The interplay of AMP-activated protein kinase and androgen receptor in prostate cancer cells

Abstract

AMP-activated protein kinase (AMPK) has recently emerged as a potential target for cancer therapy due to the observation that activation of AMPK inhibits tumor cell growth. It is well-known that androgen receptor (AR) signaling is a major driver for the development and progression of prostate cancer and that downregulation of AR is a critical step in the induction of apoptosis in prostate cancer cells. However, little is known about the potential interaction between AMPK and AR signaling pathways. In the current study, we showed that activation of AMPK by metformin caused decrease of AR protein level through suppression of AR mRNA expression and promotion of AR protein degradation, demonstrating that AMPK activation is upstream of AR downregulation. We also showed that inhibition of AR function by an anti-androgen or its siRNA enhanced AMPK activation and growth inhibition whereas overexpression of AR delayed AMPK activation and increased prostate cancer cellular resistance to metformin treatment, suggesting that AR suppresses AMPK signaling-mediated growth inhibition in a feedback mechanism. Our findings thus reveal a novel AMPK-AR regulatory loop in prostate cancer cells and should have a potential clinical significance.

Figure 1. Metformin induces AMPK activation and subsequent decrease in AR mRNA and protein levels in AR-positive prostate cancer cells

(A) LNCaP and C4-2B cells were treated with metformin at concentrations of 5, 10, 20 or 30 mM for 48 hours, followed by MTT assay. (B–C) LNCaP and C4-2B cells were treated with 30 mM metformin for up to 24 hours, followed by Western blot analysis (B) and caspase-3 activity assay (C). p-AMPKα, AMPKα phosphorylated at Thr172; p-ACC, ACC phosphorylated at Ser79. (D) LNCaP cells were treated with 30 mM metformin for up to 24 hours, followed by quantitative real-time PCR analysis. Black solid line is the quantification of AR mRNA expression after normalizing to GAPDH; data are expressed as fold change of 0 hour control (1.00) and shown as mean ± SD of triplicates. Grey dash line is the quantification of AR protein expression in the left panel of Fig. 1B after normalizing to actin.

Figure 2. Metformin in low millimolar range induces AMPK activation, AR protein decrease and apoptosis in prostate cancer cells

(A, B) LNCaP (A) and C4-2B (B) cells were treated daily with metformin at concentrations of 1.25, 2.5, 5 or 10 mM for up to 5 days, followed by MTT assay. (C) LNCaP cells were treated with 5 mM metformin for 24, 48, 72, 96 or 120 hours, followed by Western blot analysis.

Figure 3. Bicalutamide promotes metformin-induced AMPK activation, AR degradation and growth inhibition in AR-positive prostate cancer cells

(A, B) LNCaP (A) and C4-2B (B) cells were treated with various concentrations of metformin in the presence or absence of bicalutamide (Bic) at indicated concentrations for 24 hours, followed by MTT assay. (C) LNCaP cells were treated with metformin (30 mM) alone or in combination with bicalutamide (20 μM) for 0, 2, 4, 8, 12 or 24 hours, followed by Western blot analysis. p-Raptor, Raptor phosphorylated at Ser792.

Figure 4. AR negatively regulates metformin-induced AMPK activation in prostate cancer cells

(A) AR-positive LNCaP cells were transfected with AR-specific siRNA (lanes 4–6) or scramble control (lanes 1–3) for 72 hours, and then treated with 10 or 20 mM metformin for 4 hours, followed by Western blot analysis. (B) AR-negative parental PC3 cells and AR-transfected PC3-AR cells (stable transfection) were treated with metformin at 5, 10, 20 or 30 mM for 48 hours, followed by MTT assay. (C) PC3 cells were transiently transfected with AR construct (lanes 4–6) or empty vector (lanes 1–3) for 24 hours, and then treated with 30 mM metformin for 4 or 24 hours, followed by Western blot analysis.

Figure 5. Compound C prevents metformin-induced AMPK activation and AR degradation in prostate cancer cells

LNCaP cells were pretreated with either 0.1% DMSO (lanes 1–6) or 20 μM compound C (CC) (lanes 7–12) for 12 hours and then co-treated with 30 mM metformin (Met) for additional 0, 4, 8, 16, 24 or 30 hours (lanes 1–12), or treated with 20 μM CC for 0, 12, 16, 20, 28, 36 or 42 hours (lanes 13–19). Cell lysates were prepared and subjected to Western blot analysis.

Figure 6. Schematic diagram represents the regulatory loop of AR and AMPK in prostate cancer cells

See Discussion for details.