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
. 2016 Sep 13;7(37):59781-59794.
doi: 10.18632/oncotarget.10926.

Critical role of androgen receptor level in prostate cancer cell resistance to new generation antiandrogen enzalutamide

Julia Hoefer  1 , Mohammady Akbor  1   2 , Florian Handle  1 , Philipp Ofer  1 , Martin Puhr  1 , Walther Parson  3   4 , Zoran Culig  1   5 , Helmut Klocker  1 , Isabel Heidegger  1
Affiliations

Critical role of androgen receptor level in prostate cancer cell resistance to new generation antiandrogen enzalutamide

Julia Hoefer et al. Oncotarget. .

Abstract

Enzalutamide is an androgen receptor (AR) inhibitor approved for therapy of metastatic castration resistant prostate cancer. However, clinical application revealed that 30 to 40% of patients acquire resistance after a short period of treatment. Currently, the molecular mechanisms underlying such resistances are not completely understood, partly due to a lack of model systems. In the present study we established three different cellular models of enzalutamide resistance including a cell line with wild type AR (LAPC4), DuCaP cells which overexpress wild-type AR, as well as a cell which has been adapted to long term androgen ablation (LNCaP Abl) and harbors the AR T878A mutation. After 10 months of cultivation, sustained growth in the presence of enzalutamide was achieved. When compared to controls, resistant cells exhibit significantly decreased sensitivity to enzalutamide as measured with 3[H]thymidine incorporation and WST assay. Moreover, these cell models exhibit partly re-activated AR signaling despite presence of enzalutamide. In addition, we show that enzalutamide resistant cells are insensitive to bicalutamide but retain considerable sensitivity to abiraterone. Mechanistically, enzalutamide resistance was accompanied by increased AR and AR-V7 mRNA and protein expression as well as AR gene amplification, while no additional AR mutations have been identified.

Keywords: AR gene amplification; AR-V7; androgen receptor; enzalutamide resistance; prostate cancer.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST

There are no conflicts to declare.

Figures

Figure 1
Figure 1. Generation of enzalutamide resistant cell lines
A. LAPC4 cells were cultured in RPMI + 1 nM DHT and treated with increasing concentrations of enzalutamide for 48 h. mRNA expression of AR target genes PSA and FKBP5 was analyzed using RT-qPCR. B. AR reporter gene assay after treatment with R1881 [1 nM] or enzalutamide [10 μM] or both was performed with PC-3 cells ectopically expressing AR, androgen responsive elements and a TATA-box in framework of nanoluc reporter vector was measured by Nano-Glo Dual-Luciferase Assay and normalized to control luciferase activity C. Treatment scheme for the generation of enzalutamide resistant cell lines. LAPC4, DuCaP and LNCaP abl were cultured in the presence of increasing doses of enzalutamide or vehicle (EtOH) for 10 months (approximately 50 passages) until the final concentrations (8 μM for LAPC4 and DuCaP, 13 μM for LNCaP Abl) were reached. Cell lines were further cultured for 2 months in the presence of the final enzalutamide concentrations.
Figure 2
Figure 2. Enzalutamide resistant cell lines exhibit decreased sensitivity to enzalutamide
A. Representative images of vehicle and EnzaR cell lines were taken after 12 months of culturing. 4x objective. Scalebar: 500μm. B, C. Determination of the degree of enzalutamide resistance. Proliferation (B) and viability (C) in DuCaP, LAPC4 and LNCaP Abl vehicle and enzalutamide resistant cell lines after 6 days of exposure to different concentrations of enzalutamide were measured by 3[H]thymidine incorporation assay and WST assay, respectively. Ethanol was used as control. Data represent mean +SEM from at least 3 independent experiments. *;p=<0.05. **;p=<0.01. ***;p=<0.001
Figure 3
Figure 3. Determination of possible cross resistances of enzalutamide resistant cell lines with abiraterone or bicalutamide
Proliferation A. and viability B. in DuCaP, LAPC4 and LNCaP Abl vehicle and enzalutamide resistant cell lines after 6 days of exposure to 8 μM abiraterone or 10 μM bicalutamide was measured by 3[H]thymidine incorporation assay and WST assay, respectively. Ethanol was used as control. Data represent mean +SEM from at least 3 independent experiments. *;p=<0.05. **;p=<0.01. ***;p=<0.001
Figure 4
Figure 4. Enzalutamide resistant cell lines exhibit increased AR expression
A. AR mRNA expression was assessed by qRT-PCR. Data represent mean +SEM from 4 independent experiments. *;p=<0.05. **;p=<0.01. ***;p=<0.001. B. Statistical analyses and representative Western blot images of full length AR protein expression. Data represent mean +SEM from 3 independent experiments. *;p=<0.05. **;p=<0.01. ***;p=<0.001. C. Western blot of LAPC4 Veh and LAPC4 EnzaR, as well as in LAPC4 vehicle cells which were treated for 2 weeks with enzalutamide [8 μM]. D. Upper panel: Statistical analysis of AR-V7 mRNA expression. Data represent mean +SEM from 4 independent experiments. *;p=<0.05. **;p=<0.01. ***;p=<0.001. Lower panel:Representative Western blot image of AR variant observed at 70 kd size (V7). First lane represents Marker band the 75 kDa size. Last lane represents VCaP lysate as positive control for V7 expression.
Figure 5
Figure 5. Immunofluorescence staining of vehicle or enzalutamide resistant LAPC4 cells
Cells were cultured in medium containing 10% charcoal stripped FBS (SF), supplemented with vehicle (EtOH), 1 nM R1881, or 8μM enzalutamide as indicated. AR was detected using mouse anti AR (Biogenex) and visualized using AlexaFluor 488 donkey anti mouse secondary antibody. Nuclei were counterstained with DAPI. Magnification: 40x. Scalebar: 50μm.
Figure 6
Figure 6. Enzalutamide resistant LAPC4 acquired AR gene amplification
A. AR gene amplification was determined by duplex qPCR of genomic DNA amplifying an AR Exon 1 fragment (Chr Xq12) in relation a POLG Exon 3 fragment (Chr 15q25). AR/POLG copy number ratios were calculated as fold change of normal male DNA which harbors 1 copy of AR. Data represent mean +SEM from 3 independent PCR analyses. *;p=<0.05 B. Analysis of AR gene amplification over time during generation of enzalutamide resistant LAPC4 cells starting from parental LAPC4. P = Passage.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Arora V, Wongvipat J, Smith-Jones PM, Yoo D, Kwon A, Wasielewska T, Welsbie D, Chen CD, et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science. 2009;324:787–790. - PMC - PubMed
    1. Menon MP, Higano CS. Enzalutamide a second generation androgen receptor antagonist: development and clinical applications in prostate cancer. Current oncology reports. 2013;15:69–75. - 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, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. The New England journal of medicine. 2012;367:1187–1197. - PubMed
    1. Beer TM, Tombal B. Enzalutamide in metastatic prostate cancer before chemotherapy. The New England journal of medicine. 2014;371:1755–1756. - PubMed
    1. Kim W, Ryan CJ. Androgen receptor directed therapies in castration-resistant metastatic prostate cancer. Current treatment options in oncology. 2012;13:189–200. - PubMed

MeSH terms