Acetyl-CoA Counteracts the Inhibitory Effect of Antiandrogens on Androgen Receptor Signaling in Prostate Cancer Cells

Abstract

The commonly used therapeutic management of PC involves androgen deprivation therapy (ADT) followed by treatment with AR signaling inhibitors (ARSI). However, nearly all patients develop drug-resistant disease, with a median progression-free survival of less than 2 years in chemotherapy-naïve men. Acetyl-coenzyme A (acetyl-CoA) is a central metabolic signaling molecule with key roles in biosynthetic processes and cancer signaling. In signaling, acetyl-CoA serves as the acetyl donor for acetylation, a critical post-translational modification. Acetylation affects the androgen receptor (AR) both directly and indirectly increasing expression of AR dependent genes. Our studies reveal that PC cells respond to the treatment with ARSI by increasing expression of ATP-citrate lyase (ACLY), a major enzyme responsible for cytosolic acetyl-CoA synthesis, and up-regulation of acetyl-CoA intracellular levels. Inhibition of ACLY results in a significant suppression of ligand-dependent and -independent routes of AR activation. Accordingly, the addition of exogenous acetyl-CoA, or its precursor acetate, augments AR transcriptional activity and diminishes the anti-AR activity of ARSI. Taken together, our findings suggest that PC cells respond to antiandrogens by increasing activity of the acetyl-coA pathway in order to reinstate AR signaling.

Keywords: abiraterone; acetyl-coenzyme A; androgen receptor; enzalutamide; prostate cancer.

Figure 1

Treatment with enzalutamide and abiraterone increases intracellular levels of acetyl-CoA and promotes expression of ACLY and ACSS2 in PC cells. (A) LNCaP and C4-2B cells were cultured in RPMI 1640 medium supplemented with 10% FBS or treated with enzalutamide (Enz) or abiraterone (Abi) (both at 10 μM) for 24 h. Intracellular levels of acetyl-CoA were examined as described in Materials and Methods. (B) LNCaP and C4-2B\cells were cultured as described in Panel A. Gene expression was assayed by qRT-PCR. 18S gene was used for normalization. Results are expressed as the mean (n = 3) ± SD. * p < 0.01; ** p < 0.001; *** p < 0.0001. (C) Representative images of primary moderately differentiated (Gleason score 7) prostate cancer and normal prostate tissue specimens stained for ACLY and ACSS2. The images were derived from a TMA composed of human prostate cancer and normal prostate tissue specimens obtained from patients undergoing treatment at FCCC.

Figure 2

Acetyl-CoA diminishes the inhibitory effect of abiraterone and enzalutamide on AR signaling. (A) LNCaP cells were transfected with pGL3-AR-luc and phRL-TK plasmids and cultured under androgen-depleted conditions for 24 h followed by treatment with R1881 (1 nM) and the indicated concentrations of acetyl-CoA for 16 h. Samples were assayed for firefly and renilla luciferase activities using the Dual-Glo Luciferase assay. Values were normalized to Renilla activities. (B) LNCaP cells were cultured under androgen-depleted conditions for 24 h followed by treatment with R1881 (1 nM) and acetyl-CoA (0.5 mM) for 6 h. Gene expression was assayed by qRT-PCR. The 18S gene was used for normalization. (C) LNCaP cells were transfected with pGL3-AR-luc and phRL-TK plasmids and cultured under androgen-depleted conditions for 24 h followed by treatment with enzalutamide (Enz) (1 μM), abiraterone acetate (5 μM) (Abi), R1881 (1 nM), sodium acetate (10 mM), and acetyl-CoA (0.1 mM) for 16 h. (D) LNCaP and C4-2B PC cells were cultured under androgen-depleted conditions for 24 h followed by treatment with acetyl-CoA (0.1 mM) for 48 h. Expression of testosterone in cell lysates and cell culture supernatants was examined using a testosterone ELISA kit (Cayman Chemical). (E) Aliquots of LNCaP cells described in Panel C were pre-treated with or without p300/CBP inhibitors A-485 (1 μM) or C646 (25 μM). Results are expressed as the mean (n = 3) ± SD. ** p < 0.001; *** p < 0.0001; ns—non-significant.

Figure 3

Knockdown of p300 and CBP inhibits AR signaling. (A) Analysis of p300, CBP, and PSA protein expression in LNCaP cells carrying the separate or combined p300 and CBP knockdowns. Forty-eight hours after siRNA transfection, LNCaP cells were placed in medium supplemented with charcoal-stripped serum for 24 h followed by stimulation with R1881 (1 nM) for 18 h. Protein expression was examined using Western blot analysis. (B) PSA gene expression was examined in LNCaP cells carrying the separate or combined p300 and CBP knockdowns. Forty-eight hours after siRNA transfection, LNCaP cells were placed in medium supplemented with charcoal-stripped serum for 24 h followed by stimulation with R1881 (1nM) for 6 h. Gene expression was assayed by qRT-PCR. 18S gene was used for normalization. Results are expressed as the mean (n = 3) ± SD. * p < 0.01; *** p < 0.0001; ns—non-significant. Full size blots of are shown in Figure S4.

Figure 4

Effect of ACLY inhibition on the transcriptional activity of AR-FL and AR-V7. (A) Lysates from parental AR-negative and transformed PC-3 cells expressing the indicated constructs were subjected to Western blotting. (B) Parental PC-3 cells or PC-3 cells expressing either full-length AR or AR-V7 were transfected with pGL3-AR-luc and phRL-TK (Renilla) plasmids. The cells were cultured under androgen-depleted conditions (RPMI 1640 medium supplemented with 10% charcoal-stripped FBS) for 24 h followed by treatment with different combinations of BMS 303141 (BMS) (30 μM), enzalutamide (Enz) (2.5 μM), and the synthetic androgen agonist R1881 (1 nM) for 8 h. Samples were assayed for firefly and renilla luciferase activities using the Dual-Glo Luciferase assay. Values were normalized to Renilla activities. (C) PC-3 cells expressing either full-length AR or AR-V7 were transfected with pGL3-AR-luc and phRL-TK plasmids. The cells were cultured under androgen-depleted conditions for 24 h followed by treatment with different combinations of BMS 303141 (BMS) (30 μM), acetyl-CoA (0.1 mM), and R1881 (1 nM) for 8 h. Samples were assayed as described in Figure 1B. Columns, means of three different experiments; bars, SDs. ** p < 0.001; *** p < 0.0001; ns—non-significant. Full size blots of are shown in Figure S4.

Figure 5

ACLY inhibition results in the suppression of ligand-dependent and -independent PSA expression. (A) Western blot analysis of the AR expression in LNCaP, C4-2B, and 22Rv1 PC cells. (B) LNCaP, C4-2B, and 22Rv1 cells were cultured under androgen-depleted conditions for 24 h followed by treated with BMS 303141 (BMS) (30 μM), enzalutamide (Enz) (2.5 μM), and R1881 (1 nM) in for 18 hrs. Expression of PSA was examined in the cell culture supernatants using PSA ELISA kit. Columns, means of three different experiments; bars, SDs. ** p < 0.001; *** p < 0.0001. Full size blots of are shown in Figure S4.