Low β₂-adrenergic receptor level may promote development of castration resistant prostate cancer and altered steroid metabolism

Abstract

The underlying mechanisms responsible for the development of castration-resistant prostate cancer (CRPC) in patients who have undergone androgen deprivation therapy are not fully understood. This is the first study to address whether β2-adrenergic receptor (ADRB2)- mediated signaling may affect CRPC progression in vivo. By immunohistochemical analyses, we observed that low levels of ADRB2 is associated with a more rapid development of CRPC in a Norwegian patient cohort. To elucidate mechanisms by which ADRB2 may affect CRPC development, we stably transfected LNCaP cells with shRNAs to mimic low and high expression of ADRB2. Two UDP-glucuronosyltransferases, UGT2B15 and UGT2B17, involved in phase II metabolism of androgens, were strongly downregulated in two LNCaP shADRB2 cell lines. The low-ADRB2 LNCaP cell lines displayed lowered glucuronidation activities towards androgens than high-ADRB2 cells. Furthermore, increased levels of testosterone and enhanced androgen responsiveness were observed in LNCaP cells expressing low level of ADRB2. Interestingly, these cells grew faster than high-ADRB2 LNCaP cells, and sustained their low glucuronidation activity in castrated NOD/SCID mice. ADRB2 immunohistochemical staining intensity correlated with UGT2B15 staining intensity in independent TMA studies and with UGT2B17 in one TMA study. Similar to ADRB2, we show that low levels of UGT2B15 are associated with a more rapid CRPC progression. We propose a novel mechanism by which ADRB2 may affect the development of CRPC through downregulation of UGT2B15 and UGT2B17.

Keywords: ADRB2; CRPC; UGT2B15; UGT2B17; β2-adrenergic receptor.

Figure 1. ADRB2 level is positively correlated with time to CRPC development

Immunohistochemical analysis of ADRB2 expression in a TMA of transurethral resections of the prostate (TUR-P). Examples of tissue cores of Gleason score 9 tumors showing negative a. or strong staining b. intensity (original magnification 20x). Kaplan-Meier plots showing time to CRPC development c., and time to prostate cancer (PCa)- specific death d. following TUR-P in patients stratified according to strong and weak staining intensity of ADRB2.

Figure 2. ADRB2 level, receptor binding, and downstream signaling activity in LNCaP shADRB2 cell lines

a. ADRB2 mRNA levels were semi-quantitatively measured in RNA isolated from two LNCaP shADRB2 (shADRB2-1 and shADRB2-2) cell lines and a non-targeting shRNA LNCaP cell line (shCtrl) using Real-Time RT-PCR. Mean, ΔΔC_t calculated values relative to shCtrl cells are shown. b. β-adrenergic receptor level was quantified by determination of ¹²⁵I-CYP specific binding to membrane protein fractions isolated from two LNCaP shADRB2 cell lines and shCtrl cells. Bars represent β-adrenergic receptor level reported as fmol/mg protein in the membrane fraction. c. Adenylyl cyclase activities in membranes isolated from LNCaP shADRB2 and shCtrl cells treated with vehicle or 10 μM isoproterenol were measured. The bars represent mean rate of formation of cAMP normalized to total protein in the membrane fractions (fmol/mg protein/min). All experiments were performed in biological triplicates (n = 3), mean ± standard deviation (SD). Statistical significance is indicated by asterisks (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

Figure 3. LNCaP shADRB2 xenograft tumors grow more rapidly than shCtrl tumors in castrated mice

LNCaP shADRB2-2 and shCtrl cells were implanted subcutaneously into nude NOD-SCID mice. Once tumors reached 500 mm³ in size, mice were surgically castrated and taken off testosterone supplementation. Tumor volumes were measured weekly for 6 weeks. The graph a. shows mean (n = 10 for shADRB2-2 and 11 for shCtrl) tumor volumes (mm³) ± SEM. b. Box-and-whisker plot showing the percentage change in tumor volume 42 days after castration in NOD-SCID mice injected with LNCaP shADRB2-2 and shCtrl cells. Statistical significance was measured by Fischer exact test, and is indicated by asterisks (*: p < 0.05).

Figure 4. UGT2B15 and UGT2B17 mRNA, protein and effects on androgen glucuronide formation

a. UGT2B15 and UGT2B17 mRNA levels were measured in RNA isolated from LNCaP shADRB2 (shADRB2-1 and shADRB2-2) and shCtrl cells using Real-Time RT-PCR. Bars represent mean, ΔΔC_t calculated values relative to shCtrl cells (n = 3) ± SD. b. UGT2B15 and UGT2B17 protein levels were visualized in cell homogenates by immunoblotting using anti-UGT2B15 and anti-UGT2B17 antibodies. Anti-actin antibodies were simultaneously used on the same homogenates to ensure similar loading on the lanes. c.-f. Cell homogenates from two LNCaP shADRB2 cell lines (shADRB2-1 and shADRB2-2) and shCtrl LNCaP cells (shCtrl) were mixed with uridine diphosphate glucuronic acid (UDPGA) and either dihydrotestosterone (DHT), 3α-androstanediol (3α-Diol) or androsterone (AND), for one hour, and levels of glucuronidated (G) androgens (c: DHT-G; d: 3α-Diol-17G; e: 3α-Diol-3G; f: AND-G) were measured by LC-MS/MS. The results are shown as mean formed glucuronide related to total protein in the homogenates (pmol/min/mg protein) from duplicated reactions on three biological replications ± SD. Statistical significance is indicated by asterisks (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

Figure 5. LNCaP shADRB2 castrated mouse tumor characteristics and glucuronidation activity

a. Excised xenograft tumors were formalin-fixed and paraffin-embedded, and sections were stained with anti-UGT2B15 (1:500) and anti-UGT2B17 (1:500) antibodies. Frequencies of staining intensities (weak, intermediate and strong) from tumors derived from mice injected with shCtrl (n = 11) and shADRB2-2 (n = 10) LNCaP cells are shown. b.-e. Fresh frozen tumor tissue from the same mice were homogenized and added UDPGA and either dihydrotestosterone (DHT), 3α-androstanediol (3α-Diol) or androsterone (AND) in a glucuronidation assay. Formation of steroid glucuronides (c: DHT-G; d: 3α-Diol-17G; e: 3α-Diol-3G; f: AND-G) was measured by LC-MS/MS. The results are shown as a box-and-whisker plot showing formed glucuronide related to total protein in the tissue homogenates (pmol/min/mg tissue protein) from triplicate reactions on the same homogenates. Statistical significance is indicated by asterisks (**: p < 0.01; ***: p < 0.001).

Figure 6. Increased androgen responsiveness in LNCaP shADRB2 cell lines

a. LNCaP shADRB2-1, shADRB2-2 and shCtrl cells were transfected with the androgen responsive element-containing luciferase reporter construct 285-Pb-pEZX-PG04. The following day, cells were incubated in hormone-deprived medium containing 2% CSS supplemented with either 10 nM DHT or vehicle and further incubated for 48 hours. The androgen responsiveness of 285-Pb-pEZX-PG04 in LNCaP shADRB2-1, shADRB2-2, and shCtrl cells are shown relative to vehicle treated (n = 3) ± SD. b. LNCaP shADRB2-1 and shADRB2-2 were transfected with the reporter construct 285-Pb-pEZX-PG04 and either an ADRB2 expression vector (pCDNA3.1-ADRB2) or a control expression vector (pEGFP-C3). Mean androgen responsiveness relative to vehicle treated cells is shown (n = 3) ± SD. c. Cells were transfected with a reporter plasmid including the 5′-regulatory region of PSA (pGL3/PSA), and the cells were stimulated as described in (a). Androgen responsiveness is given as the relative luciferase activities from DHT-stimulated cells normalized to vehicle-treated cells from three independent experiments (n = 3) mean ± SD. d. shCtrl cells were transfected with 285-Pb-pEZX -PG04 and were either treated with 50 μM diclofenac or with vehicle and then half of the cells were stimulated with 10 nM DHT the following day and all cells were harvested after 72 hours. Mean DHT responses from three independent experiments are shown relative to un-stimulated shCtrl cells (given value 1.0) ± SD treated with either diclofenac or vehicle ± SD. Statistical significance is indicated by asterisks (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

Figure 7. Prostate-specific antigen responsiveness is higher in shADRB2 than in shCtrl cells

a., b. LNCaP shADRB2 and shCtrl cells were starved in 2% CSS for 96 hours prior to stimulation with 1 nM DHT or 1 nM R1881 for 48 hours. RNA was harvested and analyzed for PSA/KLK3 mRNA expression by Real-Time RT-PCR. Gene expression upon stimulation with a. DHT and b. R1881 relative to non-stimulated cells (vehicle) was calculated by the ΔΔC_t- method. Bars represent log₂-transformed androgen responses (n = 3) ± SEM. c., d. Secreted total PSA (TPSA) was measured in medium samples from cells stimulated with c. DHT and d. R1881 by time-resolved fluorescence, and was related to non-stimulated cells (vehicle). Bars represent androgen responses (n = 4) mean ± SD. NS: non-significant difference from shCtrl.

Figure 8. Reduced androgen glucuronidation activity affects the level of bioactive androgen in vitro

a. The basal level of testosterone was measured in shCtrl, shADRB2-1, and shADRB2-2 LNCaP cells cultured in FCS medium. Steroids were extracted from the cells, dried, reconstituted, and run on an LC-MS. Integrated, internal standard (IS)-normalized mean peak areas are shown related to equal cell pellets. b. LNCaP shADRB2-1, shADRB2-2 and shCtrl cells were transfected with pPB(−285/132)-LUC. After 72 hours incubation in FCS-medium, basal luciferase activity of the pPB(−285/132)-LUC reporter was measured and related to SEAP. The results are shown as mean, basal luciferase activities related to shCtrl (given value 1.0) ± SD from three independent experiments. c. LNCaP shADRB2-2 and shCtrl cells were treated with 50 or 100 μM diclofenac or vehicle for 48 hours. Integrated, internal standard (IS)-normalized peak areas were related to total protein content and is presented as mean relative increase compared to vehicle treated cells (given value 1.0) (nM testosterone/mg protein) from three independent experiments ± SD. Statistical significance is indicated by asterisks (*: p < 0.05, **: p < 0.01, ***: p < 0.001).