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
. 2014 Sep:143:473-80.
doi: 10.1016/j.jsbmb.2014.03.004. Epub 2014 Mar 22.

The N-terminal domain of the androgen receptor drives its nuclear localization in castration-resistant prostate cancer cells

Javid A Dar  1 , Khalid Z Masoodi  2 , Kurtis Eisermann  2 , Sudhir Isharwal  2 , Junkui Ai  2 , Laura E Pascal  2 , Joel B Nelson  3 , Zhou Wang  4
Affiliations

The N-terminal domain of the androgen receptor drives its nuclear localization in castration-resistant prostate cancer cells

Javid A Dar et al. J Steroid Biochem Mol Biol. 2014 Sep.

Abstract

Androgen-independent nuclear localization is required for androgen receptor (AR) transactivation in castration-resistant prostate cancer (CRPC) and should be a key step leading to castration resistance. However, mechanism(s) leading to androgen-independent AR nuclear localization are poorly understood. Since the N-terminal domain (NTD) of AR plays a role in transactivation under androgen-depleted conditions, we investigated the role of the NTD in AR nuclear localization in CRPC. Deletion mutagenesis was used to identify amino acid sequences in the NTD essential for its androgen-independent nuclear localization in C4-2, a widely used CRPC cell line. Deletion mutants of AR tagged with green fluorescent protein (GFP) at the 5'-end were generated and their signal distribution was investigated in C4-2 cells by fluorescent microscopy. Our results showed that the region of a.a. 294-556 was required for androgen-independent AR nuclear localization whereas a.a. 1-293 mediates Hsp90 regulation of AR nuclear localization in CRPC cells. Although the region of a.a. 294-556 does not contain a nuclear import signal, it was able to enhance DHT-induced import of the ligand binding domain (LBD). Also, transactivation of the NTD could be uncoupled from its modulation of AR nuclear localization in C4-2 cells. These observations suggest an important role of the NTD in AR intracellular trafficking and androgen-independent AR nuclear localization in CRPC cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Deletion mutagenesis analysis of AR nuclear import and export in COS1 cells upon androgen manipulation
(A) Structures of GFP fusion protein constructs: GFP-AR, GFP-DBDH-LBD, and GFP-LBD. (B) COS1 cells were transiently transfected with GFP-AR, GFP-DBDH-LBD and GFP-LBD cultured in androgen-free conditions. The subcellular signal distribution was visualized by fluorescent microscopy 16 hours post transfection (−DHT), 4 hours after treatment (+DHT) with 1.0 nM DHT for GFP-AR and GFP-DBDH-LBD or 100 nM DHT for GFP-LBD, or 12 hours after androgen withdrawal (Withdrawal). The experiment was reproduced 5 times.
Figure 2
Figure 2. Subcellular distribution of GFP-AR, GFP-DBDH-LBD and GFP-LBD in C4-2 cells
C4-2 cells were transiently transfected with GFP-AR, GFP-DBDH-LBD and GFP-LBD and stained with Hoechst. The subcellular localization (A) and quantification (B) were assessed in androgen-free conditions by fluorescent microscopy 16 hours after transfection. The results are from five transfections for each experimental group. At least 200 cells were counted for each transfection to determine the percentage of cells displaying cytoplasmic (C), even (E), or nuclear (N) localization. The experiment was reproduced 5 times.
Figure 3
Figure 3. Subcellular distribution of GFP-AR, GFP-ARΔ(294–556) and GFP-ARΔ(1–293) in C4-2 cells
(A) Diagram of different GFP-AR deletion constructs, GFP-AR, GFP-ARΔ(294–556), and GFP-ARΔ(1–293). (B) Representative fluorescent images of C4-2 cells transiently transfected with GFP-AR, GFP-ARΔ(294–556) or GFP-ARΔ(1–293) in in androgen-free conditions 16 hours after transfection. The results were derived from five transfections for each GFP-fusion protein construct. (C) Quantitative analysis of results in (B). At least 200 cells were counted for each transfection to determine the percentage of the cells displaying cytoplasmic (C), even (E), or nuclear (N) localization. The experiment was reproduced 5 times.
Figure 4
Figure 4. Subcellular localization of GFP-AR(294–556) and GFP-NTD in LAPC4, C4-2 and LNCaP cells
(A) Diagram of GFP fusion constructs GFP-NTD and GFP-AR(294–556). (B) Representative images of GFP-NTD and GFP-AR(294–556) in transiently transfected LAPC4, C4-2, and LNCaP cells. The subcellular localization was assessed in complete medium by fluorescence microscopy after 16 hours of transfection. The experiment was reproduced 5 times.
Figure 5
Figure 5. Effect of AR(294–556)on LBD sensitivity to DHT in C4-2 cells
(A) Diagram of GFP fusion constructs GFP-AR, GFP-NTD-LBD, GFP-AR(1–293)-LBD, GFP-AR(294–556)-LBD, and GFP-LBD. (B) Representative images of GFP-tagged protein localization in the absence or presence of 10 nM DHT. (C), Quantification analysis of GFP fusion constructs in C4-2 cells followed by treatment of DHT (0–100 nM) (B). At least 200 cells were counted for each transfection to determine the percentage of the cells displaying cytoplasmic (C), even (E), or nuclear (N) localization. The subcellular localization and quantification of signal were assessed by fluorescent microscopy. The experiment was reproduced 3 times.
Figure 6
Figure 6. Localization of GFP-tagged AR and AR deletion mutants in C4-2 cells in presence of Hsp90 inhibitor
C4-2 cells transfected with GFP-AR, GFP-ARΔ(294–556), GFP-ARΔ(1–293) and GFP-DBDH-LBD were treated with DMSO (control) or HSP90 inhibitor 17-AAG in androgen-free conditions. Localization was assessed by fluorescent microscopy 4 hr after the treatment.
Figure 7
Figure 7. Effect of TIF2 knockdown on AR subcellular localization and PSA expression in C4-2 cells
(A) Western blot analysis of C4-2 cells transfected with control siRNA or siTIF2 for 72 hours. The Western blots were probed with anti-TIF2, anti-PSA, or anti-GAPDH (loading control) antibodies. (B) Representative images of GFP-AR in C4-2 cells co-transfected with GFP-AR and either control siRNA or siTIF2 for 72 hours. Localization of GFP-AR was determined using fluorescence microscopy. (C) Percentage of cells displaying nuclear or even localization was determined for GFP-AR in cells co-transfected with either control siRNA or siTIF2. The experiment was reproduced 2 times.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Welsh M, et al. Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. The Journal of clinical investigation. 2008 Apr;118:1479. - PMC - PubMed
    1. Isaacs JT, Coffey DS. Etiology and disease process of benign prostatic hyperplasia. The Prostate. Supplement. 1989;2:33. - PubMed
    1. O'Malley KJ, et al. The expression of androgen-responsive genes is up-regulated in the epithelia of benign prostatic hyperplasia. The Prostate. 2009 Dec 1;69:1716. - PMC - PubMed
    1. Kozlowski JM, Ellis WJ, Grayhack JT. Advanced prostatic carcinoma. Early versus late endocrine therapy. The Urologic clinics of North America. 1991 Feb;18:15. - PubMed
    1. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA: a cancer journal for clinicians. 2010 Sep-Oct;60:277. - PubMed

Publication types

MeSH terms