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Androgens and the androgen receptor (AR) are involved both in early tumorigenesis of prostate cancer (PCa) and in androgen-refractory disease. The role of AR signalling has also been highlighted by the fusion gene TMPRSS2:ERG recently identified in the majority of PCa. Several data indicate that re-expression of AR in PCa cell lines confers a less aggressive phenotype. We observed that re-expression of AR in the AR-negative cells PC3 decreases anchorage-independent growth and Matrigel invasiveness of PC3-AR cells where plasma membrane interaction between AR and EGFR led to an interference with downstream signalling and internalization of activated EGFR. Our data evidenced a shift of EGFR internalization pathway from the clathrin-coated pit one mediating signalling and recycling of EGFR to the lipid raft-mediated one mainly involved in lysosomal degradation of EGFR. These effects involved an altered recruitment to EGFR of the adaptor proteins Grb2 and c-Cbl followed by a reduced ubiquitination of EGFR. Our preliminary results suggest that in PC3-AR cells a pool of classical AR is located within cholesterol-rich membrane microdomains (namely as lipid rafts) and a population of EGFR is within cholesterol-rich membrane microdomains too. However, AR and EGFR membrane interaction that is increased by rapid androgen signalling is not within cholesterol-rich membrane microdomains. Our data enlighten that the crosstalk between genotropic and non-genotropic AR signalling interferes with signalling of EGFR in response to ligand leading to a lower invasive phenotype of AR-positive PCa cells.

Although androgens and the androgen receptor (AR) are involved in tumorigenesis of prostate cancer (PC) in initial phases, less clear is the role played in advanced androgen-independent (AI) stages of the disease. Several recent reports indicated that re-expression of AR in PC-derived cell lines determines a less aggressive phenotype of the cells. We have previously demonstrated that re-expression of AR decreases the invasion ability of PC3 cells in vitro by affecting signalling and internalization processes of epidermal growth factor receptor (EGFR). Here, we show that reduced EGFR internalization is also a characteristic of AR positive PC cell lines LNCaP and 22Rv1. Reduced internalization in PC3-AR cells is associated to a defective interaction between the EGFR and two adaptor proteins which mediate the endocytotic process, Grb2 and c-Cbl. As a consequence of such reduced interaction, ubiquitination of the receptor, which is mainly mediated by c-Cbl, is also altered. In addition, we show that internalized EGFR co-localizes with early endosome antigen-1, a marker of clathrin-mediated endocytosis, in PC3-Neo cells but not in AR positive cell lines. Conversely, EGFR maintains co-localization with caveolin-1 after EGF stimulation in PC3-AR cells. These data suggest that expression of AR affects clathrin-mediated endocytosis pathway of EGFR, which, according to recent findings, plays an essential role in the completeness of signalling of the receptor. Taken together, these data emphasize the role of AR in the regulation of EGFR endocytotic trafficking and active signalling in PC cells. In view of the role of EGFR signalling in invasion of carcinoma cells, our data may explain the lower invasive phenotype observed in AR-positive cell lines.

Suppression of invasive phenotype is essential in developing new therapeutic tools to treat prostate cancer (PC). Evidence indicates that androgen-dependent (AD) prostate cancer cells are characterized by a lower malignant phenotype. We have demonstrated that transfection with an androgen receptor (AR) expression vector of the androgen-independent (AI) prostate cancer cell line PC3 decreases invasion of these cells through modulation of alpha6beta4 integrin expression, indicating a genotropic effect of androgens in inhibiting invasion ability of AD PC cells. Later on, we have shown that also a non-genotropic mechanism is involved in such an effect. By using immunoconfocal fluorescent microscopy, we demonstrated that AR in PC3-AR cells co-localizes with the EGFR receptors (EGFR) in PC3-AR cells. Co-immunoprecipitation studies both in PC3-AR cells and in the AD cell line LNCaP that physiologically express both receptors, confirm the occurrence of an interaction between of the two proteins. In PC3-AR cells, we demonstrated a disruption of EGFR signalling properties (reduced EGF-induced EGFR autotransphosphorylation, reduced EGF-stimulated PI3K activity as well as EGFR-PI3K interaction) contributing to the lower invasive phenotype of these cells. In another study, we investigated the effects of a new Vitamin D analogue, BXL628, on invasion in response to KGF in the androgen-independent PC cell line DU145. We found that the compound was able to reduce proliferation and invasion of the cells in response to the growth factor. In addition, we found that KGF-induced autotransphosphorylation of KGF receptor (KGFR) and PI3K activation were suppressed after short-term (5min) pre-treatment with the analogue before addition of KGF. Collectively, these studies demonstrate that a non-genotropic effect due to a direct interaction of the androgen receptor with EGFR and to a rapid effect of a Vitamin D agonist on KGFR may disrupt signalling of GF leading to decreased tumorigenicity and a less malignant phenotype of PC cells in vitro.

Recent evidence indicates that androgen-sensitive prostate cancer cells are characterized by a less pronounced malignant phenotype. We demonstrate that transfection with an androgen receptor (AR) expression vector of the androgen-independent (AI) prostate cancer cell line PC3 decreases invasion and adhesion of these cells through modulation of alpha6beta4 integrin expression. Treatment of PC3-AR cells with the synthetic androgen R1881 further reduced invasion without modifying alpha6beta4 expression on the cell surface, suggesting interference with the invasion process in response to EGF by an alternative mechanism. We investigated EGF-induced auto-transphosphorylation of EGFR in both cell lines. We found that EGFR auto-transphosphorylation was reduced in PC3-AR cells and was further decreased by administration of androgens. Since auto-transphosphorylation regulates many different functions of EGFR, including docking of kinases, ubiquitination and internalization, we next investigated all these processes in PC3-AR cells. EGF-stimulated PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881. Interestingly, EGFR-PI3K interaction was also disrupted in these cells. Furthermore, EGFR ubiquitination and internalization were found to be reduced in PC3-AR cells both in basal conditions and following treatment with androgens. According to recent findings, an endocytotic pathway may be important for EGFR signalling by controlling the specificity of the response. By using immunoconfocal fluorescent microscopy, we demonstrated that AR in PC3 cells is mainly located in cytoplasm and transmigrates in part to the nucleus following stimulation with androgens. Interestingly, immunoconfocal and immunoprecipitation experiments demonstrated also the occurrence of co-localization and interaction of AR with EGFR in PC3-AR cells and in another androgen-dependent PC cell line, LNCaP. We hypothesize a mechanism by which, through direct interaction with EGFR, the AR elicits a reduction of EGF-mediated signalling and confers a less malignant phenotype.

We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with androgens further reduced invasion of the cells without modifying alpha6beta4 expression, suggesting an interference with the invasion process by androgens. Here, we investigated EGF-mediated signal transduction processes that lead to invasion in PC3-AR cells. We show that EGF-induced EGFR autotransphosphorylation is reduced in PC3-AR cells compared to PC3 cells transfected only with the vector (PC3-Neo). EGF-stimulated PI3K activity, a key signaling pathway for invasion of these cells, and EGF-PI3K interaction are also decreased in PC3-AR cells and further reduced by treatment with androgen. Finally, we show that EGFR internalization process was reduced in PC3-AR and LNCaP cells compared to PC3-Neo. Investigations on the location of AR in PC3-AR transfected cells were also conducted. Immunoconfocal microscopy and coimminoprecipitation studies demonstrated the presence of an interaction between EGFR and AR at membrane level in PC3-AR and LNCaP cells. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less-malignant phenotype by interfering with EGFR signaling leading to invasion through a mechanism involving an interaction between AR and EGFR., (Copyright 2004 Wiley-Liss, Inc.)

Evidence indicates that androgen-sensitive prostate cancer cells have a lower malignant potential. We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells through modulation of alpha6beta4 expression. Treatment with the androgen further reduced adhesion and invasion of the cells without, however, modifying alpha6beta4. Here we investigated whether the androgen has a direct effect on alpha6beta4-EGF receptor (EGFR) interaction and signalling leading to invasion of these cells. Immunoconfocal microscopy demonstrated that in control cells (PC3-Neo), alpha6beta4 and EGFR colocalize and redistribute in response to epidermal growth factor (EGF). In PC3-AR cells colocalization and redistribution between the two molecules was reduced and abolished by pre-treatment with R1881. Co-immunoprecipitation studies demonstrated that tyrosine phosphorylation of beta4 in response to EGF was reduced in PC3-AR cells compared to PC3-Neo. Immunoconfocal and co-immunoprecipitation studies demonstrated colocalization at membrane level and co-immunoprecipitation of EGFR and AR, indicating an interaction between the two proteins. PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells in response to EGF and further reduced by treatment with R1881. EGFR internalization was strongly reduced in PC3-AR compared with PC3-Neo cells and was reduced by treatment with R1881. In conclusion, the expression of AR by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR--alpha6beta4 interaction and signalling leading to invasion through a mechanism involving an interaction between the classic AR and EGFR.

Background: Alterations in fibroblast growth factors (FGFs) production and/or FGF receptors expression have been described to play key roles in prostate tumor progression, particularly in androgen-independent tumors. However, the role of androgen receptor (AR) in altering FGF-mediated growth and survival of prostatic neoplastic cells has not been completely defined. In this study, we investigated the alterations in FGF2 production and utilization by the PC3 cell line, after transfection with a full-length AR., Methods: FGF1,2,7, FGF-binding protein (FGF-BP) production and FGF receptor (FGFR) 1-4 expression were investigated by polymerase chain reaction (PCR) and Western blot analysis., Results: De novo AR expression by PC3 cells restores FGFR2 IIIb isoform expression and sensitivity to FGF7 and FGF2. Androgen stimulation induces AR+ PC3 clones to secrete FGF-BP, likely responsible for activation and mobilization from the extracellular matrix of the high amounts of FGF2 produced by the same cells. In addition to the effects on cell proliferation, FGF2 maintains the survival of AR+ PC3 clones through a positive modulation of the Bcl-2 protein and down-modulates AR protein expression, allowing the escape of selected clones from androgen regulation., Conclusion: In the presence of an active AR, the combined production of FGF2 and FGF-BP may play an important role in the progression of prostate cancer through the selection of AR- clones expressing high levels of Bcl-2., (Copyright 2002 Wiley-Liss, Inc.)

Prostate cancer cells may lose androgen-sensitivity after androgen ablation therapy, becoming highly invasive and metastatic. The biological mechanisms responsible for higher tumurogenicity of androgen-independent prostate carcinomas are not entirely known. We demonstrate that androgen receptor regulation of adhesion and invasion of prostate cancer cells through modulation of alpha6beta4 integrin expression may be one of the molecular mechanisms responsible of this phenomenon. We found that protein and gene expressions of alpha6 and beta4 subunits were strongly reduced in the androgen-sensitive cell line LNCaP respect to the androgen-independent PC3 and that transfection of PC3 cells with a full-length androgen receptor expression vector resulted in a decreased expression of alpha6beta4 integrin, reduced adhesion on laminin, and suppressed Matrigel invasion. Growth in soft agar was also suppressed in androgen receptor-positive PC3 clones. Treatment of androgen receptor positive clones with the synthetic androgen R1881 further reduced alpha6 and beta4 messenger RNA expression as well as adhesion on laminin and Matrigel invasion. Our results indicate that androgens regulate cell-extracellular matrix adhesion and invasion by modulation of integrin expression and function, thus keeping a low invasive phenotype of prostate cancer cells.