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Génétique des Eucaryotes et Endocrinologie Moléculaire, UMR 6547 CNRS, Equipe Physiologie Comparée et Endocrinologie Moléculaire, Université Blaise Pascal, Campus Universitaire des Cézeaux, 24 Avenue des Landais, 63177 Aubière Cedex, France
1 UMR 8161 Institut de Biologie de Lille/CNRS/Université de Lille 1/Université Lille2/Institut Pasteur de Lille, 1 rue Calmette, BP 447, 59021 Lille Cedex, France
2 Laboratoire d’Immunologie, Centre Jean Perrin, 58 rue Montalembert, BP 392, 63011 Clermont-Ferrand, France
(Requests for offprints should be addressed to L Morel; Email: laurent.morel{at}univ-bpclermont.fr)
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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During early foetal life, AR is present only in mesenchymal cells of the urogenital tract suggesting that androgens may regulate epithelial morphogenesis and growth via mesenchymal paracrine-acting factors (Cunha et al. 1992). This idea is strengthened by ontogenic studies, which have demonstrated that androgen-responsive mesenchyme in the prostate can elicit a tissue-specific morphologic development of the epithelium, assessed to be negative for AR expression (Cunha 1996). Altogether, these findings suggest that androgen-induced epithelial cell proliferation in the male reproductive tract could be regulated at least by an indirect pathway involving paracrine mediators produced by stromal cells, such as insulin-like growth factor, fibroblast growth factor (FGF) and epidermal growth factor (EGF; Cunha & Donjacour 1989, Byrne et al. 1996).
Nevertheless, in the developing prostate as well as in the Wolffian-derived sex accessory organs (vas deferens, epididymis and seminal vesicles), epithelium growth and terminal differentiation give raise to a mature non-dividing cell population that expresses AR. These epithelial cells are highly dependent on androgenic content for the maintenance of their differentiated functions and survival. Several studies have contributed to the elucidation of the mechanisms of AR actions in these different processes. These mechanisms include crosstalk between the AR-signalling pathway and growth factor receptor pathways: EGF activation of the AR promoter (Culig et al. 1994), androgen-mediated EGFR up-regulation (Myers et al. 1999, Torring et al. 2003), activation of mitogen-activated protein kinase (MAPK) by androgen-activated AR (Peterziel et al. 1999), enhancement of AR transcriptional activity by peptide growth factors (Orio et al. 2002) and AR interactions with the MAPK/extracellular signalling-regulated kinase kinase kinase-1 (MEKK1) and the epidermal growth factor-1 receptor (EGFR; Abreu-Martin et al. 1999, Bonaccorsi et al. 2004b). These findings suggest that the interaction between these two pathways may be crucial for the acquisition and the maintenance of androgen sensitivity.
However, most of these studies were conducted in prostate tumour cells so that both the AR contribution to a normal differentiation process and the crosstalk between androgens and EGF-signalling pathways in differentiated cells still remain unclear.
In previous studies, we showed in non-transformed vas deferens epithelial cells that endogenous AR expression as well as AR transcriptional activity were regulated by insulin and EGF through an activation of the PI3K transduction pathway (Manin et al. 1992, 2000, 2002). We now report that androgens do not play a major role in the cell cycle arrest associated with cell differentiation, but that the acquisition of androgen-dependent activities is a consequence of this process. In these conditions, EGF-activated MAPK signalling cascade interferes with AR functions to down-regulate androgen responsiveness. Up-regulation of AR following the onset of differentiation correlates with inhibition of ERK1/2 activity and, in differentiated cells cultured to a non-saturating density, exposure to EGF quickly restores pERK1/2 levels while AR is partially lost. Moreover, the blockade of ERK restores AR functions in these cells and abolishes EGF-mediated inhibition of AR-dependent activities. Finally, our data show that when active in differentiated cells, androgen signalling is able to reduce phospho-ERK1/2 independently of EGFR phosphorylation, thus pointing to a disconnection between the EGFR and the MAPK cascade in response to androgens.
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Materials and methods |
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Life Technologies (Cergy Pontoise, France) supplied the Dulbecco’s modified Eagle (DMEM)/Ham’s F12 medium, glutamine, HEPES, transferrin, PBS and gentamicin. Bovine insulin, EGF, cholera toxin, dibutyryl cAMP, selenium, hydrocortisone, dihydrotestosterone (DHT) and matrigel were from Sigma–Aldrich. Microporous PET membranes were from Becton Dickinson Labware (Ozyme, Saint-Quentin en Yvelines, France). Rabbit polyclonal anti-AR (PG-21) antibody, mouse monoclonal anti-phospho-MAP kinase (12D4), anti-phosphotyrosine PY20 (clone 4G10) and anti-phospho-EGFR (Tyr1173, clone 9H2 and Tyr1068, clone 1H12) antibodies were purchased from Upstate Biotechnology (Euromedex, Mundolsheim, France), rabbit polyclonal anti-EGFR (Ab-17) antibody from Lab Vision Corporation (Interchim, Montlu
on, France), mouse monoclonal anti-Rb antibody (G3-245) from PharMingen (Ozyme, Saint-Quentin en Yvelines, France), sheep polyclonal anti-p53 antibody (Ab-7) from Oncogene Research (Merck Eurolab), rabbit polyclonal anti-p27Kip1 (C-19) and anti-p21Cip1 (C-19) antibodies from Santa Cruz Biotechnology (Tebubio, Le Perray en Yvelines, France) and rabbit polyclonal anti-MAP kinase (M5670) and anti-ß-actin (C-11) antibodies were from Sigma–Aldrich. Rabbit polyclonal anti-aldo-keto reductase 1B7 (AKR1B7) antibody (IL-3) has been described elsewhere (Lefrancois-Martinez et al. 2004).
Cell culture conditions
Mouse vas deferens epithelial cells (VDEC) were grown as previously described (Baron et al. 2004) but with the following minor modifications. Briefly, the medium is made of a basal mixture of DMEM/Ham F12 (1/1 v/v) containing transferrin (10 g/ml), cholera toxin (10 ng/ml), selenium (17.3 ng/ml), cAMP (1.5 g/ml) glutamine (2 mM), ethanolamine (0.6 g/ml), insulin (5 g/ml), HEPES (20 mM) and gentamicin (50 g/ml) supplemented or not with EGF 1 ng/ml. Cells maintained in proliferation were seeded on serum fibronectin-coated plastic in medium supplemented with 1 ng/ml EGF. Differentiation of VDEC was allowed for 3 days after seeding onto matrigel-coated microporous membranes in six-well plates at confluent density (1.5 x 106 cells/well) in basal medium supplemented (high cell density culture) or not (low cell density culture) with EGF and/or DHT. As the cell monolayer response to hormonal treatments is heterogeneous (Manin et al. 1992), DHT was mostly used at a concentration of 100 nM, which gives an optimal proportion of responding cells in short-term induction experiments without any harmful effect on the cell monolayer. The medium was changed every 2 days and experiments were repeated thrice.
Western blot analysis
Cells were harvested, washed in cold ice PBS buffer, lysed in NaCl buffer (0.42 M NaCl, 20 mM HEPES, 1.5 M MgCl2, 0.2 M EDTA, 25% glycerol, 0.1% NP-40, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 µg/ml apoprotinin, 1 µg/ml leupeptin), sonicated and centrifuged at 15 000 g for 30 min at 4 °C. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad). Total proteins (40 µg) were resolved through SDS-polyacrylamide gels and transferred to nitrocellulose (Hybond-C extra, Amersham Biosciences). Membranes were probed with antibodies specific for the indicated proteins and subsequently with horseradish peroxidase-conjugated donkey anti-rabbit (PARIS Biotech, Compiègne, France), sheep anti-mouse (Amersham Biosciences) or rabbit anti-sheep (Upstate Biotechnology) immunoglobulin G secondary antibodies, followed by enhanced chemiluminescence according to manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Northern blot analyses and real-time PCR
Total RNA was isolated from VDEC cells using the Trizol method according to the manufacturer’s instructions (Invitrogen). For northern blot analyses, total cellular RNA (30 µg) was electrophoresed on 1% denaturing formaldehyde–agarose gels and transferred onto positively charged nylon membranes. Filters were hybridised with [32P]dCTP-labelled probes (akr1b7, AR, GAPDH and 18S). For real-time PCR, cDNA was synthesised with Moloney-Murine Leukemia Virus Reverse Transcriptase (Promega) and random hexamer primers (Promega) according to the manufacturer’s recommendations. The real-time PCR was performed using an iCycler (Bio-Rad). Four microlitres of 1 out of 50 diluted cDNA templates were amplified by 0.75 U of HotMaster Taq DNA polymerase (Eppendorf, Brumath, France) using SYBR Green dye to measure duplex DNA formation. Sequence primers used for QPCR are the following: cyclophilin forward, 5'-GGA GAT GGC ACA GGA GGA A-3'; cyclophilin reverse, 5'-GCC CGT AGT GCT TCA GCT T-3'; mAR forward, 5'-CCA CTG AGG ACC CAT CCC AGA A-3'; mAR reverse, 5'-CGG CAC ACA CCA CTC CTG GCT C-3'; mAKR1B7 forward, 5'-CCC TCA CGC ATA CAG GAG AA-3'; mAKR1B7 reverse, 5 '-GCC ATG TCC TCC TCA CTC AA-3'.
SiRNA transfections
Cells were seeded onto microporous membrane to initiate a differentiation process, then transfected 2 h later with 20 nM anti-AR siRNA duplexes (5'-GACT-CAGCTGCCCCATC CA-3') using Metafectene (Biontex, Martinsried/Planegg, Germany). As a control, we used a duplex of 19 nt and two-dT overhang against the Green Fluorescent Protein at 20 nM (5'-ACT ACC AGC AGA ACA CCC CTT-3'). Cells were grown to a differentiated state for 70 h then treated or not with DHT 100 nM and collected for western blot analysis.
Cell cycle arrests and fluorescence-activated cell sorting analysis
VDEC were seeded in six-well plates under conditions allowing cells to proliferate or differentiate and cell cycle progression was measured in the presence or the absence of DHT. At the desired time points, cells were trypsinised for 10 min, washed with PBS and cell samples (107 cells/ml) were suspended in 50 µg/ml propidium iodide (PI; Sigma–Aldrich) and 500 µg/ml ribonuclease A (Sigma–Aldrich). The cells were stained for 30 min at 4 °C in the dark and then filtered through a 40 µm nylon mesh just before analysis. The flow cytometric analysis of cell DNA content was performed using an Epics XL (Coulter, Hialeah, FL, USA). Fluorescence attributable to PI was determined using excitation by an argon laser, operating at 488 nm and at a power output of 15 mW. A minimum of 15 000 events was acquired in list mode for each sample. For each DNA histogram, the cell cycle distribution was calculated using the Multicycle Software program (Phoenix, Flow Systems, San Diego, CA, USA).
Immunoprecipitation
VDEC were lysed in IP buffer (150 mM NaCl, 0.2% triton, NP40 0.25%, 20 mM Tris–HCl pH 7.4, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4). Cell lysates were centrifuged and 500 µg of total proteins were subjected to immunoprecipitation using rabbit polyclonal anti-EGFR (Ab-17, Interchim) or purified IgG as control overnight at 4 °C. The immune complexes were captured using Protein A sepharose (Amersham-Pharmacia Biotech) during 3 h at 4 °C. After four washes with the IP buffer, the immune complexes were eluted in Laemmli buffer and analysed by western blotting with appropriate antibodies as previously described.
Statistical analysis
Statistical comparisons were performed by a one-way ANOVA followed by post hoc pairwise comparisons with a Fisher’s probability of at least significant difference (PLSD) test. Values of P < 0.01 were considered significant and are presented in the results section.
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Results |
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Androgen-sensitive mouse VDEC are able to grow, polarise and then express a specific androgen-dependent marker (AKR1B7/MVDP) when seeded at confluency onto matrigel-coated microporous membrane (Manin et al. 1992, 2000). Morphological changes associated with cell polarisation are observable under inverse microscope. In the present study, we analysed changes in the cell cycle status during this differentiation process. As shown in Fig. 1a
, cell differentiation was associated with an increasing number of cells blocked in the G0/G1 phase as assessed by flow cytometric analysis. Differentiated cells were also characterised by a 90, 70 and 60% decrease in hyper-phosphorylated retinoblastoma (ppRb), p21Cip1 and p53 proteins respectively. This decrease was associated with a parallel increase in the accumulation of the p27Kip1 protein (Fig. 1b), thus assessing cell cycle arrest at the molecular level. Interestingly, the presence of DHT in the culture medium neither significantly changed the expression levels nor the phosphorylation status of the cell cycle regulators. This was also true during the time course of differentiation (not shown). This suggests that androgens do not play an important role in growth arrest and are dispensable for promoting cell differentiation.
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, a marked decrease leading to a complete inhibition of ERK1 basal phosphorylation, and to a lesser extend of ERK2, was observed when VDEC are differentiated. Interestingly, this was correlated to a strong increase in AR expression that mainly resulted from an increase in AR mRNA levels (Fig. 1c). In accordance with the previous observation, this was independent regardless of the presence of androgens. All these data then suggest that the down-regulation of the MAPK pathway could directly or indirectly allow the AR expression. AR expression is dependent on the establishment of tight junctions
The proliferation rate of non-transformed cells is known to be controlled by the cell density and the establishment of cell–cell contacts. This mechanism, referred to as contact inhibition, could thus be considered as a key regulator of the MAPK pathway and the subsequent AR expression. Knowing that cell density determines the establishment of tight junctions between adjacent polarised cells (Day et al. 1999) and that E-cadherin-mediated adhesion can inhibit ligand-dependent activation of the EGF receptor and the subsequent EGF signalling (Takahashi & Suzuki 1996, St Croix et al. 1998, Qian et al. 2004), VDEC were grown to differentiation in the absence or presence of an anti-E-cadherin antibody to test whether this signalling could interfere with the AR expression. As expected, the blockade of E-cadherin led to an important and significant increase in the phosphorylation level of ERK1 and to a lesser extent to that of pERK2 (Fig. 2a and b). Interestingly, this gain in ERK activity was correlated to a complete decrease in AR accumulation thus assessing a functional link between the MAPK activity and the AR expression. Similar data were obtained by incubating differentiated cells for 5 h with EGTA 2 mM (not shown), which is known to alter the dynamics of tight junctions (Rothen-Rutishauser et al. 2002).
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As AR expression is correlated to a decrease in the MAPK activity, we then tested the ability of EGF to reverse both the AR signalling and the cell cycle status. For this, VDEC were cultured in a medium devoid of EGF at a seeding density sufficient for the assembly of tight junctions and cell polarisation (low-density cell cultures). In these conditions, they remain sensitive to the mitogenic effects of EGF, while cells seeding at a same density but cultured in the presence of EGF go on proliferation until they reach a saturated density that arrest growth and polarisation. These latest cultures (high-density cell cultures) are not able to proliferate again in response to an additional EGF stimulation (not shown). Thus, 24 h stimulation with EGF of low-density differentiated cells induces the whole differentiated monolayer to proliferate again as attested by their morphological features observable under inverse microscope (Fig. 3a). Re-entry into the cell cycle was evaluated by FACS analysis of the cell population. Their proportion in S phase rose significantly from 12 to 23% (P < 0.01), while it decreased from 70 to 55% in the G1/G0 phase (Fig. 3b). Consistent with these observations, EGF treatment totally restored ppRb expression, increased both p21Cip1 and p53 protein levels and decreased p27Kip1 (Fig. 3c).
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). Effect of EGF was then evaluated at the mRNA level. As usually observed and inversely to AR protein, AR mRNA levels decreased in the presence of DHT. This hormone is known to exert its effect mainly at the post-transcriptional level through the mobilisation of AR mRNA to polyribosomes, the increase in translation efficiency and the stabilisation of AR protein (Burnstein et al. 1995). When cells were stimulated with EGF, both basal and DHT-induced AR mRNA levels dramatically decreased suggesting that this growth factor may act by inhibiting AR gene transcription and/or by increasing AR mRNA degradation (Fig. 3e). As a consequence, the EGF-treated differentiated cells did not express anymore Akr1b7, a well-characterised, androgen-regulated gene in the mouse vas deferens.
Since ERK1/2 activation is regulated by the upstream kinase MEK, we next examined the effect of PD098059, a pharmacological inhibitor of MEK1, on AR accumulation. Blockade of MAPK activity with 20 µM PD098059 both decreased the phosphorylation levels of ERK1/2 proteins and enhanced the basal and the DHT-induced AR expression (Fig. 3f).
These data clearly indicate that AR expression is negatively correlated to the EGF-induced activity of the MAPK pathway and cell cycle activation. They also suggest that the EGF-signalling pathway interferes with yet undefined events that trigger AR accumulation as well as androgen-dependent transcriptional activity in differentiated cells.
Androgens down-regulate ERK activity in differentiated cells
Cells present a full active androgen signalling capacity when they have completed their differentiation process, as attested by the high AR expression. Interestingly, EGF-induced pERK1/2 levels were reproducibly lowered in differentiated cells in presence of DHT (Fig. 4a and b, lane 4 vs 3), whereas no significant effect of androgens could be observed on the basal ERK phosphorylation in these cells as it is already very weak (Fig. 4a and b, lane 2 vs 1).
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, AR expression increased in a dose-dependent manner. Conversely, phosphorylation levels of ERK1/2 decreased dramatically in response to DHT from 10 nM concentration. It is likely that androgens could act as a supplemental down-regulator of the EGF-signalling pathway in differentiated cells to the one that is dependent on the differentiation process itself. The androgen-dependent control of ERK phosphorylation does not involve EGF-R
To test this hypothesis, we next determined the EGF receptor phosphorylation status on tyrosine residues 1068 (not shown) and 1173 (Fig. 5), whose phosphorylation is mainly linked to the activation and to the inhibition of the MAPK pathway respectively (Batzer et al. 1994, Sturla et al. 2005). To do so, we used low-density cells grown to differentiation in presence of androgens. As expected, ERK1/2 phosphorylation levels remained low in response to DHT, while phosphorylation levels of both Tyr1068 and Tyr1173 on EGFR were greatly increased when compared with untreated cells (Fig. 5a, lane 2 vs 1). In the absence of DHT, cells stimulated for 5 min with 10 ng/ml EGF exhibited increased phosphorylation of both EGFR and ERK1/2 (Fig. 5a, lane 3). However, addition of DHT to such EGF-treated cells led to a high steady-state phosphorylation level of EGFR on both residues but to a reduced phosphorylation level of ERK1/2 (Fig. 5a, lane 4 vs 3). Such a discrepancy could not be explained by the Tyr1173-dependent inhibition of ERK1/2 as the activating Tyr1068 was also phosphorylated. It is likely that phospho-EGFR increased levels and phospho-ERK1/2 down-regulated levels in response to DHT are two independent events, suggesting that EGFR and ERK pathway might be disconnected depending on the hormone or the growth factor treatment.
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, lane 4 vs 2). To assess this result, AR expression was knocked down using specific siRNA. As shown in Fig. 5c, siRNA-dependent decrease in AR expression was associated with a parallel decrease in EGFR phosphorylation that confirms that AR is involved in androgen-dependent phosphorylation of EGFR. To test whether or not the two proteins could physically interact as proposed by Bonaccorsi et al. (2004b), we performed co-immuno-precipitation experiments but did not detect the partner protein using either AR antibody or EGFR antibody (not shown).
Since there are almost 20 other known phosphorylation sites on EGFR, the effect of DHT on the global phosphorylation status of EGFR was further analysed from immunoprecipitated EGFR using a total anti-phosphotyrosine antibody (PY20–clone 4G10; Fig. 5d and e). As expected, the global phosphorylation level of EGFR was found to be highly increased in response to EGF stimulation. In accordance with what was observed using the anti-phospho-Tyr1173 antibody (Fig. 5a), DHT alone was able to slightly increase the global phosphorylation of EGFR but did not modulate the phosphorylation status of EGFR obtained in response to EGF.
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Discussion |
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In the prostate tumour LNCaP (lymph node cancer prostate) cells, androgens were shown to repress proliferation via the induction of p27kip1, which in turn controls the cell cycle arrest by inhibiting the Cdk2 activity (Kokontis et al. 1998). On the contrary, our data show that androgens do not significantly influence the cell cycle arrest in VDEC, since they could not regulate the expression of cell cycle regulators nor favour cells to exit the cell cycle. Unexpectedly, the amount of the p21Cip1 and p53 proteins decreased when VDEC were induced to differentiate and a treatment with the proteasome inhibitor MG-132 was able to restore both protein levels (not shown). Thus, p21Cip1 expression in differentiated cells is down-regulated by a specific proteasome-mediated pathway as already documented in a number of cell lineages (Maki et al. 1996, Maki & Howley 1997, Cayrol & Ducommun 1998, Rousseau et al. 1999, Sheaff et al. 2000, Fukuchi et al. 2002). The protein p21Cip1 was originally known as a mediator of p53-induced growth arrest; however, there is increasing evidence that, depending on the tissue, this protein is rather involved in cell proliferation, as has been shown in keratinocytes (Devgan et al. 2006) and in intestinal epithelial cells using p27kip1 and p21Cip1 conditional knockout mice (Stehr et al. 2005). The association of ß-catenin within cell–cell junctions in differentiated cells ongoing conformational changes (Manin et al. 2000) may explain the reduction of functionally competent p53 through an ubiquitin-mediated proteolysis, since ß-catenin can no longer interact with p53 and interfere with the normal proteasomal degradation (Damalas et al. 1999). Of interest, the E-cadherin/ ß-catenin axis is also known to be a major regulatory pathway involved in the contact-dependent inhibition of cell proliferation (Motti et al. 2005) through the stimulation of p27kip1 expression and the subsequent inhibition of Cdk2 activity (St Croix et al. 1998). As the down-regulation in the binding capacity of several growth factors, such as EGF, platelet-derived growth factor or FGF, were described as a function of cell density associated with cell–cell junction formation (Rizzino et al. 1988, Takahashi & Suzuki 1996, Qian et al. 2004) or with the activation of protein tyrosine phosphatases (Mansbridge et al. 1992, Sorby & Ostman 1996), it was of particular interest to investigate the EGFR–MAPK pathway status. In that way, we have shown that the disruption of cell–cell junctions by incubating cells either with anti-E-cadherin antibodies or with EGTA, is associated with a fall in AR expression and a parallel increase in the MAPK pathway activity. Moreover, when cells were seeded in medium devoid of EGF but at a density which allows intercellular junctions and cell polarisation, they were able to re-enter the cell cycle after an EGF stimulation, thus leading to a sustained ERK phosphorylation stimulation of MAPK and a loss in AR expression. The EGF-mediated AR down-regulation in VDEC is linked to a reduction of AR mRNA accumulation suggesting a regulation at transcriptional or post-transcriptional level that is consistent with reported studies on tumoural cell sublines (Mizokami et al. 1992, Hakariya et al. 2006). Contrarily to what was observed in LNCaP cells by Hakarya et al., EGF-induced AR signalling repression is abolished in the presence of a MAPK inhibitor such as PD98059 in VDEC, suggesting the existence of cell type differences in the crosstalk between intracellular signalling pathways, which could be altered in tumoural cells. These data clearly indicate a close inverse correlation between MAPK activity and AR expression, and we therefore hypothesise that EGFR signalling may play a crucial role as a negative regulator of AR expression during epithelial cell proliferation.
This interplay between AR and EGFR is bidirectional since exposure to DHT was able to down-regulate ERK activity in androgen-responsive differentiated cells similarly to that observed in other cell lines such as granulosa cells (Kayampilly & Menon 2004) or osteo-blasts (Wiren et al. 2004). We thus hypothesised that androgens negatively interact with EGFR signalling as already proposed (Gravina et al. 2004); however, in differentiated VDEC, the androgen-dependent decrease in ERK phosphorylation was firstly found associated with an increase in EGFR phosphorylation when evaluated using an anti-pTyr1173 and pTyr1068 EGFR antibodies. This DHT-induced phosphorylation of EGFR was abolished by inhibiting AR signalling using a specific AR antagonist (bicalutamide) or specific antiAR siRNA suggesting its dependence on an active AR. Further analyses using immunoprecipitation with an anti-EGFR antibody followed by total phosphotyrosine detection, revealed that androgens are able to increase EGFR global phosphorylation status although the great variations in efficiency of EGFR immunoprecipitation in the presence of androgens made the differences not statistically significant. We cannot exclude that the differences observed between results obtained using anti-phospho-Tyr1068 and Tyr1173 EGFR and anti-PY following EGFR immunoprecipitation may result from cross-reaction of anti phospho-EGFR antibodies with other related members of the ErbB family members. However, our data show that androgen can inhibit the EGFR–MAPK cascade in differentiated cells to maintain the differentiated state by a mechanism which remains to be determined. Indeed, if we could show that EGFR phosphorylation is dependent on AR action, ERK phosphorylation was found unaffected by anti-androgen or siRNA treatments suggesting other mechanism of androgen action. Androgens may inhibit the MAPK pathway either through the crosstalk with other intracellular signalling pathways or through the activation of phosphatases such as SHP-1, already described to decrease the EGFR–MAPK signalling (Keilhack et al. 1998) or through the involvement of novel membrane ARs. Indeed, it has been recently demonstrated in glial cells, that MAPK and Akt pathways are differentially modulated by androgens whether they act through the nuclear or a novel membrane AR (Gatson et al. 2006). Interestingly, uncoupling EGFR signalling from the ras–MAPK pathway has been demonstrated to be exerted by CEACAMI, a protein known as a tumour suppressor and which is involved in cell adhesion and cell differentiation (Abou-Rjaily et al. 2004). Moreover, the CEACAM1 protein has been shown to be expressed in the epithelia of the genital tract and regulated by androgens (Phan et al. 2001). There is evidence indicating that deregulation of the EGFR–MAPK pathway plays a critical role in cancer progression from an androgen-dependent to an androgen-independent state in patients who undergo hormonal therapy (Gioeli et al. 1999, Bonaccorsi et al. 2004a) but that the presence of a functional AR in prostate cancer cells induces the down-regulation of the EGFR–MAPK cascade (Gravina et al. 2004) and reduces malignant and invasion potential (Bonaccorsi et al. 2000, 2006). The above-mentioned data support the idea that abrogation of androgen action by anti-androgen therapy may contribute to the conversion of androgen-sensitive tumours to a hormone refractory state and increased proliferation by growth factors. Our data suggest that androgens do not play a significant role in the growth arrest associated with the differentiation process in epithelial cells, but rather act as essential supports in maintaining differentiated functions and, as recently proposed by Algarte-Genin et al.(2004), a restored androgenic status might prevent the initiation of PCa in elderly men.
It has also been shown that androgen-induced disruption of EGFR signalling can occur through direct interaction between AR and EGFR (Bonaccorsi et al. 2004b). In our immunoprecipitation studies, we did not found any direct interaction between AR and EGFR, suggesting that it may not occur in differentiated cells or may be specific to tumoural cells or that only over-expression of AR allows this interaction. The mechanisms by which androgens can disrupt the EGFR signalling from the MAPK pathway appear mediated by complex crosstalks and need further studies to be elucidated.
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Acknowledgements |
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Received in final form 29 May 2007
Accepted 12 June 2007
Made available online as an Accepted Preprint 14 June 2007
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