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. 2015 Apr 1;75(7):1413-22.
doi: 10.1158/0008-5472.CAN-14-3080. Epub 2015 Feb 3.

Intracrine Androgens and AKR1C3 Activation Confer Resistance to Enzalutamide in Prostate Cancer

Chengfei Liu  1 , Wei Lou  1 , Yezi Zhu  2 , Joy C Yang  1 , Nagalakshmi Nadiminty  1 , Nilesh W Gaikwad  3 , Christopher P Evans  4 , Allen C Gao  5
Affiliations

Intracrine Androgens and AKR1C3 Activation Confer Resistance to Enzalutamide in Prostate Cancer

Chengfei Liu et al. Cancer Res. .

Abstract

The introduction of enzalutamide and abiraterone has led to improvement in the treatment of metastatic castration-resistant prostate cancer. However, acquired resistance to enzalutamide and abiraterone therapies frequently develops within a short period in many patients. In the present study, we developed enzalutamide-resistant prostate cancer cells in an effort to understand the mechanisms of resistance. Global gene-expression analysis showed that the steroid biosynthesis pathway is activated in enzalutamide-resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide-resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide-resistant prostate xenograft tumors. LC/MS analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly upregulated in enzalutamide-resistant prostate cancer cells compared to the parental cells. Knockdown of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide-resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance; targeting intracrine androgens and AKR1C3 will overcome enzalutamide resistance and improve survival of advanced prostate cancer patients.

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Figures

Figure 1
Figure 1
C4-2B MDVR cells are resistant to enzalutamide in vitro and in vivo. A, C4-2B parental and C4-2B MDVR cells were treated with different concentrations of enzalutamide for 48 hours, total cell numbers were counted and cell survival rate was calculated. B, The clonogenic ability of C4-2B parental and C4-2B MDVR cells treated with 10 μM or 20 μM enzalutamide was analyzed. Enzalutamide significantly inhibited clonogenic ability of C4-2B parental cells. . C, C4-2B parental and C4-2B MDVR cells were injected orthotopically into the prostates of SCID mice and treated with 25mg/Kg enzalutamide or vehicle control. Tumors were harvested and weighed at 3 weeks. D, LNCaP, LN-95 and CWR22Rv1 cells were treated with different concentrations of enzalutamide for 2 days, total cell numbers were counted and cell survival rate (%) was calculated. * p>0.05.
Figure 2
Figure 2
Intracrine androgen synthesis pathway activated in enzalutamide resistant prostate cancer cells. A, Expression of transcripts encoding genes involved in steroid hormone biosynthesis was analyzed by gene set enrichment. Genes that were regulated 1.3 fold between C4-2B parental cells and C4-2B MDVR cells were enriched and heat map was generated by Subio platform. B, C4-2B parental cells and C4-2B MDVR cells were cultured in RPMI 1640 media containing 10% FBS for 3 days, total RNAs were extracted and CYP17A1, HSD3B1, HSD3B2, HSD17B3, SRD5A1, AKR1C1/2 or AKR1C3 mRNA levels were analyzed by qRT-PCR. AKR1C3, HSD3B and CYP17A1 protein levels were examined by western blot (right panel).
Figure 3
Figure 3
AKR1C3 is highly expressed in metastatic prostate tumors and enzalutamide resistant xenografts . A, C4-2B parental, C4-2B MDVR, VCaP, CWR22Rv1 , LNCaP and LN-95 cells were harvested and whole lysates were subjected to Western blotting. B, AKR1C3 expression level was analyzed by IHC staining in C4-2B parental, C4-2B MDVR and CWR22Rv1 xenografts. C, Gene expression analysis using the Oncomine database showing the relative expression levels of AKR1C3 in two datasets comparing normal prostate tissue and prostate cancer. Vanaja: normal, n = 8; cancer, n = 32. Singh: normal, n = 50; cancer, n = 52. Data are presented as mean ± S.E. of normalized expression units according to Oncomine output (upper). AKR1C3 gene expression analysis using the GEO database in two datasets comparing benign, primary or metastatic prostate cancer. GSE27616: Benign, n = 4; primary prostate cancer, n = 5; and metastatic prostate cancer, n = 4; GSE32269: primary prostate cancer, n = 22; and metastatic prostate cancer, n = 29. Data were extracted and analyzed by Subio platform (bottom). D, Gene expression analysis using the Oncomine database showed that AKR1C3 expression was correlated with prostate cancer progression and recurrence in two independent datasets (Glinsky and Singh prostate).
Figure 4
Figure 4
Intracrine androgens were up regulated in enzalutamide resistant prostate cancer cells. A, C4-2B parental and C4-2B MDVR cells were cultured in serum free and phenol red free RPMI1640 medium for 5 days, and levels of steroids in the cell extracts were analyzed by LC-MS. Representative testosterone and estradiol chromatograms generated by MassLynx 4.1 software are shown. B, the difference between levels of androgen metabolites in C4-2B parental and C4-2B MDVR cells was analyzed and quantified. C, the represented steroid metabolites between C4-2B parental and C4-2B MDVR cells were quantified. D, the androgen metabolism pathway was up regulated in enzalutamide resistant prostate cancer cells.
Figure 5
Figure 5
AKR1C3 confers resistance to enzalutamide in prostate cancer cells. A, CWR22Rv1 cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561 and #694), following treatment with 20 μM enzalutamide and cell numbers were determined on 3 days. B, C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561 and #694), following treatment with 20 μM enzalutamide and cell numbers were determined on 3 days. C, CWR22Rv1 or C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561 and #694), cells were collected on 3 or 5 days, and whole cell lysates were subjected to Western blotting. D, LNCaP-neo or LNCaP-AKR1C3 cells were treated with different concentrations of enzalutamide for 2 days, total cell numbers were counted and cell survival rate (%) was calculated. E, LNCaP-neo or LNCaP-AKR1C3 cells were treated with 10 μM enzalutamide and clonogenic assay was performed, the colony size pictures were taken under a microscope. F, Colonies were counted and results are presented as means ± SD of 2 experiments performed in duplicate. * p<0.05. Enza: Enzalutamide.
Figure 6
Figure 6
Indomethacin, an inhibitor of AKR1C3activity, overcomes enzalutamide resistance. A, CWR22Rv1 cells were treated with 10 μM or 20 μM indomethacin with or without 20 μM enzalutamide for 2 days, total cell numbers were counted (left), and clonogenic assay was performed, the colony size pictures were taken under a microscope (middle). Colonies were counted and results are presented as means ± SD of 2 experiments performed in duplicate (right). B, C4-2B MDVR cells were treated with 10 μM or 20 μM indomethacin with or without 20 μM enzalutamide for 2 days, total cell numbers were counted (left), and clonogenic assay was performed; the colony size pictures were taken under a microscope (middle). Colonies were counted and results are presented as means ± SD of 2 experiments performed in duplicate (right). C, Mice bearing CWR22Rv1 xenografts were treated with vehicle control, enzalutamide (25 mg/Kg p.o), Indomethacin (3mg/Kg i.p) or their combination for 3 weeks, tumor volumes were measured twice weekly and the tumors were collected and weighed. D, IHC staining of Ki67 and H/E staining in each group was performed and quantified as described in methods. * p<0.05. Enza: Enzalutamide. Indocin: Indomethacin.
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