Paracrine Wnt signaling is necessary for prostate epithelial proliferation

Abstract

Introduction: The Wnt proteins play key roles in the development, homeostasis, and disease progression of many organs including the prostate. However, the spatiotemporal expression patterns of Wnt proteins in prostate cell lineages at different developmental stages and in prostate cancer remain inadequately characterized.

Methods: We isolated the epithelial and stromal cells in the developing and mature mouse prostate by flow cytometry and determined the expression levels of Wnt ligands. We used Visium spatial gene expression analysis to determine the spatial distribution of Wnt ligands in the mouse prostatic glands. Using laser-capture microscopy in combination with gene expression analysis, we also determined the expression patterns of Wnt signaling components in stromal and cancer cells in advanced human prostate cancer specimens. To investigate how the stroma-derived Wnt ligands affect prostate development and homeostasis, we used a Col1a2-CreER^T2 mouse model to disrupt the Wnt transporter Wntless specifically in prostate stromal cells.

Results: We showed that the prostate stromal cells are a major source of several Wnt ligands. Visium spatial gene expression analysis revealed a distinct spatial distribution of Wnt ligands in the prostatic glands. We also showed that Wnt signaling components are highly expressed in the stromal compartment of primary and advanced human prostate cancer. Blocking stromal Wnt secretion attenuated prostate epithelial proliferation and regeneration but did not affect cell survival and lineage maintenance.

Discussion: Our study demonstrates a critical role of stroma-derived Wnt ligands in prostate development and homeostasis.

Keywords: Wls; Wnt; homeostasis; prostate cancer; prostate stromal cells.

Figure 1.

Spatiotemporal analysis of Wnt ligand expression during prostate development and homeostasis. (A) qRT-PCR analysis of 19 Wnt ligands in FACS-isolated epithelial and mesenchymal cells from E17 pelvic UGS tissues of C57BL/6 mice. Dot graphs represent means from three independent experiments. Genes shown in areas shaded in red and green are expressed at a relatively higher level in stromal cells and epithelial cells, respectively. (B) qRT-PCR analysis of 19 Wnt ligands in FACS-isolated basal, luminal, and stromal cells from adult prostates of C57BL/6 mice. Dot graphs represent means from four independent experiments. Genes shown in areas shaded in red and green are expressed at a relatively higher level in stromal cells and basal/luminal cells, respectively.

Figure 2.

Visium spatial gene expression analysis of Wnt ligands in adult mouse prostate. (A) Violin graph shows the average expression levels of 11 Wnt ligands in the distal and proximal ducts of anterior prostatic lobes from 10-week-old C57BL/6 mice. Other Wnt ligands were undetected in the Visium spatial gene expression assay and not shown in the violin graph. Statistical analysis was performed based on four independent experiments. (B) Representative images of spatial gene expression levels in the anterior prostatic lobe and urethra for Wnt4, Wnt5a, Wnt7b, Wnt9a and Wnt10a.

Figure 3.

Ablating Wls in prostate stromal cells decreases epithelial proliferation during early prostate development. (A) Schematic illustration of the experimental design. A pair of UGS tissues from Col1a2-Wls and control groups were transplanted into the capsules of two kidneys in each SCID/Beige male host, respectively. Tmx: tamoxifen. (B) qRT-PCR analysis of Axin2 in FACS-isolated epithelial and stromal cells from the xenografts of Col1a2-Wls and control groups. Data represent means ± SD from four and six independent experiments for the analysis of epithelial and stromal cells, respectively. (C) Transillumination images of the xenografts after tamoxifen treatment. Scale bars, 1 mm. Dot graph shows the weight of seven pairs of xenografts analyzed by paired t test. (D) Co-immunostaining of BrdU and Nkx3.1 in the xenografts of Col1a2-Wls and control groups. Scale bars, 50 μm. Dot graph shows the percentage of BrdU⁺ cells in Nkx3.1⁺ prostate cells from five pairs of xenografts analyzed by paired t test. (E) H&E staining of the xenografts of Col1a2-Wls and control groups. Scale bars, 50 μm. (F) Co-immunostaining of Trp63/AR and Krt5/Krt8 in the xenografts of Col1a2-Wls and control groups. Scale bars, 50 μm.

Figure 4.

Blockage of Wnt ligands secretion from prostate stromal cells impairs the prostate epithelial homeostasis in adult mice. (A) Schematic illustration of the experimental design. Tmx: tamoxifen. (B) qRT-PCR analysis of Axin2 in FACS-isolated prostate basal, luminal and stromal cells from the tamoxifen-treated Col1a2-Wls and control mice. Dot graphs show means ± SD from five independent experiments. (C) Dot graphs show the quantification of prostate weight of five Col1a2-Wls and seven control mice. (D) Co-immunostaining of BrdU and Krt5 in anterior prostate lobes of the Col1a2-Wls and control mice. Scale bars, 50 μm. Dot graph shows means ± SD of the percentage of BrdU⁺ cells in prostate epithelial cells from four pairs of mice. (E) Co-immunostaining of αSMA and CC3 in anterior prostate lobes of the Col1a2-Wls and control mice. Scale bars, 50 μm. (F) H&E staining of anterior prostate lobes of the tamoxifen-treated Col1a2-Wls and control mice. Scale bars, 50 μm.

Figure 5.

Knockout of stromal Wntless reduces the proliferation of prostate epithelial cells during androgen-induced prostate regrowth. (A) Schematic illustration of the experimental design. Tmx: tamoxifen. Cas: castration. Reg: regeneration. (B) qRT-PCR analysis of Axin2 in FACS-isolated prostate basal, luminal and stromal cells from the Col1a2-Wls and control mice. Dot graphs show means ± SD from six independent experiments. (C) Dot graphs show the quantification of prostate weight of 8 pairs of Col1a2-Wls and control mice. (D) Co-immunostaining of BrdU and Krt5 in anterior prostate lobes of the Col1a2-Wls and control mice. Scale bars, 50 μm. Dot graph shows means ± SD of the percentage of BrdU⁺ cells in prostate epithelial cells from eight pairs of mice. (E) Co-immunostaining of Krt5 and CC3 in anterior prostate lobes of the Col1a2-Wls and control mice. Scale bars, 50 μm. (F) Co-immunostaining of Krt8 and AR in anterior prostate lobes of the Col1a2-Wls and control mice. Scale bars, 50 μm. (G) H&E staining of anterior prostate lobes of the Col1a2-Wls and control mice after androgen-induced regrowth. Scale bars, 50 μm.

Figure 6.

A systematic analysis of Wnt-related genes in human advanced prostate cancer. (A) qRT-PCR analysis of 19 Wnt ligands in laser captured cancer and stromal cells from locally invasive PCa, lung metastasis, liver metastasis, lymph node metastasis and bone metastasis specimens. (B) qRT-PCR analysis of Wnt-related receptors/co-receptors in laser captured cancer and stromal cells from locally invasive PCa, lung metastasis, liver metastasis, lymph node metastasis and bone metastasis specimens. (C) qRT-PCR analysis of Secreted frizzled-related proteins (SFRPs) and Dickkopf-related proteins (DKKs) in laser captured cancer and stromal cells from locally invasive PCa, lung metastasis, liver metastasis, lymph node metastasis and bone metastasis specimens. (D) Comparison of expressions of 19 Wnt ligands in laser captured stromal cells from locally invasive PCa with those in stroma of lung metastasis, liver metastasis, lymph node metastasis and bone metastasis specimens. Dot graphs represent means from 10 locally invasive PCa specimens, 7 lung metastasis specimens, 8 liver metastasis specimens, 6 lymph node metastasis specimens and 4 bone metastasis specimens. In all figures, genes shown in the areas shaded in red and green are expressed at a relatively higher level in stromal cells and cancer cells, respectively.