Sox2 is necessary for androgen ablation-induced neuroendocrine differentiation from Pten null Sca-1+ prostate luminal cells

Abstract

Prostate adenocarcinoma undergoes neuroendocrine differentiation to acquire resistance toward antihormonal therapies. The underlying mechanisms have been investigated extensively, among which Sox2 has been shown to play a critical role. However, genetic evidence in mouse models for prostate cancer to support the crucial role of Sox2 is missing. The adult mouse prostate luminal cells contain both castration-resistant Sox2-expressing Sca-1⁺ cells and castration-responsive Sca-1^- cells. We show that both types of the luminal cell are susceptible to oncogenic transformation induced by loss of function of the tumor suppressor Pten. The tumors derived from the Sca-1⁺ cells are castration resistant and are more inclined to develop castration-induced neuroendocrine differentiation. Genetic ablation of Sox2 suppresses neuroendocrine differentiation but does not impact the castration-resistant property. This study provides direct genetic evidence that Sox2 is necessary for androgen ablation-induced neuroendocrine differentiation of Pten null prostate adenocarcinoma, corroborates that the lineage status of the prostate cancer cells is a determinant for its propensity to exhibit lineage plasticity, and supports that the intrinsic features of cell-of-origin for prostate cancers can dictate their clinical behaviors.

Figure 1.. Both Sca-1⁺ and Sca-1⁻ luminal cells can serve as targets for transformation induced by Pten loss.

(A) H&E staining of proximal and distal anterior prostate of K8-Pten mice at 1 and 6 months after tamoxifen treatment (N=8 and 9, respectively). Scale bars:100 μm. (B-C) Co-immunostaining of pAKT/K8 (B), K5/K8 (C, upper), Ar/K14 (C, middle), and Nkx3.1/Smooth muscle actin (SMa) (C, lower) in proximal and distal prostates of K8-Pten mice 6 months after tamoxifen treatment. Scale bars:50 μm. Dot plots show integrated density of staining of Ar (left) and Nkx3.1 (right) (N=8, each dot represents average value from 3 representative images from each mouse). (D) Co-immunostaining of Sox2/K8 in proximal and distal prostates of K8-Pten mice 6 months after tamoxifen treatment. Scale bars:50 μm. Dot plot shows integrated density of staining of Sox2 (N=8, each dot represents average value from 3 representative images from each mouse). (E) Co-immunostaining of Ki67/K5 in proximal and distal prostates of K8-Pten mice 6 months after tamoxifen treatment. Scale bars:50 μm. (F) Dot plot shows means ± SD of percentage of Ki67⁺ cells in proximal and distal prostates of K8-Pten mice 6 months after tamoxifen treatment (N=8, each dot represents average value from 6 representative images of individual mouse). N.S.: no significance.

Figure 2.. Prostate cancer derived from Sca-1⁺ luminal cells displays castration-resistance.

(A) Schematic illustration of experimental design. Tmx: tamoxifen. (B) H&E staining of proximal and distal anterior prostates of castrated K8-Pten mice. Scale bar: 25 μm. (C) Co-immunostaining of Ar/K8 (upper panels) in proximal and distal prostates of intact and castrated K8-Pten mice. Lower panels show Ar staining only. Scale bar: 50 μm. Dot plot shows integrated density of Ar staining (N=8, each dot represents average value from 3 representative images from each mouse). (D-E) Immunostaining of Ki67 (E) and cleaved caspase 3 (CC3) (F) in proximal and distal regions of androgen depleted K8-Pten mice. Scale bars:50 μm. Dot plots show means ± SD of percentage of positively stained cells (N=8, each dot shows average value from 6 representative images of individual mice). N.S.; no significance.

Figure 3.. Transplantation experiment corroborates that prostate cancer derived from Sca-1⁺ luminal cells displays castration-resistance.

(A) Schematic illustration of experimental design. Tmx: tamoxifen. (B) Representative transillumination images of tumors outgrown from distal and proximal prostate chunks of tamoxifen-treated K8-Pten mice in host SCID mice underwent mock surgery and androgen deprivation-replacement. Scale bars:5 mm. (C) Dot plot shows means ± SD of weight of tumors (N=6). (D) H&E staining of transplanted tissues in intact or castrated SCID mice. Scale bars:100 μm. (E) Co-immunostaining of pAKT/K8 (upper) and Nkx3.1/Trp63 (lower) of transplants outgrown from proximal and distal prostate of tamoxifen-treated K8-Pten mice in intact and castrated SCID hosts. Scale bars:50 μm. (F) Dot plot shows means ± SD of percentage of Ki67⁺ cells (N=6, each dot shows average value from 6 representative images from each mouse). (G) Dot plot shows means ± SD of percentage of CC3⁺ cells (N=6, each dot shows average value from 6 representative images from each mouse). (H) Co-immunostaining of Ar/K8 of transplants outgrown from proximal prostate tissues of tamoxifen-treated K8-Pten mice in intact SCID hosts. Scale bar: 50 μm.

Figure 4.. Androgen deprivation drives neuroendocrine differentiation of prostate cancer cells at proximal ducts of K8-Pten mice.

(A) Immunostaining of Chga in anterior prostates of K8-Pten mice at 3 months after mock surgery (intact, upper panel) or castration (castration, lower panel) and 6 months after tamoxifen treatment. Scale bar:200 μm. (B) Pie charts show quantification of size of NE colonies (Chga⁺ or Syp⁺) in proximal and distal regions of intact (N=9) and castrated (N=10) K8-Pten mice (results summarized from 10–20 representative images per mouse). (C) Co-immunostaining of Sox2/Syp of tumors outgrown from transplanted proximal and distal prostate of tamoxifen-treated K8-Pten mice under kidney capsules of castrated SCID mice. Scale bars:25 μm. Pie charts show quantification of size of NE colonies (Chga⁺ or Syp⁺) (N=6, results summarized from 16–30 representative images per mouse). (D) Co-immunostaining of pAKT/Syp (upper) and Pten/Syp (lower) of proximal prostates of castrated K8-Pten mice. Scale bars:25 μm. (E) Co-immunostaining of Ki67/Syp in proximal prostates of castrated K8-Pten mice. Scale bar: 50 μm.

Figure 5.. Sox2 is not essential for the castration resistant property.

(A) Dot plot shows means ± SD of prostate weight of intact and castrated K8-Pten and K8-Pten-Sox2 mice (N=8). N.S.: no significance. (B-C) H&E staining (B) and co-immunostaining of Sox2/Trp63 (C, upper panels) and pAKT/K8 (C, lower panels) in intact and castrated K8-Pten and K8-Pten-Sox2 mice. Black bars:100 μm; white bars:25 μm. Dot plot shows the integrated density of Sox2 staining (N=8, each dot represents average value from 3 representative images from each mouse). (D-E) Dot plots show means ± SD of percentages of Ki67⁺ (D) and CC3⁺ (E) cells in proximal and distal regions of intact and castrated K8-Pten and K8-Pten-Sox2 mice (N=8, each dot indicates average value from 6 representative images per individual mouse). N.S.: no significance.

Figure 6.. Sox2 is necessary for androgen deprivation-induced neuroendocrine differentiation of Pten-null prostate tumor cells.

(A) Co-immunostaining of Chga/Syp in proximal and distal prostates of intact and castrated K8-Pten and K8-Pten-Sox2 mice. White arrows point to Chga⁺/Syp⁺ cells. Scale bars:25 μm. (B) Pie charts show quantification of size of neuroendocrine colony in proximal prostates of intact and castrated K8-Pten and K8-Pten-Sox2 mice (N=8, results summarized from 8–15 representative images per mouse). (C) Co-immunostaining of Chga/Syp of transplanted tissues in intact and castrated SCID mice. Arrows point to neuroendocrine cells. Scale bars=25 μm. (D) Pie charts show quantification of neuroendocrine colony size. N=5–7. Results summarized from 13–25 representative images per mouse.