Inhibition of AKR1C3 Activation Overcomes Resistance to Abiraterone in Advanced Prostate Cancer

Abstract

Abiraterone suppresses intracrine androgen synthesis via inhibition of CYP17A1. However, clinical evidence suggests that androgen synthesis is not fully inhibited by abiraterone and the sustained androgen production may lead to disease relapse. In the present study, we identified AKR1C3, an important enzyme in the steroidogenesis pathway, as a critical mechanism driving resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. We found that overexpression of AKR1C3 confers resistance to abiraterone while downregulation of AKR1C3 resensitizes resistant cells to abiraterone treatment. In abiraterone-resistant prostate cancer cells, AKR1C3 is overexpressed and the levels of intracrine androgens are elevated. In addition, AKR1C3 activation increases intracrine androgen synthesis and enhances androgen receptor (AR) signaling via activating AR transcriptional activity. Treatment of abiraterone-resistant cells with indomethacin, an AKR1C3 inhibitor, overcomes resistance and enhances abiraterone therapy both in vitro and in vivo by reducing the levels of intracrine androgens and diminishing AR transcriptional activity. These results demonstrate that AKR1C3 activation is a critical mechanism of resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. Furthermore, this study provides a preclinical proof-of-principle for clinical trials investigating the combination of targeting AKR1C3 using indomethacin with abiraterone for advanced prostate cancer. Mol Cancer Ther; 16(1); 35-44. ©2016 AACR.

Figure 1. Overexpression of AKR1C3 confers resistance to abiraterone

A. AKR1C3 is involved in the canonical, 5-diol and backdoor pathways of androgen synthesis and activates AR. B. LNCaP-neo and LNCaP-AKR1C3 cells were treated with different concentrations of abiraterone for 2 days. Total cell numbers were counted and cell survival rate (%) was calculated. Whole cell lysates from LNCaP-neo and LNCaP-AKR1C3 cells were subjected to western blot (Inside panel). C. CWR22Rv1, C4-2B MDVR and VCaP cells were transiently transfected with AKR1C3 shRNA (#561 and #694). Following treatment with 10 μM abiraterone for 3 days, total cell numbers were counted and cell survival rate (%) was calculated. D. CWR22Rv1 cells, C4-2B MDVR and VCaP cells were transiently transfected with AKR1C3 shRNA (#561 and #694) for 3 days, AKR1C3, AR-V7 and AR expression were examined by western blot. * p<0.05.

Figure 2. AKR1C3 inhibition suppressed testosterone level and AR activity in prostate cancer

A. C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561, #694) for 2 days. Total RNA was extracted and AKR1C3 mRNA level was examined by qRT-PCR. B. C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561, #694), Total RNA was extracted, PSA and NKX3.1 mRNA levels were examined by qRT-PCR, and the supernatants were collected and subjected to PSA ELISA. C. C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561, #694) with PSA-E/P-luc reporter. The luciferase activity was detected by dual luciferase reporter system (left) and whole cell lysates were subjected to ChIP assay (right). D. C4-2B MDVR cells were transiently transfected with PSA-E/P-luc reporter, followed by treatment with DMSO or 20 μM Indocin for 3 days. Luciferase activity was detected by dual luciferase reporter system (left) and whole cell lysates were subjected to ChIP assay (right). E. C4-2B MDVR (top) and CWR22Rv1 (bottom) cells were treated with DMSO or 20 μM Indocin in serum free, phenol red free RPMI1640 medium for 3 days. Fifty million cells were collected per treatment and testosterone level was examined by LC-MS. * p<0.05. Indocin: Indomethacin.

Figure 3. Exogenous AKR1C3 promotes testosterone production and regulates AR transcriptional activity in prostate cancer

A. LNCaP-neo and LNCaP-AKR1C3 cells were cultured in serum free, phenol red free RPMI1640 medium for 3 days. One hundred million cells were collected per group and testosterone level was examined by LC-MS. B. LNCaP-neo and LNCaP-AKR1C3 cells were cultured in CS-FBS conditions for 3 days, total RNA was extracted and PSA mRNA levels were examined by qRT-PCR, C. The supernatants were collected and PSA level was examined by PSA ELISA. D. LNCaP-neo and LNCaP-AKR1C3 cells were cultured in CS-FBS conditions for 3 days, whole cell lysates were subjected to ChIP assay. *p<0.05.

Figure 4. Abiraterone resistant prostate cancer cells express higher levels of AKR1C3

A. C4-2B parental cells and C4-2B AbiR cells were treated with different concentration of abiraterone acetate in RPMI 1640 media containing 10% FBS, total cell numbers were counted and cell survival rate was calculated on day 3. B. The clonogenic ability of C4-2B parental and C4-2B AbiR cells treated with 2.5 μM, 5 μM or 10 μM abiraterone acetate. The colonies were counted and results are presented as means ± SD of 2 experiments performed in duplicate, C. C4-2B parental cells and C4-2B MDVR cells were cultured in RPMI 1640 media containing 10% FBS for 3 days, total RNA was extracted and AKR1C3, CYP17A1, HSD3B2 and AR mRNA levels were analyzed by qRT-PCR. Whole cell lysates were immunoblotted with the indicated antibodies. D. C4-2B AbiR cells were transiently transfected with AKR1C3 shRNA (#561 and #694). Following treatment with 5 μM abiraterone acetate for 3 days, total cell numbers were counted and cell survival rate (%) was calculated. Knockdown effects were examined by western blot. Abi: Abiraterone. * P<0.05

Figure 5. Indocin enhances abiraterone treatment in vitro and in vivo

A. CWR22Rv1cells were treated with 20 μM Indocin with or without 10 μM abiraterone for 2 days and total cell numbers were counted and cell survival rate was calculated. B. CWR22Rv1cells were treated with 20 μM Indocin with or without 10 μM abiraterone and clonogenic assays were performed; colonies were counted and results are presented as means ± SD of 2 experiments performed in duplicate, the representative pictures were taken under a microscope, C. Mice bearing CWR22Rv1 xenografts were treated with vehicle control, abiraterone acetate (200 mg/Kg p.o), Indocin (3mg/Kg i.p) or their combination for 3 weeks. Tumor volumes were measured twice weekly. D. Each group of tumors was weighed. E. IHC staining of Ki67 in each group was performed and quantified. *p<0.05. Abi: Abiraterone, Indocin: Indomethacin.