The Activity of SN33638, an Inhibitor of AKR1C3, on Testosterone and 17β-Estradiol Production and Function in Castration-Resistant Prostate Cancer and ER-Positive Breast Cancer

Abstract

AKR1C3 is a novel therapeutic target in castration-resistant prostate cancer (CRPC) and estrogen receptor (ER)-positive breast cancer because of its ability to produce testosterone and 17β-estradiol intratumorally, thus promoting nuclear receptor signaling and tumor progression. A panel of CRPC, ER-positive breast cancer and high/low AKR1C3-expressing cell lines were treated with SN33638, a selective inhibitor of AKR1C3, in the presence of hormone or prostaglandin (PG) precursors, prior to evaluation of cell proliferation and levels of 11β-PG F2α (11β-PGF2α), testosterone, 17β-estradiol, and prostate-specific antigen (PSA). A meta-analysis of AKR1C3 mRNA expression in patient samples was also conducted, which revealed that AKR1C3 mRNA was upregulated in CRPC, but downregulated in ER-positive breast cancer. 11β-PGF2α and testosterone levels in the cell line panel correlated with AKR1C3 protein expression. SN33638 prevented 11β-PGF2α formation in cell lines that expressed AKR1C3, but partially inhibited testosterone formation and subsequently cell proliferation and/or PSA expression only in high (LAPC4 AKR1C3-overexpressing cells) or moderate (22RV1) AKR1C3-expressing cell lines. SN33638 had little effect on 17β-estradiol production or estrone-stimulated cell proliferation in ER-positive breast cancer cell lines. Although SN33638 could prevent 11β-PGF2α formation, its ability to prevent testosterone and 17β-estradiol production and their roles in CRPC and ER-positive breast cancer progression was limited due to AKR1C3-independent steroid hormone production, except in LAPC4 AKR1C3 cells where the majority of testosterone was AKR1C3-dependent. These results suggest that inhibition of AKR1C3 is unlikely to produce therapeutic benefit in CRPC and ER-positive breast cancer patients, except possibly in the small subpopulation of CRPC patients with tumors that have upregulated AKR1C3 expression and are dependent on AKR1C3 to produce the testosterone required for their growth.

Keywords: 11β-prostaglandin F2α; 17β-estradiol; AKR1C3; ER-positive breast cancer; SN33638; castration-resistant prostate cancer; prostate-specific antigen; testosterone.

Figure 1

Chemical structures of AKR1C3 inhibitors.

Figure 2

AKR1C3 mRNA expression is upregulated in tumor samples from CRPC patients but downregulated in breast cancer patient tumors. (A) Meta-analysis of AKR1C3 mRNA expression in normal prostate (n = 54), primary prostate carcinoma (n = 133), and CRPC (n = 100) tissue in patients from seven Oncomine prostate cancer datasets. The upper dotted line indicates 75th percentile for CRPC. (B) Correlation of NQO1 mRNA expression with AKR1C3 mRNA expression in CRPC patients from (A). (C) Meta-analysis of AKR1C3 mRNA expression in normal breast (Normal, n = 257), premenopausal ER-positive breast cancer (ER+ pre, n = 281), post-menopausal ER-positive breast cancer (ER+ post, n = 1337), premenopausal ER-negative breast cancer (ER− pre, n = 189), and post-menopausal ER-negative breast cancer (ER− post, n = 278) patients in seven Oncomine breast cancer datasets. The line in each box represents the median, the lower, and upper boundaries represent the 25th and 75th percentiles, and the whiskers show the 5th and 95th percentiles. (D) AKR1C3 mRNA expression in paired normal and ER-positive breast cancer samples in post-menopausal women (n = 23) from (C). Significance of differences was evaluated by one-way ANOVA with Dunnett’s multiple comparison analysis (A,C) or by Student’s t-test (D).

Figure 3

PGD₂ 11-ketoreductase activity correlates with AKR1C3 protein expression and can be inhibited by SN33638. (A) AKR1C3 and α-tubulin protein expression by western blotting in the cell line panel. (B) Correlation between 11β-PGF_2α formation following stimulation with 28 nM PGD₂ (n = 2–6) and AKR1C3 protein expression (average of two to three independent lysates) in the cell line panel. (C) 11β-PGF_2α formation in the cell line panel after stimulation with PGD₂ in the presence or absence of 10 μM SN33638 (n = 2–6). (D) EC₅₀ plot of inhibition of 11β-PGF_2α production by SN33638 following stimulation with PGD₂ in HCT116 AKR1C3 cells (n = 2–3). Bars or symbols represent the mean ± SEM. Statistical significance of correlation analysis was evaluated by Spearman’s rank-order correlation and of differences between mean values by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001 vs. no inhibitor controls.

Figure 4

Testosterone production correlates with AKR1C3 protein expression and can be partially inhibited by SN33638 in high AKR1C3-expressing cell lines. Testosterone levels were determined by ELISA 24 h after stimulation with 28 nM androstenedione. (A) Correlation between testosterone levels (n = 3–6) and AKR1C3 protein expression (average of 2–3 independent lysates) in the prostate cancer, HCT116, and NCI-H460 cell lines. (B) Concentration of testosterone in the cell lines in the presence or absence of 10 μM SN33638 (n = 3–6). (C) EC₅₀ plot of the inhibition of testosterone production by SN33638 in HCT116 AKR1C3 cells (n = 3). (D) Testosterone levels in HCT116 AKR1C3, NCI-H460, 22RV1, and LAPC4 cell lines following treatment with 10 μM SN33638 and 1 μM 17β-HSD3 inhibitor alone or in combination (n = 3–6). Bars or symbols represent the mean ± SEM. Statistical significance of correlation analysis was evaluated by Spearman’s rank-order correlation and of differences between mean values by Student’s t-test (B) or two-way ANOVA with Tukey’s multiple comparison analysis (D).*P < 0.05; **P < 0.01; ***P < 0.001 vs. no inhibitor controls. Statistically significant differences between treatment groups are marked as indicated.

Figure 5

SN33638 has little effect on estrone reduction to 17β-estradiol in ER-positive breast cancer cell lines. 17β-estradiol levels were determined by ELISA 24 h after stimulation with 28 nM estrone. (A) Correlation between 17β-estradiol levels (n = 3–4) and AKR1C3 protein expression (average of 2–3 independent lysates) in ER-positive breast cancer, HCT116, and NCI-H460 cell lines. (B) Concentration of 17β-estradiol in the cell lines in the presence and absence of 10 μM SN33638 (n = 3–4). (C) 17β-estradiol levels in T47D and T47D AKR1C3 cells following treatment with 10 μM SN33638 and 30 μM apigenin alone or in combination (n = 3). Bars or symbols represent the mean ± SEM. Statistical significance of correlation analysis was evaluated by Spearman’s rank-order correlation and of differences between mean values by Student’s t-test (B) or one-way ANOVA with Dunnett’s multiple comparison analysis (C). *P < 0.05; **P < 0.01 vs. no inhibitor controls.

Figure 6

SN33638 partially inhibits PSA expression and cell proliferation in LAPC4 AKR1C3 cells, but has limited activity on low-moderate AKR1C3-expressing CRPC cells and ER-positive breast cancer cells. (A) PSA levels in CRPC cell lines 24 h after stimulation with androstenedione in the presence or absence of 10 μM SN33638 (n = 3). Cell proliferation in (B) CRPC cell lines and (C) breast cancer cell lines 5 days after stimulation with androstenedione or estrone and treatment with or without 10 μM SN33638 (n = 3–4). Bars represent the mean ± SEM. Significance of differences was evaluated by Student’s t-test. *P < 0.05; ***P < 0.001 vs. no inhibitor controls.