-type AP (M,P) contains tall columnar epithelium arranged in papillary tufts (arrow), the heterozygous AP (N,Q) contains hyperplastic epithelium with mildly dysplastic regions (arrow) and reduced lumenal space and secretory protein, and the homozygous AP (O,R) contains severely hyperplastic epithelium and regions of dysplasia (arrows). (S–U) At 45 weeks of age, the wild-type DLP (S) contains columnar epithelium and lumenal secretions, the heterozygous DLP (T) contains areas of mild dysplasia (arrow), and the homozygous DLP (U) contains severely dysplastic epithelium (arrows). (V–X) Ki67 immunoreactivity in the anterior prostates of wild-type (V), heterozygous (W), and homozygous (X) Nkx3.1 mice at 6 weeks of age. Arrows indicate Ki67-labeled nuclei. In total, 55 Ki67-labeled nuclei were observed out of 3767 total nuclei (1.5%) in wild type; 207 of 2991 (6.9%) in heterozygotes; and 315 of 3573 (8.8%) in homozygotes. (A–L and V–X) Scale bars, 50 μm; (M–U) scale bars, 100 μm.

Figure 6

Model for Nkx3.1 activities in prostate development, maturation, and carcinogenesis. The model is described in the text; expression of Nkx3.1 is shown in blue.