Androgens regulate prostate cancer cell growth via an AMPK-PGC-1α-mediated metabolic switch

Abstract

Prostate cancer is the most commonly diagnosed malignancy among men in industrialized countries, accounting for the second leading cause of cancer-related deaths. Although we now know that the androgen receptor (AR) is important for progression to the deadly advanced stages of the disease, it is poorly understood what AR-regulated processes drive this pathology. Here we demonstrate that AR regulates prostate cancer cell growth via the metabolic sensor 5'-AMP-activated protein kinase (AMPK), a kinase that classically regulates cellular energy homeostasis. In patients, activation of AMPK correlated with prostate cancer progression. Using a combination of radiolabeled assays and emerging metabolomic approaches, we also show that prostate cancer cells respond to androgen treatment by increasing not only rates of glycolysis, as is commonly seen in many cancers, but also glucose and fatty acid oxidation. Importantly, this effect was dependent on androgen-mediated AMPK activity. Our results further indicate that the AMPK-mediated metabolic changes increased intracellular ATP levels and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-mediated mitochondrial biogenesis, affording distinct growth advantages to the prostate cancer cells. Correspondingly, we used outlier analysis to determine that PGC-1α is overexpressed in a subpopulation of clinical cancer samples. This was in contrast to what was observed in immortalized benign human prostate cells and a testosterone-induced rat model of benign prostatic hyperplasia. Taken together, our findings converge to demonstrate that androgens can co-opt the AMPK-PGC-1α signaling cascade, a known homeostatic mechanism, to increase prostate cancer cell growth. The current study points to the potential utility of developing metabolic-targeted therapies directed toward the AMPK-PGC-1α signaling axis for the treatment of prostate cancer.

Figure 1

AMPK is required for androgen-mediated prostate cancer cell growth. A and B, LNCaP (A) or VCaP (B) cells were transfected with mock or siRNAs targeting a β-lactamase negative control (siControl) or AMPKα1 (#1-3). The following day, cells were treated for 72 hours ± 10 nM R1881 (synthetic androgen). Whole cell extracts were subjected to Western blot analysis using GAPDH as a loading control. C and D, LNCaP (C) and VCaP (D) cells were transfected the same as in A and B and then treated for 7 days ± R1881. Cells were then lysed and the relative number of cells was quantified using a fluorescent DNA-binding dye. Results are expressed as mean relative cell number + SE (n = 3). ^*, significant changes from vehicle-treated cells.

Figure 2

Phosphorylated/Activated AMPK correlates with clinical prostate cancer progression. A, examples of IHC with anti-phospho-AMPK antibody (Thr172). The staining index for benign and cancer epithelial cells is indicated under each figure. Original magnification: 100X. Box plot of staining index of prostate cancer (n = 61) and benign tissues (n = 65) is also shown. The difference in staining index was highly statistically significant (p < .001, Mann-Whitney). B, comparison of staining index of non-recurrent (n = 29) and recurrent (n = 32) tumors following radical prostatectomy. Staining was significantly higher in the recurrent group (p = .017, Mann-Whitney).

Figure 3

Androgens promote glycolysis and OXPHOS in prostate cancer cells. A and B, LNCaP (A) or VCaP (B) cells were treated for 72 hours with increasing concentrations of R1881 (0, 0.01, 0.1, 1, and 10 nM). Extracellular acidification rates (ECARs) were then measured using a Seahorse XF Analyzer and values were normalized to cell numbers. Results are expressed as ECAR normalized to cell numbers + SE (n = 3). C, LNCaP cells were treated and analyzed for ECAR as in A but were also tested for effects on ECAR in the sequential presence of oligomycin, FCCP and a combination of rotenone and antimycin A. D and E, LNCaP (D) and VCaP (E) cells were treated the same as in A and B. At the same time ECAR rates were being measured, simultaneous oxygen consumption rates (OCRs) were measured and normalized the same as in A and B. F, LNCaP cells were treated and analyzed for OCR as in D but were also tested for effects on OCR in the sequential presence of oligomycin, FCCP and a combination of rotenone and antimycin A. G, LNCaP cells were treated for 72 hours with increasing doses of R1881 (0, 0.1, 1, and 10 nM). Acetyl-CoA levels were then measured using MS/MS and normalized to total protein. Results are expressed as acetyl-CoA levels normalized to total protein + SE (n = 3). H, LNCaP cells were treated as in G. TCA cycle intermediate levels were then measured using GC/MS and normalized to total protein. Results are expressed as metabolite levels normalized to total protein + SE (n = 2). I, LNCaP cells were treated as in A and then incubated with ¹⁴C-labeled glucose and subjected to a CO₂ trap assay to measure complete glucose oxidation and normalized to cell numbers. Results are expressed as fold counts per minute (CPM) normalized to cell numbers + SE (n = 3). J, LNCaP cells were cotreated for 7 days with increasing doses of R1881 in the presence of vehicle (DMSO) or the β-oxidation inhibitor etomoxir (100 μM). Relative cell numbers were then determined as in Fig. 1. K and L, LNCaP cells were treated and subjected to a CO₂ trap assay the same as in I, but using ¹⁴C-labeled palmitate (K) or oleate (L) to quantitate the levels of fatty acid oxidation. ^*, significant changes from vehicle-treated cells.

Figure 4

Androgen-mediated OXPHOS requires AMPK. A and B, LNCaP (A) or VCaP (B) cells were transfected and treated as in Figs. 1A and B. OCRs were then measured using the Seahorse Analyzer and normalized to cell numbers as described in Fig. 3. C and D, LNCaP (C, E, G) or VCaP (D, F, H) cells were transfected and treated as described in A and B. Oxidation of radiolabeled glucose (C and D), palmitate (E and F) and oleate (G and H) were then measured using CO₂ trap assays and normalized to cell numbers as in Fig. 3. ^*, significant changes from vehicle-treated cells.

Figure 5

Androgens increase intracellular ATP levels through AMPK. A, LNCaP cells were treated the same as in Fig. 3G. ATP levels were then quantitated using a luciferase-based assay and data were normalized to cell numbers. Results are expressed as relative ATP levels normalized to cell numbers + SE (n = 3). B, cells were transfected and treated as in Fig. 1A. ATP levels were then quantitated and normalized as in A. ^*, significant changes from vehicle-treated cells.

Figure 6

AR-AMPK signaling increases PGC-1α levels. A-D, prostate cancer cells were treated with increasing concentrations of R1881 for 72 hours. A left, representative LNCaP Western blots following treatment (0, 0.1, 1 and 10 nM R1881). A right, LNCaP immunoblot densitometry values. PGC-1α levels were normalized to GAPDH (n = 4). B left, representative VCaP Western blots following treatment (0, 0.01, 0.1, 1 and 10 nM R1881). B right, VCaP immunoblot densitometry values. PGC-1α levels were normalized to GAPDH (n = 4). C and D, LNCaP (C) and VCaP (D) cells treated for 72 hours with 0, 0.01, 0.1, 1 or 10 nM R1881. After treatment, cells were lysed and RNA was isolated and reverse transcribed. The expression of PGC-1α was assessed using qPCR (n = 3). E, cell/tumor lysates from untreated parental LNCaP and CRPC-derivative C4-2 cells or LAPC9-derived androgen-dependent (LAPC9-AD) and CRPC (LAPC9-AI) tumor xenografts were subjected to Western blot analysis. F and G, LNCaP cells were transfected and treated as described in Fig. 1A. Cells were then subjected to immunoblot (F) or qPCR (G) analysis (n = 3). Densitometry values for F are presented in Supplementary Fig. S8B. ^*, significant changes from vehicle-treated cells. H, C4-2 cells stably expressing shRNAs targeting either scramble control (shControl) or PGC-1α (shPGC-1α) were subjected to a 3-day cell growth/viability assay. Inset, Western blot control demonstrating PGC-1α stable knockdown. ^*, significant change from shControl cells.

Figure 7

AR-AMPK signaling increases PGC-1α -mediated mitochondrial biogenesis, OXPHOS and cell growth. A, LNCaP cells were treated ± 10 nM R1881 for 72 hours and then subjected to TEM analysis. Shown are representative images (left) and results from blinded scoring of mitochondrial numbers/section (right). B-D, VCaP (Figs. 7B and C) and LNCaP (Fig. 7D and Supplementary Fig. S10) cells were transfected with indicated siRNAs and treated as in Figs. 1A and B. Mitochondrial volume was then assessed using fluorescence confocal microscopy and subsequent image analysis. Shown are representative images (Fig. 7B and Supplementary Fig. S10) and quantitated morphometric analysis (Figs. 7C and D). Scale bar, 10 μm. E, LNCaP cells were transfected with indicated siRNAs and treated as described in D. Oxidation of radiolabeled glucose was then measured using a CO₂ trap assay and normalized to cell numbers as in Fig. 4. ^*, significant changes from vehicle-treated cells. F, LNCaP cells were transfected as in E and then treated for 7 days ± R1881. Relative cell numbers were determined using the assay described in Fig. 1. ^*, significant changes from vehicle-treated cells.

Figure 8

Proposed model of the AR-AMPK-PGC-1α signaling axis. Androgens, through AR, increase AMPK activity. AMPK then potentiates PGC-1α. Increased PGC-1α can then promote mitochondria biogenesis and function.