(A) Pb-Cre⁺;Pten^L/L mutants were castrated (Cx) at 2, 4 or 6 weeks and aged to 10 weeks. (B) Mutants Cx at 2 weeks were evaluated for carcinoma, PI3K (P-AKT, P-S6) activation coinciding with PTEN loss and invasiveness based on smooth muscle actin (SMA) loss but maintained E-Cadherin expression (arrow). Bar=100 μm (C) Cell proliferation, Ki67+; Cyclin D1+ (Cyc D1) cells, in 2 and 6 week Cx cohorts in comparison to WT Cx controls (**, p<0.01) Bar = 150 μm. Error bars, mean +/− SD. See also .

(A, B) Expression profile (means +/− s.e.m) of AR activated (A) and suppressed (B) genes in WT and Pten-null murine prostates after castration. (C) Heat map of expression ratios of androgen responsive genes in WT (14-day post castration) and intact Pten-null mutants with respect to intact WT mice. (D) Variation in expression of androgen-responsive genes based on PTEN copy number (CN) in human CaP samples. Left, summary of human samples based on PTEN CN (the numbers inside parentheses indicates the number of metastatic cases); right, a comparative analysis of AR activated (red circles) and suppressed (blue circles) gene expression values in two human CaP datasets. (E) Top, gene expression and NCA derived activities of EGR1, JUN, and AR transcription factors in induced PTEN expression in Pten^−/− cells. Bottom, the activity of AR in murine models when PTEN/AKT/mTOR pathway was manipulated genetically or pharmacologically. (F) Expression (means +/− SD) of Ezh2 (left), and AR and EZH2 co-target genes (right) in PIN and cancer (CAN) stages of Pten-null prostate. *, p<0.05; **, p<0.005. See also and .

(A) Deletion of epithelial AR in the anterior lobe of Pten-null CaP (Pb-Cre⁺;Pten^L/L;Ar^L/Y) mutants and the impact on cell proliferation (Ki67+ cells), apoptosis (red arrow) and cancer formation (bar, top = 200 μm; bottom = 50 μm). (B) Cell proliferation (left) and apoptotic indexes (right) in Cre⁻ (C⁻), Pb-Cre⁺;Ar^L/Y (C⁺;Ar); Pb-Cre⁺;Pten^L/L and (C⁺;Pt) and Cre⁺;Pten^L/L;Ar^L/Y (C⁺;Pt;Ar) mutants. (C) Frequency of invasiveness based on smooth muscle actin (SMA) break down in Pb-Cre⁺;Pten^L/L and Cre⁺;Pten^L/L;Ar^L/Y mutants during progression. Error bars, means +/− SD. See also .

(A) Evaluating the impact of cre-mediated deletion of Pten and Ar on histopathology and PI3K signaling. The top panel shows the outline of the experiment and the bottom panels show results of tissues stained as indicated. Bar = 100 μm. (B) Evaluating AR deletion and myristoylated-AKT expression in primary, Pb-Cre⁺;Ar^L/Y mutants and the impact on prostate histopathology and PI3K signaling. The top panel shows the outline of the experiment and the bottom panels show results of tissues stained as indicated. Bars, top = 200 μm, bottom = 50 μm. See also .

(A) PI3K activation (P-AKT, P-S6) in AR+ regions (arrows) versus AR− regions (arrow heads) in castrate, Pb-Cre⁺;Pten^L/L;Ar^L/Y mutants. (Bar, low mag = 150 μm, high mag = 75 μm). (B) Effect of AR deletion on FKBP5 expression in Pb-Cre⁺Pten^L/L;Ar^L/Y mutants (bar = 50 μm). (C, D) Expression of FKBP5, PHLPP and P-AKT in AR⁺ regions (arrow heads) compared to AR-null regions (arrows) in castrated Pb-Cre⁺;Pten^L/L;Ar^L/Y mutants at 2 days (C) and 4 weeks (D) after castration (bar = 75 μm). See also .

(A) PTEN and AR expression in human CaP (bar = 500 μm). (B) PI3K pathway components (P-AKT, P-S6, P-4EBP1) in Pten⁻;AR⁻ regions of Pb-Cre⁺;Pten^L/L;Ar^L/Y mice and in PTEN-negative;AR-low/negative regions of human CaPs. Error bars, means +/− SD. (C) Unsupervised clustering analysis of PTEN, PHLPP, AR, and FKBP5 in human TMA samples (N=91) (left). Chi squared test P-values* (N = 91) were used to quantitate the strength of association between each pair (right, table). Protein level was categorized to high (IHC> 1) and low (< 1) levels). See also .

(A) In vitro response of Pten-null;Ar⁺ mouse (CaP8) and human (LNCaP) CaP cells to AR knockdown (sh-AR) or pharmacological inhibition of AR (MDV3100, 10 μM) with and without rapamycin (R: 1 nM) treatment. (Sc = control sh oligo). (B, D) In vivo response to treatments with castration, MDV3100, rapamycin or their combinations as measured by cell proliferation (Ki67⁺ cells) and (C, D) tumor burden in Pb-Cre⁺;Pten^L/L and Pb-Cre⁺;Pten^L/L:Ar^L/Y mutants (C, bar = 2 mm; D, bar = 200 μm; D, inset bar = 75 μm). Error bars = means +/− SD. See also .

By regulating EGR1, c-JUN and EZH2 expression and activities, PTEN loss suppresses AR transcription factor activity and output, leading to reduced prostate epithelial differentiation and survival. Collaboratively, PTEN loss activates the PI3K/AKT signaling pathway and reduces the AR-regulated FKBP5-PHLPP negative feedback loop to AKT activation, further enhances AKT activation, leading to androgen/AR-independent prostate epithelial proliferation.