The authors have declared that no competing interests exist.
Conceived and designed the experiments: JC FC. Performed the experiments: FC IA EE. Analyzed the data: FC JC. Contributed reagents/materials/analysis tools: JEK JPB. Wrote the paper: FC JC.
Androgen receptor (AR) signaling pathway remains the foremost target of novel therapeutics for castration-resistant prostate cancer (CRPC). However, the expression of constitutively active AR variants lacking the carboxy-terminal region in CRPC may lead to therapy inefficacy. These AR variants are supposed to support PCa cell growth in an androgen-depleted environment, but their mode of action still remains unresolved. Moreover, recent studies indicate that constitutively active AR variants are expressed in primary prostate tumors and may contribute to tumor progression. The aim of this study was to investigate the impact of constitutively active AR variants on the expression of tumor progression markers. N-cadherin expression was analyzed in LNCaP cells overexpressing the wild type AR or a constitutively active AR variant by qRT-PCR, Western blot and immunofluorescence. We showed here for the first time that N-cadherin expression was increased in the presence of constitutively active AR variants. These results were confirmed in C4-2B cells overexpressing these AR variants. Although N-cadherin expression is often associated with a downregulation of E-cadherin, this phenomenon was not observed in our model. Nevertheless, in addition to the increased expression of N-cadherin, an upregulation of other mesenchymal markers expression such as
Prostate cancer (PCa) is the most common cancer in men over 50 years of age and the second cause of male mortality due to cancer in Europe. Androgens signaling plays a key role in PCa cells proliferation or survival
Genetic and splicing events affecting the androgen receptor (AR) gene have been linked to CRPC. Constitutively active AR variants, lacking the carboxy-terminal region that encompasses the ligand binding domain and the activation function 2, might contribute to the progression of PCa into castration resistance. These constitutively active AR variants result from premature stop codons due to nonsense mutations as reported for the ARQ640X
The role of constitutively active AR variants in CRPC has been shown in many studies
Recent studies suggest that constitutively active AR variants could play a role in tumor progression. Indeed, although these constitutively active AR variants are already expressed in primary prostate tumors, their expression is all the more expressed in bone metastasis
N-cadherin, which belongs to cadherin superfamily, is located at adherens junctions in nervous, endothelial or mesenchymal cells and is involved in tumor progression
More recently, studies have shown that castration-resistant PCa is associated with an upregulation of N-cadherin expression in cellular models as well as PCa xenografts and clinical samples of CRPC
The aim of this work was to show a possible link between the presence of constitutively active AR variants and the expression of tumor progression markers. More particularly, we focused on the impact of constitutively active AR variants on the expression of N-cadherin and other mesenchymal markers. In the present study, we have shown that
The human prostate carcinoma LNCaP cell line, clone FGC and the 22Rv1 cell line (ECACC, Salisbury, United Kingdom) was maintained in RPMI-1640 complete medium containing 10% fetal calf serum (FCS), 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (Sigma-Aldrich, France) and 1mM sodium pyruvate (Invitrogen, Fisher Scientific, France).
C4-2B cell line (ViroMed Laboratories, Minnetonka, MN, USA) was maintained in DMEM medium supplemented with 20% Ham's F12, 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 13.65 pg/mL triiodo-thyronine, 4.4 µg/mL apo-transferrin human, 0.244 µg/mL d-biotin and 12.5 µg/mL adenine (Sigma-Aldrich, France).
For immunofluorescence experiments, the wild type androgen receptor (AR) (AR-WT) and the constitutively active AR Q640X and AR Q670X
For transfections, the JetPEITM transfection reagent (Polyplus Transfection, Ozyme, France) was used according to the manufacturer's protocol. LNCaP cells were seeded in 10 cm dishes at 1×106 cells/dish or in 6-wells plate at 2×105/well. Three days later, the medium was changed and cells were transfected with 10 µg of the indicated plasmid using 20 µl of JetPEI transfection reagent for 10 cm dishes or with 3 µg of plasmid using 6 µl of JetPEI for 6-wells plate. Medium was changed 48 h after and cells were incubated up to 9 days according to the experiments. The medium was changed every two days and for incubations beyond 4 post-transfection days, cells were incubated in the presence of 400 µg/mL geneticin (Invitrogen, France).
LNCaP cells were seeded in 6-wells plate in complete medium and transfected as previously described. Twenty four hours later, medium was changed to phenol red free RPMI-1640 supplemented with 5% dextran-coated charcoal-stripped FCS (DCC-FCS) and with the indicated concentration of dihydrotestosterone (DHT) (Sigma-Aldrich, France) or vehicle (ethanol).
For experiment with MDV3100, transfected LNCaP cells were incubated in RPMI-1640 supplemented with 5% DCC-FCS containing the indicated DHT dose and 100 nM MDV3100 (Enzalutamide, Selleck Chemicals, Euromedex, France) or vehicle (dimethyl sulfoxide, DMSO). To confirm the effects of androgens on N-cadherin expression, 22Rv1 cells were grown in RPMI-1640 with 100 nM or 1 µM MDV3100, or DMSO.
LNCaP cells were seeded in 10cm dishes at 1×106 cells/dish and were transfected with pEGFP-ARWT or pEGFP-ARQ640X. Four days after transfection, cells were trypsinized and sorted thanks to the green fluorescence (EGFP) with a BD FACSAria-II cell sorter (BD Biosciences, Le Pont de Claix, France). Total RNA was extracted from EGFP negative (non-transfected) and EGFP positive (transfected) cells and was used to analyze gene expression by qRT-PCR.
Total cellular RNA was extracted from cell lines using NucleoSpin® RNA II assay (Macherey-Nagel, France) according to the manufacturer's procedure. RNA concentrations and purity were quantified measuring the absorbance at 260 nm and 280 nm (GeneQuant pro, GE Healthcare, France). The reverse transcription was performed from 400 ng or 1 µg RNA using RT Omniscript assay (Qiagen, Courtaboeuf, France). RNA were diluted into 13 µL and denatured at 65°C during 5 minutes. A 7 µL reaction mix containing 1×RT template, 0.5 mM of each dNTP, 1 µM oligo dT, 10U RNase inhibitor and 4U Omniscript Reverse Transcriptase was added and the reaction was incubated 1 h at 37°C. The reaction was stopped by heating to 93°C for 5 minutes.
Genbank | QuantiTect reference | Hybridization temperature (°C) | Amplicon length (bp) | Amplified exons |
QT01680476 | 55/60 | 104 | NA | |
QT00080143 | 55 | 84 | 5/6 | |
QT00063196 | 60 | 102 | 14/15 | |
QT00014462 | 55 | 107 | 7/8/9 | |
QT00011956 | 55 | 127 | 1/2 | |
QT00010010 | 60 | 131 | 2/3 | |
QT00095795 | 60 | 94 | 2/3 | |
QT01888446 | 58 | 105 | 2/3/4 |
Cells were lysed in buffer containing 10 mM Tris-HCl pH7, 140 mM NaCl, 3 mM MgCl2, 0.5×Igepal, 5 mM DTT, 1× phosphatase inhibitor, and 1× protease inhibitor. Protein concentration for each sample was quantified using BCA Protein Assay (Pierce Biotechnology, Inc., Rockford, IL, USA) according to the manufacturer's procedure. A quantity of 15 µg to 100 µg of total proteins was loaded on 7,5% SDS-PAGE. After migration and transfer to nitrocellulose membrane, membranes were saturated with PBS/0.1%Tween/2%ECL and incubated at 4°C overnight with 0.1 µg/mL mouse monoclonal anti N-cadherin (catalog no. 610920, BD Biosciences, France) or 1 µg/mL mouse monoclonal anti AR (catalog no. 554225, BD Biosciences, France) antibody. β-actin (0.2 µg/mL) (catalog no. sc-47778, Tebu-bio, France) was used as internal control. After washes, immunocomplexes were detected with 0.2 µg/mL HRP-conjugated goat anti mouse (catalog no. sc-2005, Tebu-bio, France), or 0.5 µg/mL rat anti mouse IgG2a secondary antibodies (catalog no. 553391, BD Biosciences, France), and finally revealed by chemiluminescence (ImmobilonTM Western, Millipore, Molsheim, France).
Lab-Tek II chamber slides (2 wells) were coated with LNCaP medium for two hours and 1×105 LNCaP cells/well were seeded. LNCaP cells were transfected with 2 µg of pEGFP-WT, pEGFP-ARQ640X or pEGFP-ARQ670X 3 days later and incubated for 4 days. LNCaP cells were rinsed in PBS and fixed with 2% paraformaldehyde. Cells were blocked and permeabilized by 0.1% Triton/1% Bovine Serum Albumin (BSA)/PBS for 30 min at room temperature. Cultures were incubated with 2.5 µg/mL anti N-cadherin mouse monoclonal antibody (catalog no. 610920, BD Biosciences, France) or isotypic antibody (Sigma-Aldrich, Saint-Quentin Fallavier, France) at 4°C overnight. After washing in PBS, LNCaP cells were incubated with 2 µg/mL Alexa Fluor 568-conjugated goat anti mouse (Invitrogen, Fisher Scientific, France) for 1 h and nuclei were stained with 0.1 µg/mL DAPI solution for 20 min at 30°C. Images were captured with the Leica LAS AF6000 fluorescence microscope using LAS AF software (Leica).
Constitutively active AR variants have been associated with CRPC. Moreover, some studies showed that CRPC is also associated with an upregulation of N-cadherin expression
A). N-cadherin expression was assessed by qRT-PCR in LNCaP cells overexpressing the constitutively active AR Q640X or AR-V7, or the AR-WT and in cells transfected with the empty plasmid (C3). Cells were grown in complete medium for 9 days after transfection. Parental LNCaP cells were used as control. y-Axis represents the relative fold change compared with control (parental LNCaP cells).
To confirm these data from transient transfection, a cell-sorting analysis was performed after LNCaP transfection to demonstrate that N-cadherin expression was restricted to cells expressing a constitutively active AR.
LNCaP cells were transiently transfected with pEGFP-ARWT or pEGFP-ARQ640X plasmid and were sorted 4 days after. A).
Taken together, these data strongly suggest that constitutively active AR variants upregulate N-cadherin expression in PCa cells.
A recent study reported that constitutively active AR variants might require a full-length AR (AR-FL) to activate endogenous target genes. To explore the effect of the endogenous AR-FL present in LNCaP cells on the ability of constitutively active AR variants to induce N-cadherin expression, LNCaP cells overexpressing AR-WT or a constitutively androgen variant were incubated in the presence of 100 nM DHT or vehicle, and N-cadherin expression was analyzed by qRT-PCR. In accordance with our previous results, no N-cadherin expression was observed in cells overexpressing AR-WT. Interestingly, a 1.4-fold decrease in N-cadherin expression level was observed when cells overexpressing AR Q640X or AR-V7 were cultured in the presence of 100 nM DHT compared to vehicle (
A). LNCaP cells were grown in RPMI-1640 containing 5% DCC-FCS and 100 nM of DHT or vehicle (EtOH). N-cadherin expression was analyzed by qRT-PCR in LNCaP cells 4 days after transfection with AR-WT or the constitutively active AR Q640X or AR-V7 expression plasmid. B). N-cadherin expression level in LNCaP cells was investigated by qRT-PCR 4 days after transfection with AR Q640X expression plasmid in the presence of different DHT concentrations (10 nM, 25 nM and 50 nM) or vehicle. C). N-cadherin expression induced by constitutively active AR variants (AR variants) was negatively regulated when LNCaP cells were grown in the presence of DHT. We hypothesize that endogenous AR-FL present in LNCaP cells and AR variants could act differently. In this model, DHT-stimulated endogenous AR-FL represses N-cadherin expression whereas AR variants upregulate its expression. D). LNCaP cells overexpressing AR-WT, AR Q640X and AR-V7 were cultured in DCC-FCS medium supplemented with 100 nM of DHT and in the presence of 100 nM of MDV3100 or DMSO as control during 3 days. N-cadherin expression was analyzed by qRT-PCR 4 days after transfection, and was normalized to
All together, these data suggest that DHT-activated AR-FL could compete with constitutively active androgen receptor for regulating N-cadherin expression.
It is widely known that in tumor cells, the expression of mesenchymal markers is associated with a down-regulation of epithelial markers. We hypothesized that the upregulation of N-cadherin expression observed in the presence of constitutively active AR variants is accompanied by a decreased expression of E-cadherin. To test this hypothesis, we analyzed E-cadherin expression in LNCaP transfected with ARQ640X, AR-V7 or the wild type AR expression plasmid, or the empty plasmid as control.
LNCaP cells were transfected with the ARWT, ARQ640X or AR-V7 expression plasmid or the empty plasmid (C3). A).
We also investigated whether constitutively active AR expression in PCa cells is associated with other mesenchymal markers. Expression levels of
Although Twist1 is known to induce
The AR signaling is very important for proliferation and survival of prostate cancer cells. The AR pathway remains activated during the progression of PCa towards a castration-resistant disease and the emergence of constitutively active AR variants lacking the ligand-binding domain is now considered as a major event in CRPC. In spite of some studies suggest that constitutively active AR variants have an impact on tumor progression, their function remains so far unresolved.
In this study, we have shown that N-cadherin is upregulated in LNCaP cells expressing constitutively active AR variants, but not in LNCaP cells overexpressing a full-length AR. These data suggest for the first time that constitutively active AR variants can induce N-cadherin expression. This finding should be connected to recent studies reporting a correlation between CRPC and N-cadherin upregulation
These findings again highlight the link between AR signaling pathway and N-cadherin expression. Recent studies suggest that AR negatively regulates N-cadherin expression
However, constitutively active AR variants could also indirectly control
These hypotheses deserve to be studied in further studies to understand how constitutively active AR variants regulate
In this study, we have also shown that constitutively active AR variants were associated with an increased expression of mesenchymal markers as
The expression of mesenchymal markers reported here in the presence of constitutively active AR variants was not associated with a downregulation of E-cadherin in our model. The inverse correlation between N-cadherin upregulation and E-cadherin downregulation is still debated. Indeed, McKeithen and colleagues, and more recently Tiwari and colleagues report a co-expression of both E- and N-cadherins in tumor cells
Further studies are warranted to understand functional consequences of N-cadherin and other mesenchymal markers upregulation in the presence of constitutively active AR variants. N-cadherin expression is widely associated with tumor progression notably owing to its role in migration and invasion. Indeed, N-cadherin favors the migration of cancer cells via cytoskeleton reorganization and lamellipodia formation
There is presently great interest in the mode of action of constitutively active AR variants in CRPC. In this study, we have shown for the first time that constitutively active AR variants induce N-cadherin expression and other mesenchymal markers in PCa. These findings support the hypothesis that these constitutively active AR variants could contribute to systemic dissemination of PCa cells, and reinforce the importance to target these AR variants in PCa.
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We would like to thank Claudine Ebel (IGBMC, Illkirch, France) for her technical assistance for cell sorting, and Pr. Etienne Weiss (ESBS, Illkirch, France) for cell imaging.