Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer

Abstract

Prostate cancer is characterized by its dependence on androgen receptor (AR) and frequent activation of PI3K signaling. We find that AR transcriptional output is decreased in human and murine tumors with PTEN deletion and that PI3K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinases. Similarly, AR inhibition activates AKT signaling by reducing levels of the AKT phosphatase PHLPP. Thus, these two oncogenic pathways cross-regulate each other by reciprocal feedback. Inhibition of one activates the other, thereby maintaining tumor cell survival. However, combined pharmacologic inhibition of PI3K and AR signaling caused near-complete prostate cancer regressions in a Pten-deficient murine prostate cancer model and in human prostate cancer xenografts, indicating that both pathways coordinately support survival.

Figure 1. Pre-clinical trial of PI3K pathway inhibition in GEM models of prostate cancer

(A) Target inhibition was confirmed with BEZ235 treatment (45 mg/kg/day) by analyzing the expression of pAkt and pS6 in the Pten^lox/lox mice (microscopy at 400x). (B) Histologic and immunohistochemical staining for ki67 in PB-MYC and Pten^lox/lox mice treated with vehicle control or BEZ235 (microscopy at 200x). (C) Quantification of ki67 staining (mean of 3 HPF +/− SD) for PB-MYC and Pten^lox/lox mice treated with vehicle and BEZ235. (D) MRI images of representative PB-MYC and Pten^lox/lox mice at initiation and completion of study evaluating PI3K pathway inhibition with BEZ235. (E) Waterfall plot depicting proportional change in tumor response for PB-MYC (n=4) and Pten^lox/lox (n=4) mice treated with vehicle and BEZ235. See also Figure S1.

Figure 2. Inhibition of the PI3K pathway restores, in part, androgen responsive signaling in PTEN loss prostate cancers

(A) AR protein levels by Western blot analysis in wild-type, Pten^lox/lox, and Pten^lox/lox mice treated with BEZ235. Ventral prostates from individual 8-week old mice were analyzed. (B) Western blot analysis of AR, pAKT, pS6, pERK in the LNCaP cell line treated for 24 hours with DMSO, BEZ235 (500nM), and RAD001 (100nM). (C) Expression analysis (qRT-PCR) of the androgen regulated genes Pbsn, Nkx3.1, and Psca in wild-type, Pten^lox/lox, and Pten^lox/lox mice treated with BEZ235, normalized to Hprt and wild-type intact mice. Ventral prostates from individual 8-week old mice were analyzed and mean fold-change +/− SD is reported. (D) Luciferase mean fold-change +/− SD measurements in the LNCaP AR-ARE-Luciferase cell line treated for 24 hours with DMSO, BEZ235 (500nM), RAD001 (100nM), and AKT1/2 inhibitor (1uM) normalized to DMSO. (E) Western blot analysis of HER2, HER3, AR, and PSA in the LNCaP cell line treated with DMSO and BEZ235 (500nM) for 24 hours and Pten^lox/lox mice treated with BEZ235. (F) Western blot analysis for HER3, AR, pAKT, pERK in LNCaP cells treated with BEZ235 or siRNA AKT1/2 for 24 hours. (G) Luciferase mean fold-change +/− SD measurements in the LNCaP AR-ARE-Luciferase cell line treated for 24 hours with DMSO, BEZ235 (500nM), RAD001 (100nM), with or without PKI166 (5uM) or PD0325901 (1uM) normalized to DMSO.

Figure 3. PTEN loss prostate cancer is associated with reduced androgen responsive gene signaling

(A) Mean summed z-score +/− SD for an androgen responsive gene signature in human PTEN loss and PTEN normal prostate cancer showing PTEN loss tumors have a reduced androgen responsive gene signature with corresponding heat map. Significance was determined by the Student’s t-test. GSEA showing the (B) Hieronymus and (C) Nelson androgen responsive gene signatures revealed that androgen responsive gene signaling is reduced in PTEN loss human prostate cancers. (D) Mean ERBB2 expression levels +/− SD in PTEN loss and PTEN normal prostate cancer. Significance was determined by the Student’s t-test. (E) Gene signature enrichment analysis (GSEA) of a murine androgen responsive gene set in Pten loss prostate cancers compared to an intact wild-type mice. See also Figure S2.

Figure 4. Pre-clinical trial of combined androgen blockade in GEM models of prostate cancer

(A) Waterfall plot depicting proportional change in tumor response for PB-MYC (n=5) and Pten^lox/lox (n=5) mice treated with castration and MDV3100 (30 mg/kg/day). (B) Histologic and immunohistochemical staining for AR and ki67 and Pten^lox/lox mice treated with vehicle control or castration and MDV3100 (microscopy at 200x). (C) Western blot analysis of prostates from wild-type and Pten^lox/lox mice treated with surgical castration and/or BEZ235 for 7-days. Ventral prostates from individual 8-week old mice were analyzed. (D) Western blot analysis for PHLPP, FKBP5, pAKT in LNCaP treated with MDV3100 (10uM) or siRNA AR for 24 hours. (E) Western blot analysis for PHLPP, FKBP5, pAKT in LNCaP transfected with siRNA FKBP5. Western blot analysis for PHLPP, FKBP5, pAKT in LNCaP transfected with vector control, treated with MDV3100 (10uM) or transfected with CMV-FKBP5 and treated with MDV3100. (F) Phlpp immunohistochemistry of intact and castrate Pten^lox/lox mice. See also Figure S3.

Figure 5. Therapeutic efficacy of combined androgen blockade and PI3K pathway inhibition in pre-clinical models of prostate cancer

(A) LNCaP cell proliferation assay reported as mean cell count +/− SD following treatment with DMSO, MDV3100 (10uM), BEZ235 (500nM) and combinations. (B) Western blot analysis of LNCaP cells treated with DMSO, MDV3100, BEZ235, and BEZ235/MDV3100 for cleaved-PARP, pAKT, AKT, and pS6. (C) LNCaP cell proliferation assay reported as mean cell count +/− SD following treatment with DMSO, MDV3100 (10uM), PKI166 (5uM) and combinations. (B) Western blot analysis of LNCaP cells treated with DMSO, PKI166, BEZ235, and BEZ235/PKI166 for cleaved-PARP, pAKT, pERK, pS6, and PSA. (E) MRI images of representative Pten^lox/lox mice at initiation and completion of study evaluating combined AR and PI3K pathway inhibition with castration, MDV3100 (30 mg/kg/day), and BEZ235 (45 mg/kg/day). (F) Waterfall plot depicting proportional change in tumor response for Pten^lox/lox (n=4) and PB-MYC (n=4) mice treated with combined AR and PI3K pathway inhibition. (G) Model of PI3K pathway and AR crosstalk demonstrating activation of the PI3K pathway in the setting of PTEN loss leads to up-stream feed-back inhibition on receptor tyrosine kinases (HER2/3) leading to repressed AR activity. Inhibition of the PI3K pathway stimulates up-stream HER2/3 resulting in activation of AR, while blockade of AR reduces FKBP5 levels impairing PHLPP function leading to up-regulation of pAKT. See also Figure S4.