Activins and related proteins in the establishment of pregnancy.

Activin A and related proteins (inhibins, follistatin [FS], follistatin-related gene [FLRG], endometrial bleeding associated factors [ebaf]) are involved in the complex mechanisms allowing the establishment and the maintenance of pregnancy. As a consequence of ovarian progesterone stimuli, activin A is expressed and secreted by the stromal endometrial cells, which locally induces the decidualization process, a prerequisite for implantation. Moreover, activin A does influence the implantation phase, also enhancing cytotrophoblast differentiation, indirectly, by increasing the expression of other molecules involved in embryo implantation, such as matrix metalloproteinases (MMPs) and leukemia inhibitory factor (LIF). The local derangement of activin A pathway in some pregnancy disorders (incomplete and complete miscarriages, recurrent abortion, and ectopic pregnancy [EP]) further sustains the hypothesis that activin A and its related proteins play a relevant role in the establishment of pregnancy.

Androgen receptor regulation of the seladin-1/DHCR24 gene: altered expression in prostate cancer.

Prostate cancer (CaP) represents a major leading cause of morbidity and mortality in the Western world. Elevated cholesterol levels, resulting from altered cholesterol metabolism, have been found in CaP cells. Seladin-1 (SELective Alzheimer Disease INdicator-1)/DHCR24 is a recently described gene involved in cholesterol biosynthesis. Here, we demonstrated the androgen regulation of seladin-1/DHCR24 expression, due to the presence of androgen responsive element sequences in its promoter region. In metastatic androgen receptor-negative CaP cells seladin-1/DHCR24 expression and cholesterol amount were reduced compared to androgen receptor-positive cells. In tumor samples from 61 patients who underwent radical prostatectomy the expression of seladin-1/DHCR24 was significantly higher with respect to normal tissues. In addition, in cancer tissues mRNA levels were positively related to T stage. In tumor specimens from 23 patients who received androgen ablation treatment for 3 months before surgery seladin-1/DHCR24 expression was significantly lower with respect to patients treated by surgery only. In conclusion, our study demonstrated for the first time the androgen regulation of the seladin-1/DHCR24 gene and the presence of a higher level of expression in CaP tissues, compared to the normal prostate. These findings, together with the results previously obtained in metastatic disease, suggest an involvement of this gene in CaP.

Prostate cancer: a model of integration of genomic and non-genomic effects of the androgen receptor in cell lines model.

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.

Altered endocytosis of epidermal growth factor receptor in androgen receptor positive prostate cancer cell lines.

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.

The vitamin D analogue BXL-628 inhibits growth factor-stimulated proliferation and invasion of DU145 prostate cancer cells.

PURPOSE: Suppression of the invasive phenotype is essential in developing new therapeutic tools to treat advanced prostate cancer (PC) indicating that androgen-independent prostate cancer (AI-PC) is characterized by increased metastatic potential. In the present study, we have investigated the effect of the nonhypercalcemic vitamin D analogue BXL-628 on proliferation and invasive properties of the human PC cell line DU145. In particular, the effect of the analogue was tested following stimulation with a potent growth factor, keratinocyte growth factor (KGF), which stimulates both proliferation and invasion of these cells. We have also evaluated the effect of the analogue on KGF stimulation of PI3K/AKT signaling pathway. METHODS: Cell proliferation was determined by cell counting. Invasion through Matrigel was evaluated using Boyden chambers. PI3K activity was measured by immunokinase assay and AKT phosphorylation was evaluated by western blot analysis. Keratinocyte growth factor receptor (KGFR) autotransphosphorylation was evaluated by western blot after immunoprecipitation of the receptor. RESULTS: BXL-628 is able to inhibit both proliferation and invasion of DU145 cells in basal conditions and in response to KGF. Following stimulation with KGF, the inhibition is due to suppression of KGFR autotransphosphorylation and downstream PI3K/AKT activation, both achieved following a brief (5 min) incubation with the analogue. This effect on KGFR autophosphorylation was still present when cells were treated with the alpha-amanitin, an inhibitor of RNA transcription, indicating a rapid, nongenomic effect. CONCLUSIONS: Our results demonstrate that the vitamin D analogue BXL-628 is able to suppress KGF-induced proliferation and invasion of AI-PC cells in vitro, prospecting a possible use of the drug, which is currently in phase II clinical studies for benign prostatic hyperplasia, in the treatment of advanced prostate cancer.

The androgen receptor and prostate cancer invasion.

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.

Non-genomic effects of the androgen receptor and vitamin D agonist are involved in suppressing invasive phenotype of prostate cancer cells.

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.

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen-sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR).

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.

Gefitinib ('IRESSA', ZD1839) inhibits EGF-induced invasion in prostate cancer cells by suppressing PI3 K/AKT activation.

PURPOSE: Androgen-independent prostate cancer (AI-PC) is characterized by a higher invasive potential compared to hormone-responsive prostate cancer. A therapeutic option for AI-PC should thus be targeted to suppress not only cell proliferation, but also the invasive ability of the cells. Here, we investigated the effect of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib ('IRESSA', ZD1839) on EGF-stimulated invasion and proliferation in two androgen-independent prostate cancer cell lines PC3 and DU145. In addition, we determined the effect of the compound on EGF-stimulated PI3 K/AKT pathway activation, in view of the key role exerted by this pathway in carcinoma cell invasion. METHODS: Cell proliferation was determined by thymidine incorporation in the nuclei. Cell cycle analysis was performed by flow cytometry. Invasion through matrigel in vitro was measured by using Boyden chambers. PI3 K activity was measured by immunokinase assay and AKT phosphorylation was evaluated by Western blot analysis. RESULTS: Gefitinib inhibits invasion through matrigel and collagen in response to EGF in both cell lines. In addition, we confirm the inhibitory effect of the compound on basal and EGF-induced cell proliferation. Such an effect was accompanied by accumulation of the cells in the G0/G1 phase of the cell cycle. The effect of the compound is due, as expected, to suppression of EGF-induced autotransphosphorylation of EGFR. In addition, we demonstrate here that gefitinib inhibits EGF-induced activation of PI3 K/AKT pathway in both cell lines. CONCLUSION: Overall, our results demonstrate that gefitinib is able to suppress invasion and proliferation of AI-PC cells by suppressing EGF-stimulated activation of the PI3 K/AKT pathway and support a possible use of the drug in the treatment of advanced PC to limit not only proliferation but also invasion to other districts.

Androgen receptor and prostate cancer invasion.

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.

Androgen receptor expression induces FGF2, FGF-binding protein production, and FGF2 release in prostate carcinoma cells: role of FGF2 in growth, survival, and androgen receptor down-modulation.

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.

Characterization of membrane nongenomic receptors for progesterone in human spermatozoa.

Rapid, nongenomic actions of steroid hormones have been characterized only recently. They may be mediated by interaction with a poorly characterized membrane receptor, by classic receptor located to the plasma membrane, or by interaction of the classic receptor with other signaling effectors. Among these, rapid effects of progesterone on human spermatozoa have been shown to be mediated by interaction with one or more membrane receptors. Two proteins, respectively of 57 and 28 kDa, representing the possible surface progesterone receptors in human spermatozoa, have been identified by our group employing an antibody (c-262) directed against the progesterone binding domain of the genomic receptor. The two proteins have been immunoprecipitated using c-262, isolated by 2D gel electrophoresis and analyzed by Maldi-Tof. Preliminary results of the analysis in data bank of the obtained masses suggest that the two proteins represent previously unidentified ones since they do not match with any protein in the database. We have also performed RT-PCR analysis with RNA extracted from human spermatozoa, utilizing various oligoprimers in different regions of the human progesterone genomic receptor. Results indicate the presence of transcripts for the complete genomic receptor. However, several previously published studies in the literature indicate the absence of expression of the genomic receptor in human spermatozoa. In this light posttranscriptional/posttraductional modifications of the receptor can be hypothesized. Interestingly, with primers amplifying in the DNA-binding domain of the progesterone receptor gene, we detected a higher molecular weight transcript when compared to the placenta. Further studies are needed to determine whether the sequences of the transcripts obtained by RT-PCR analysis of human sperm RNA match exactly with the human genomic receptor gene and to define the sequence of the higher molecular weight transcript detected in the DNA-binding region.

Vitamin D3 analogue inhibits keratinocyte growth factor signaling and induces apoptosis in human prostate cancer cells.

BACKGROUND: Prostate cancer is a worldwide significant health care problem, due to its high incidence and mortality. In particular, androgen-independent tumors have the worst prognosis, because they are refractory to almost all kinds of available therapy. Hence, there is the need of new treatment opportunities targeting androgen-independent, growth factor-mediated, tumor signaling. One of these new promising opportunities is vitamin D3 and its related analogues. METHODS: We investigated the effect of a vitamin D3 analogue, analogue (V), on proliferation of several human prostate cancer cells in basal condition and after treatment with KGF, one of the intraprostatic growth factors that might participate in the progression of prostate cancer. In addition, in the androgen-independent cell line DU 145, we also studied the effect of analogue (V), KGF, and their mutual interaction on protein tyrosine phosphorylation, bcl-2 expression and apoptosis. RESULTS: Overall, we found that analogue (V) dose-dependently decreased basal and KGF-induced prostate cancer cell growth, although to a different extent. Maximal effect was obtained in DU 145 cells. In these cells, KGF stimulated tyrosine phosphorylation of a protein corresponding to its receptor, induced bcl-2 expression, and prolonged cell survival. Analogue (V) not only counteracted all these KGF-mediated events, but also decreased basal bcl-2 expression, therefore, allowing DU 145 cells to undergo an apoptotic program. CONCLUSIONS: Our results indicated that in prostate cancer cells analogue (V) decreased basal and KGF-induced cell proliferation. This effect, at least in DU 145 cells, is in part mediated by negative interactions with cell survival and KGF signaling.

Signal transduction pathways in human spermatozoa.

Following ejaculation, sperm functions are regulated by interactions with the environment found in the female genital tract. Here spermatozoa become 'capacitated' (i.e. they acquire the capability of completing the acrosome reaction and successfully fertilizing the egg), through a series of surface and intracellular transformations occurring during a process known as capacitation. The limited number of spermatozoa that eventually reach the oocyte must then cross the surrounding cumulus cells before contacting the zona pellucida, undergoing the acrosome reaction and finally fertilizing the oocyte. All the interactions between sperm and their diverse environments are mediated by intracellular signaling pathways. Virtually all the signaling systems known to operate in somatic cells, with the exception of those implying nuclear activity, have been found in spermatozoa, although in some cases the function(s) of the pathway has not been clearly proved.