Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 24;8(4):6796-6808.
doi: 10.18632/oncotarget.14296.

A novel nonsense mutation in androgen receptor confers resistance to CYP17 inhibitor treatment in prostate cancer

Dong Han  1 , Shuai Gao  1   2 , Kevin Valencia  1 , Jude Owiredu  1 , Wanting Han  1 , Eric de Waal  3 , Jill A Macoska  1 , Changmeng Cai  1   2
Affiliations

A novel nonsense mutation in androgen receptor confers resistance to CYP17 inhibitor treatment in prostate cancer

Dong Han et al. Oncotarget. .

Abstract

The standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that blocks transcriptional activity of androgen receptor (AR). However, ADT invariably leads to the development of castration-resistant PCa (CRPC) with restored activity of AR. CRPC can be further treated with CYP17 inhibitors to block androgen synthesis pathways, but most patients still relapse after a year of such treatment. The mechanisms that drive this progression are not fully understood, but AR activity, at least in a subset of cancers, appears to be restored again. Importantly, AR mutations are more frequently detected in this type of cancer. By analyzing tumor biopsy mRNA from CRPC patients who had developed resistance to CYP17 inhibitor treatment, we have identified a novel nonsense mutation (Q784*) at the ligand binding domain (LBD) of AR, which produces a C-terminal truncated AR protein that lacks intact LBD. This AR-Q784* mutant is transcriptionally inactive, but it is constitutively expressed in the nucleus and can bind to DNA in the absence of androgen. Significantly, our results show that AR-Q784* can heterodimerize with, and enhance the transcriptional activity of, full-length AR. Moreover, expressing AR-Q784* in an AR positive PCa cell line enhances the chromatin binding of endogenous AR and the recruitment of p300 coactivator under the low androgen condition, leading to increased cell growth. This activity of AR-Q784* mimics the function of some AR splice variants, indicating that CYP17 inhibitor treatment in CRPC may select for LBD-truncated forms of AR to restore AR signaling.

Keywords: CYP17 inhibitor; abiraterone; androgen receptor; androgen receptor mutation; prostate cancer.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. AR-Q784* produces a C-terminal truncated form of AR protein
A. Predicted protein structure of AR-FL, AR-V7, or AR-Q784*. B. Immunoblotting for AR (using antibody against N-terminal region) in COS-7 cells transfected with AR-FL, AR-V7, AR-Q784*, or empty vector. C. Immunoblotting for AR (N-terminal) in PC-3 cells transfected with AR-FL, empty vector, or AR-Q784*, and treated with vehicle or 10nM DHT (24h).
Figure 2
Figure 2. AR-Q784* mutant is deficient for androgen stimulated transcription activity
A, B, C. Dual-luciferase reporter assays were carried out in PC-3 cells transfected with empty vector, AR-FL, AR-V7, and AR-Q784* using various androgen-regulated reporters driven by (A) ARE4, (B) MMTV promoter, or (C) Probasin promoter, and treated with vehicle or 10nM DHT (24h). Reporter activity was normalized to the cotransfected CMV-Renilla-Luc. D. Endogenous FKBP5 mRNA expression was examined by qRT-PCR in PC-3 cells transfected with empty vector, AR-FL, or AR-Q784*, and then treated with 10nM DHT (24h). E, F, G. Dual-luciferase reporter assays were performed in COS-7 cells transfected with empty vector, AR-FL, or AR-Q784*, and treated with the indicated small molecules using reporters driven by (E) ARE4, (F) Probasin promoter, or (G) PSA enhancer.
Figure 3
Figure 3. AR-Q784* localizes in nucleus and binds to chromatin independent of androgen stimulation
A. Immunofluorescence staining for AR (N-terminal) (green) in COS-7 cells transfected with AR-FL or AR-Q784* and treated with 10nM DHT for 24h. Nuclear compartment was determined by DAPI staining (blue). B. ChIP-qPCR for measuring AR binding (N-terminal) at an FKBP5 enhancer in PC-3 cells transfected with empty vector, AR-FL, AR-V7, or AR-Q784*, and treated with vehicle or 10nM DHT (4h).
Figure 5
Figure 5. AR-Q784* heterodimerizes with AR-FL and enhances its transcriptional activity
A, B. Dual-luciferase reporter assays were carried out in PC-3 cells transfected with empty vector, AR-FL, AR-Q784*, or AR-FL+AR-Q784* using androgen-regulated reporters driven by (A) ARE4 or (B) PSA enhancer, and treated with vehicle or 10nM DHT (24h). C. Immunoprecipitation for HA in COS-7 cells cotransfected with HA-tagged AR-FL plus FLAG-tagged AR-FL or AR-Q784*, and treated with vehicle or 10nM DHT (24h), followed by immunoblotting for FLAG or HA. D. COS-7 cells transfected with empty vector, AR-FL, AR-Q784*, or AR-FL plus AR-Q784*, and treated with vehicle or DHT were fractionated into nuclear and cytoplasmic extracts, followed by immunoblotting for AR (N-terminal). GAPDH is a loading control for whole cell extracts and cytoplasmic fraction. Lamin B1 is a loading control for nuclear fraction.
Figure 4
Figure 4. Loss of intact NES contributes for the nuclear localization of AR-Q784*
A. Immunofluorescence staining for AR (N-terminal) (green) in COS-7 cells transfected with AR-K823* and treated with 10nM DHT for 24h. Nuclear compartment was determined by DAPI staining (blue). B. COS-7 cells transfected with AR-FL or AR-K823* and treated with vehicle or DHT were fractionated into nuclear and cytoplasmic extracts, followed by immunoblotting for AR (N-terminal). GAPDH is a loading control for whole cell extracts and cytoplasmic fraction. Lamin B1 is a loading control for nuclear fraction.
Figure 6
Figure 6. AR-Q784* enhances the transcription activity of endogenous AR-FL in PCa cells stimulated by low levels of androgen
A. Dual-luciferase reporter assays were carried out in LNCaP cells transfected with empty vector, AR-FL, AR-V7, or AR-Q784* using PSA enhancer-driven reporter, and treated with vehicle or 10nM DHT (24h). B, D. Immunoblotting for total AR expression (N-terminal) in LNCaP cells stably infected by lentiviral vectors containing doxycycline-inducible (B) AR-Q784* (LNCaP-AR-Q784*) or (D) AR-FL (LNCaP-AR-FL), and treated with doxycycline (0, 0.02, 0.1, or 0.5μg/ml) for 2d. (C, E) qRT-PCR for the mRNA expression of endogenous PSA in (C) LNCaP-AR-Q784* or (E) LNCaP-AR-FL cells pretreated with doxycycline for 2d and then stimulated by 10nM DHT for 24h. F. qRT-PCR for PSA expression in LNCaP-AR-Q784* pretreated with doxycycline for 2d and then stimulated by 0-1nM DHT for 24h. G. Flow cytometry analysis (PI staining) for measuring the proliferation of the LNCaP stable cell line with empty vector or LNCaP-AR-Q784* treated with vehicle, 0.1nM DHT, or 0.1nM DHT+10μM enzalutamide for 0-4d. Both control cell line and LNCaP-AR-Q784* cell line were pretreated with 0.1μg/ml doxycycline.
Figure 7
Figure 7. AR-Q784* stabilizes AR-FL chromatin binding and enhances the recruitment of p300 coactivator
A, B. ChIP-qPCR for measuring (A) AR-FL binding (using antibody against C-terminal region) or (B) p300 binding at an FKBP5 enhancer in LNCaP-AR-Q784* cells pretreated with vehicle or doxycycline and then stimulated by DHT (0, 0.1 or 1 nM) for 4h.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Green SM, Mostaghel EA, Nelson PS. Androgen action and metabolism in prostate cancer. Mol Cell Endocrinol. 2012;360:3–13. - PMC - PubMed
    1. Yuan X, Cai C, Chen S, Chen S, Yu Z, Balk SP. Androgen receptor functions in castration-resistant prostate cancer and mechanisms of resistance to new agents targeting the androgen axis. Oncogene. 2014;33:2815–2825. - PMC - PubMed
    1. de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, Chi KN, Jones RJ, Goodman OB, Saad F, Jr, Staffurth JN, Mainwaring P, Harland S, Flaig TW, Hutson TE, Cheng T, et al. Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011;364:1995–2005. - PMC - PubMed
    1. Ryan CJ, Molina A, Griffin T. Abiraterone in metastatic prostate cancer. N Engl J Med. 2013;368:1458–1459. - PubMed
    1. Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, de Wit R, Mulders P, Chi KN, Shore ND, Armstrong AJ, Flaig TW, Flechon A, Mainwaring P, Fleming M, Hainsworth JD, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012;367:1187–1197. - PubMed

MeSH terms