Regulation of epithelial branching morphogenesis and cancer cell growth of the prostate by Wnt signaling

Abstract

Although Wnt signaling has been shown to be important for embryonic morphogenesis and cancer pathogenesis of several tissues, its role in prostatic development and tumorigenesis is not well understood. Here we show that Wnt signaling regulated prostatic epithelial branching morphogenesis and luminal epithelial cell differentiation in developing rat prostate organ cultures. Specifically, Wnt signaling regulated the proliferation of prostate epithelial progenitor cells. Assessment of the expression levels of a Wnt pathway transcriptional target gene, Axin2, showed that the Wnt pathway was activated in the developing prostate, but was down-regulated in the adult. Castration resulted in an upregulation of Axin2 whereas androgen replacement resulted in a down regulation of Axin2. Such dynamic changes of Wnt activity was also confirmed in a BAT-gal transgenic mouse line in which beta-galactosidase reporter is expressed under the control of beta-catenin/T cell factor responsive elements. Furthermore, we evaluated the role of Wnt signaling in prostate tumorigenesis. Axin2 expression was found upregulated in the majority of human prostate cancer cell lines examined. Moreover, addition of a Wnt pathway inhibitor, Dickkopf 1 (DKK1), into the culture medium significantly inhibited prostate cancer cell growth and migration. These findings suggest that Wnt signaling regulates prostatic epithelial ductal branching morphogenesis by influencing cell proliferation, and highlights a role for Wnt pathway activation in prostatic cancer progression.

Figure 1. Wnt3a and DKK1 regulate prostatic epithelial branching morphogenesis.

Whole mount ventral prostates were prepared from P2 rats and maintained for 7 days in serum-free medium in the absence (A) or presence of 50 nM of Wnt3a (B) or 400 nM of DKK1 (C). Similar patterns were consistently seen in 3 repeat experiments of 5–6 prostate organs per group in each individual experiment. Note that addition of either Wnt3a or DKK1 to the culture resulted a change in epithelial branching morphogenesis. Quantification of the cultures by measuring the diameter of the cultured prostates (D) and the ductal tips (E) using Axiovision software and the branching points (F) were done by analyzing 4 randomly selected cultures per group. Data are expressed as mean + SEM (t-test, compared to control cultures). Bar, 400 µm.

Figure 2. Wnt signaling prevents prostatic epithelial cell differentiation. (A,B,C) p63 immunocytochemistry (red) of the P2 rat ventral prostate organ cultures maintained for 7 days in the absence (A) or presence of 50 nM of Wnt3a (B) or 400 nM of DKK1 (C).

The tissue sections were counterstained with DAPI (blue). While p63 positive cells (purple) represent basal cells where progenitor cells reside, the blue cells (arrows) that are negative to p63 in the epithelium are differentiated luminal cells. (D) Quantification of p63 positive cells over total epithelial cells. Data were collected from randomly selected 22–27 ductal units from sections of the organ cultures per group and are expressed as mean + SEM (t-test). Note that while Wnt3a led to a significant increase in the number of basal cells, DKK1 resulted in a reduction in basal cells. Bar, 50 µm for A-C.

Figure 3. Activation of Wnt signaling enhances prostate epithelial cell proliferation.

(A-F) Shown are anti-BrdU antibody (B,D,F) and DAPI (A,C,E) double labeling of the P3 rat ventral prostate organ cultures maintained for 3 days in the absence (A,B) or presence of 50 nM of Wnt3a (C,D) or 400 nM of DKK1(E,F). (G). Quantification of BrdU-positive cells in a given visual field of 434 µm×322 µm. (H) Quantification of Ki67-positive cells in the P2 prostate cultures maintained for 7 days, which was normalized to the control cultures. Cell counts (for G and H) were performed from randomly selected visual fields of 5 organ cultures per group and data were expressed as mean+SEM (t-test). Note that while Wnt3a significantly enhanced progenitor cell proliferation, DKK1 inhibited progenitor cell proliferation. (I). Expression level change of cyclin B2 in P2 rat prostate organ cultures.. Data were collected from 4 organ cultures maintained for 2 days per group and are expressed as mean+SEM (t-test). Note that expression of cyclin B2 was upregulated by wnt3a, but down-regulated by DKK1. Bar, 100 µm for A-F.

Figure 4. TaqMan RT-PCR analyses of Axin2 expression during prostate development and regrowth following androgen deprivation and replacement.

(A) A gradual downregulation of Axin2 from newborn to adulthood.nbsp;(B) Axin2 was upregulated following castration, but returned to normal low levels after androgen replacement. Data were collected from 4–6 samples per group and are expressed as mean+SEM (t-test, compared to normal adult prostates). Abbreviation: N, normal prostate; C3, 3 days after castration, C17; 17 days after castration; C14+T. 14 days after castration+3 days of testosterone treatment.

Figure 5. β-galactosidase expression in BAT-gal transgenic prostates.

Double immunostaining of prostate tissue sections with anti-β-galactosidase (green in A, D, G, J) and anti-p63 (red, in B, E, H, K) antibodies, prepared from mice of P5 (A-C), adult (D-F), 14 days post-castration (G-I) and 14 days after androgen replacement (J-L). The sections were counterstaining with DAPI (blue). While arrows indicate cells that co-express p63 and β-galactosidase, arrowhead (Fig. 1A-C) shows a cell that is β-gal positive but p63 negative in the developing prostate. Note that β-galactosidase-expressing cells are located exclusively in the epithelium, and mainly within the basal cell compartment in the adult prostate, although a noticeable number of the are seen in the luminal cell layer in developing prostate. Bar, 50 µm. (M) Quantification of β -gal positive cells per epithelial ductal unit, which is determined in the tissue sections based on DAPI counterstaining and the presence of an epithelial lumen. (N) Cell counts of β -gal positive cells over total basal cells (p63 positive cells). Data were collected from 14–20 randomly selected ductal units in the sections from 4–5 prostates per group and are expressed as mean+SEM (t-test, compared to normal adult prostates).

Figure 6. Wnt signaling is activated in prostate cancer cells.

TaqMan RT-PCR analysis of Axin2 expression in various human prostate epithelial cells and cancer cells. Data were collected from triplets and are expressed as mean+SEM (t-test). Note that Axin2 expression is much higher in prostate cancer cell lines (LNCaP, Du145, PC3) and human prostate tumor xenografts (LuCaP35, LuCaP77) as compared to primary prostate epithelial cells (PrEC) or non-tumorigenenic immortalized prostate epithelial cells (BPH1).

Figure 7. DKK1 inhibits proliferation and migration of prostate cancer cells.

(A) Tritiated thymidine incorporation in PC3 cultures in the absence or presence of Wnt3a or DKK1. While high concentration of Wnt3a (10nM) enhanced PC3 cell proliferation, DKK1 inhibited PC3 proliferation in a dose-dependent manner. (B) Regulation of PC3 cell migration by Wnt signaling. Data were collected from 6–8 cultures per group and are expressed as mean+SEM (t-test). Note that Wnt3a increased the number of migrated cells, whereas DKK1 inhibited cell migration.