Bmi-1 is a crucial regulator of prostate stem cell self-renewal and malignant transformation

Abstract

The Polycomb group transcriptional repressor Bmi-1 is often upregulated in prostate cancer, but its functional roles in prostate stem cell maintenance and prostate cancer are unclear. Loss- and gain-of-function analysis in a prostate sphere assay indicates that Bmi-1 expression is required for self-renewal activity and maintenance of p63(+) stem cells. Loss of Bmi-1 blocks the self-renewal activity induced by heightened β-catenin signaling, suggesting that Bmi-1 is required for full activity of another self-renewal pathway. In vivo, Bmi-1 expression is necessary for normal prostate tubule regeneration. Altered self-renewal and proliferation through Bmi-1 modulation diminishes the susceptibility of prostate cells to transformation. In an in vivo prostate regeneration system, Bmi-1 inhibition protects prostate cells from FGF10-driven hyperplasia and slows the growth of aggressive Pten-deletion-induced prostate cancer. We conclude that Bmi-1 is a crucial regulator of self-renewal in adult prostate cells and plays important roles in prostate cancer initiation and progression.

Figure 1. Bmi-1 is expressed in prostate stem cell enriched regions and cell fractions

(A) FACS plots of the luminal (CD49f^loSca-1⁻, blue) and ‘LSC’ basal/stem (CD49f⁺Sca-1⁺, red) cell populations. Graph shows Bmi-1 mRNA levels in the luminal and LSC cells of adult mouse prostate. (B) Quantification of the Bmi-1⁺ cells in the proximal and distal regions of adult prostate. Representative images show Bmi-1 staining in these regions. Bar=100μm. (C) Image shows representative area from the proximal region co-stained with Bmi-1 and cytokeratin (CK) 5. Scale bar=50μm (D) Bmi-1 mRNA levels in intact and castrated 8 week old prostates. Data shown as mean +/− SEM

Figure 2. Bmi-1 expression controls sphere-formation and self-renewal in adult prostate stem cells

(A) Diagram of shRNA and Bmi-1^Res lentivector constructs. (B) Western blot of scrambled (Scr) shRNA, Bmi-1 shRNA, and Bmi-1 shRNA/Bmi-1^Res infected prostate cell lysates. (C) Frequency of primary prostate spheres formed from infected cells. Sphere counts are normalized to mock-infected spheres. (D) Representative sphere images from each condition – transilluminating (TI), and fluorescence images show viral infection with respective shRNA (red), and Bmi-1^Res (green) virus. Bar=100μm. (E) Schematic of sphere passaging. (F) Scrambled and Bmi-1 shRNA infected prostate spheres were dissociated and equal numbers of cells were passaged for 4 generations. Spheres counts are normalized to the first generation scrambled shRNA spheres. See also Figure S1A, B and S2. (G) Frequency of spheres from GFP and Bmi-1-GFP infected P53−/− prostate cells. Spheres were dissociated and passaged at equal cell numbers for 4 generations. Sphere counts are normalized to generation 1 GFP infected spheres. Data represented as mean +/− SEM. See also Figure S1C and D.

Figure 3. Bmi-1 regulates the depletion and expansion of self-renewing cells in prostate spheres

(A) p63 and DAPI stains of scrambled (left) and Bmi-1 shRNA (right) infected spheres. (B) Percentage of p63⁺ cells in 12 representative scrambled and Bmi-1 shRNA infected spheres. (C) p63 and DAPI stains of GFP (left column) and Bmi-1 (right column) infected p53^−/− prostate spheres. (D) Percentage of p63⁺ cells in 12 representative GFP and Bmi-1 infected spheres. (E) Number of daughter spheres formed from single dissociated scrambled and Bmi-1 shRNA infected and (G) GFP and Bmi-1 infected spheres. (F) Merged images of representative daughter spheres formed from Bmi-1 shRNA (red) infected and (H) Bmi-1-GFP (green) infected spheres. Data represented as mean +/− SEM. Bar=200μm. See also Figures S3, S4, and Table S1.

Figure 4. Bmi-1 is required for β-catenin mediated self-renewal

(A) Diagram of experimental approach. (B). Number of spheres formed in two generations of RFP and Cre infected Catnb^{+/Lox(Ex 3)} prostate cells. (C) Western blot and (D) Q-PCR analysis of β-catenin, β-catenin target genes, and Bmi-1 expression levels in RFP infected (WT) and Cre infected (Ex3 KO) cells. (E) Frequency of 1° and 2° spheres from RFP, Cre/scrambled, and Cre/Bmi-1 shRNA infected Catnb^{+/Lox(Ex 3)} cells normalized to generation 1 RFP spheres. Data represented as mean +/− SEM. (F) Images of representative Cre/scrambled and Cre/Bmi-1 shRNA infected spheres. Bar=200μm. See also Figures S1 and S5.

Figure 5. Loss of Bmi-1 decreases in vivo prostate tubule formation

(A) Schematic of experimental approach. (B) (Top) TI images of regenerated grafts and fluorescent images of RFP signal that represents infection. (Bottom) H&E and fluorescent images of representative grafts of scrambled and Bmi-1 shRNA infected primary prostate cells. RFP shows integrated virus. Bars=200μm. (C) Average number of RFP⁺ tubules per viewing field, calculated from 3 viewing fields from 12 different tissue sections of scrambled and Bmi-1 shRNA infected grafts (9 grafts/condition from 3 experiments were analyzed), represented as mean +/− SEM. See also Figure S6 and Table S2.

Figure 6. Bmi-1 inhibition protects primary prostate cells from hyperplasia driven by FGF10 signaling

(A) Schematic shows experimental approach. (B) Average weight of 6 scrambled shRNA/FGF10 UGSM and Bmi-1 shRNA/FGF10 UGSM grafts each from 2 experiments. (C) TI (top) and RFP fluorescence images (bottom) of scrambled shRNA/FGF10 (left) and Bmi-1 shRNA/FGF10 (right) grafts. (D) H&E sections show representative tubules from Bmi-1 and scrambled shRNA infected grafts (top). Fluorescence images show representative tubules and their infection status (red) from Bmi-1 and scramble shRNA infected tubules. Bars=200μm. (E) Average percentage of hyperplastic and normal appearing RFP⁺ tubules calculated from 10 representative sections from 3 grafts of each condition, represented as mean +/− SEM. (F) H&E and fluorescence images show viral infection status (red), AR (green), p63 (green), and Ki67 (green) staining of a Scrambled shRNA infected region(red) (top) and a Bmi-1 shRNA infected tubule (red) surrounded by non-infected epithelium (non-colored) (bottom). Bars=100μm. See also Figure S7A.

Figure 7. Bmi-1 inhibition attenuates tumor growth from Pten-null prostate cells

(A) Schematic of experimental approach. (B) Average weight of Cre/scrambled and Cre/Bmi-1 shRNA infected grafts from 6 grafts each and 2 experiments, represented as mean +/− SEM. (C and D) Representative images and H&E sections (top) show the grafts and microscopic phenotypes of regenerated Cre/scrambled (C) and Cre/Bmi-1 shRNA (D) infected tumors. Fluorescent images (bottom) show Ki67 (red) and TUNEL (green) staining of representative sections. Bars=200μm. See also Figure S7B and C.