Androgen receptor (AR) suppresses normal human prostate epithelial cell proliferation via AR/β-catenin/TCF-4 complex inhibition of c-MYC transcription

Abstract

Introduction: Physiologic testosterone continuously stimulates prostate stromal cell secretion of paracrine growth factors (PGFs), which if unopposed would induce hyperplastic overgrowth of normal prostate epithelial cells (PrECs).

Methods: Lentiviral shRNA stable knock down of c-MYC, β-catenin, or TCF-4 completely inhibits normal (i.e., non-transformed) human PrECs growth. c-MYC enhancer driven reporter expression and growth is inhibited by two chemically distinct molecules, which prevent β-catenin signaling either by blocking TCF-4 binding (i.e., toxoflavin) or by stimulating degradation (i.e., AVX939). Recombinant DKK1 protein at a dose, which inhibits activation of canonical Wnt signaling does not inhibit PrEC growth. Nuclear β-catenin translocation and PrEC growth is prevented by both lack of PGFs or Akt inhibitor-I. Growth inhibition induced by lack of PGFs, toxoflavin, or Akt inhibitor-I is overcome by constitutive c-MYC transcription.

Results: In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen binding to AR suppressing c-MYC transcription, resulting in G0 arrest/terminal differentiation independent of Rb, p21, p27, FoxP3, or down regulation of growth factors receptors and instead involves androgen-induced formation of AR/β-catenin/TCF-4 complexes, which suppress c-MYC transcription. Such suppression does not occur when AR is mutated in its zinc-finger binding domain.

Discussion: Proliferation of non-transformed human PrECs is dependent upon c-MYC transcription via formation/binding of β-catenin/TCF-4 complexes at both 5' and 3' c-MYC enhancers stimulated by Wnt-independent, PGF induced Akt signaling. In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen-induced formation of AR/β-catenin/TCF-4 complexes, which retains binding to 3' c-MYC enhancer, but now suppresses c-MYC transcription.

Keywords: TCF-4; androgen receptor; c-MYC, β-catenin; human prostate epithelial cells.

Fig. 1

Androgen mediated AR inhibition of the growth of human prostate epithelial cells (PrECs) and PrEC-hTERT cells in response to AR stimulation using physiologic levels of the synthetic androgen R1881 (i.e., 1nM). Expression and ligand activation of AR in both PrEC-LV-AR and PrEC-hTERT-LV-AR cells results in a significant growth suppression over a 1-week observation period, which is inhibited by co-administration of the anti-androgen Casodex (10 μM). An AR DNA-binding mutant (A573D) failed to inhibit growth when ligand-activated [* indicates a P-value < 0.05].

Fig. 2

Exogenous c-MYC expression (RV-c-MYC) overrides growth inhibition of PrEC-hTERT-LV-AR cells induced by both AR-signaling and toxoflavin induced β-catenin inhibition [* indicates a P-value < 0.05].

Fig. 3

(A) Western blot determination of efficiency of lentiviral shRNA knockdown of c-MYC, β-catenin, or TCF-4 protein expression in human PrECs. (B) Growth response to such specific protein knock down[* indicates a P-value < 0.05].

Fig. 4

Western blot documents a lack of FOXP3 nuclear translocation in response to AR-mediated growth suppression of PrEC-hTERT-LV-AR cells. Cyt. ext, cytoplasmic extract; Nu. ext, nuclear extract.

Fig. 5

(A) β-Catenin and ligand (R1881)-bound wild type AR, but not DNA-binding AR mutant A573D, co-immunoprecipitate (IP) with nuclear TCF-4 in PrEC-hTERT-LV-AR cells. Non-specific IgG antibody was used as a negative control. (B) Immuno-histochemical staining of active β-catenin in PrEC-hTERT-LV-AR cells.

Fig. 6

(A) Chromatin immunoprecipitation (ChIP) documents that TCF-4 continues to bind the 3′ c-MYC enhancer elements of the c-MYC gene even in the presence of ligand-activated AR, but there is a decrease binding to the 5′ c-MYC enhancer element. (B) Ligand-activated AR and toxoflavin inhibition of β-catenin/TCF-4 complex formation inhibits the transcriptional activity of the 5′ plus 3′ c-MYC enhancer element using appropriate luciferase reporter constructs. (C) Toxoflavin dose-dependent growth inhibition of non-immortalized PrEC cells. (D) Growth response of PrEC-hTERT-LVAR cells to growth factor removal, Wnt inhibition (DKK1), or AKT inhibition (AKT-inhibitor) alone and in combination with constitutive c-MYC expression (RV-c-MYC), and to XAV939 induced inhibition of β-catenin signaling.

Fig. 7

Phosphorylation status of β-catenin in nuclear extracts of PrEC-hTERT-LV-AR cells growth arrested by treatment with AKT-inhibitor (AIN), removal of pararcrine growth factors (−GF), or addition of androgen (+R1881) compared to growing cells (i.e., growth factor +).

Fig. 8

Nuclear AR, c-MYC, and β-catenin are expressed in different populations in the normal human prostate. Upper Panel-IHC staining for active (i.e., N-terminal hypo-phosphorylated) nuclear β-catenin. Arrowheads indicated basal cells, which are usually negative for active nuclear β-catenin. Secretory-luminal cells are nearly universally positive for nuclear active β-catenin. Lower Left Panel-Immuno-fluorescent (IF) staining for nuclear c-MYC and DAPI counter-stain. Arrows indicate occasionally c-MYC positive basal cells; arrowheads usual c-MYC negative basal cells. Lower Middle Panel-IF staining for AR and DAPI counter-stain. Arrows indicate occasionally c-MYC positive basal cells, which are AR negative; arrowheads usual c-MYC negative basal cells, which are also AR negative. Lower Right Panel-Dual IF staining for c-MYC plus AR and DAPI counter-stain. Arrows and arrowheads are as described in left and middle panels.