Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth

David J Mulholland, Linh M Tran, Yunfeng Li, Houjian Cai, Ashkan Morim, Shunyou Wang, Seema Plaisier, Isla P Garraway, Jiaoti Huang, Thomas G Graeber, Hong Wu, David J Mulholland, Linh M Tran, Yunfeng Li, Houjian Cai, Ashkan Morim, Shunyou Wang, Seema Plaisier, Isla P Garraway, Jiaoti Huang, Thomas G Graeber, Hong Wu

Abstract

Alteration of the PTEN/PI3K pathway is associated with late-stage and castrate-resistant prostate cancer (CRPC). However, how PTEN loss is involved in CRPC development is not clear. Here, we show that castration-resistant growth is an intrinsic property of Pten null prostate cancer (CaP) cells, independent of cancer development stage. PTEN loss suppresses androgen-responsive gene expressions by modulating androgen receptor (AR) transcription factor activity. Conditional deletion of Ar in the epithelium promotes the proliferation of Pten null cancer cells, at least in part, by downregulating the androgen-responsive gene Fkbp5 and preventing PHLPP-mediated AKT inhibition. Our findings identify PI3K and AR pathway crosstalk as a mechanism of CRPC development, with potentially important implications for CaP etiology and therapy.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1. Early castration does not prevent…
Figure 1. Early castration does not prevent initiation of Pten-null CaP
(A) Pb-Cre+;PtenL/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 Figure S1.
Figure 2. PTEN loss can suppress androgen-responsive…
Figure 2. PTEN loss can suppress androgen-responsive gene expression
(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 Figure S2 and Table S1.
Figure 3. Epithelial AR is not required…
Figure 3. Epithelial AR is not required for the initiation of Pten-null CaP
(A) Deletion of epithelial AR in the anterior lobe of Pten-null CaP (Pb-Cre+;PtenL/L;ArL/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+;ArL/Y (C+;Ar); Pb-Cre+;PtenL/L and (C+;Pt) and Cre+;PtenL/L;ArL/Y (C+;Pt;Ar) mutants. (C) Frequency of invasiveness based on smooth muscle actin (SMA) break down in Pb-Cre+;PtenL/L and Cre+;PtenL/L;ArL/Y mutants during progression. Error bars, means +/− SD. See also Figure S3.
Figure 4. Epithelial AR is not required…
Figure 4. Epithelial AR is not required for transformation by Pten deletion or myristoylated Akt in regeneration assays
(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+;ArL/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 Figure S4.
Figure 5. AR down regulates AKT activity…
Figure 5. AR down regulates AKT activity by stimulating FKBP5 and PHLPP-mediated, AKT dephosphorylation
(A) PI3K activation (P-AKT, P-S6) in AR+ regions (arrows) versus AR− regions (arrow heads) in castrate, Pb-Cre+;PtenL/L;ArL/Y mutants. (Bar, low mag = 150 μm, high mag = 75 μm). (B) Effect of AR deletion on FKBP5 expression in Pb-Cre+PtenL/L;ArL/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+;PtenL/L;ArL/Y mutants at 2 days (C) and 4 weeks (D) after castration (bar = 75 μm). See also Figure S5.
Figure 6. Heterogeneous AR expression in human…
Figure 6. Heterogeneous AR expression in human CaPs correlating with PI3K/AKT signaling and FKBP5 and PHLPP levels
(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+;PtenL/L;ArL/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 Figure S6.
Figure 7. Cooperative effects of AR and…
Figure 7. Cooperative effects of AR and mTOR inhibition in vitro and in vivo
(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+;PtenL/L and Pb-Cre+;PtenL/L:ArL/Y mutants (C, bar = 2 mm; D, bar = 200 μm; D, inset bar = 75 μm). Error bars = means +/− SD. See also Figure S7.
Figure 8. PTEN loss promotes CRPC development…
Figure 8. PTEN loss promotes CRPC development by two collaborative mechanisms
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.

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