Inhibition of the SH3 domain-mediated binding of Src to the androgen receptor and its effect on tumor growth (original) (raw)
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Molecular Cancer Research, 2007
This report offers direct evidence that association of the estradiol receptor (ER) with Src triggered by steroid agonists or growth factors controls breast and prostate cancer cell growth. This association is abolished in whole cells and in vitro by a six-amino-acid peptide that mimics the sequence around the phosphotyrosine residue in position 537 of the human ERα. The phosphorylated peptide, at nanomolar concentrations, is taken up by MCF-7 and LNCaP cells derived from human mammary and prostate cancers, respectively. In addition, to block the ER/Src interaction, the phosphopeptide inhibits Src/Erk pathway, cyclin D1 expression, and DNA synthesis induced by estradiol or androgen or triggered by epidermal growth factor. In contrast, no inhibition of the Src-mediated epidermal growth factor action on DNA synthesis is detectable in human mammary cancer cells that do not express ER (MDA-MB231), indicating that the peptide specifically targets the ER-associated Src. Remarkably, the pep...
2005
Under conditions of short-term hormone deprivation, epidermal growth factor (EGF) induces DNA synthesis, cytoskeletal changes, and Src activation in MCF-7 and LNCaP cells. These effects are drastically inhibited by pure estradiol or androgen antagonists, implicating a role of the steroid receptors in these findings. Interestingly, EGF triggers rapid association of Src with androgen receptor (AR) and estradiol receptor A (ERA) in MCF-7 cells or ERB in LNCaP cells. Here, we show that, through EGF receptor (EGFR) and erb-B2, EGF induces tyrosine phosphorylation of ER preassociated with AR, thereby triggering the assembly of ER/AR with Src and EGFR. Remarkably, experiments in Cos cells show that this complex stimulates EGF-triggered EGFR tyrosine phosphorylation. In turn, estradiol and androgen antagonists, through the Src-associated receptors, prevent Src activation by EGF and heavily reduce EGFR tyrosine phosphorylation and the subsequent multiple effects, including DNA synthesis and cytoskeletal changes in MCF-7 cells. In addition, knockdown of ERa or AR gene by small interfering RNA (siRNA) almost abolishes EGFR tyrosine phosphorylation and DNA synthesis in EGF-treated MCF-7 cells. The present findings reveal that steroid receptors have a key role in EGF signaling. EGFR tyrosine phosphorylation, depending on Src, is a part of this mechanism. Understanding of EGF-triggered growth and invasiveness of mammary and prostate cancer cells expressing steroid receptors is enhanced by this report, which reveals novel aspects of steroid receptor action. (Cancer Res 2005; 65(22): 10585-93) Note: A. Migliaccio and M. Di Domenico contributed equally to this work. Requests for reprints: Ferdinando Auricchio, Dipartimento di Patologia Generale,
The EMBO …, 2000
Treatment of human prostate carcinoma-derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor b with Src, activates the Src/Raf-1/ Erk-2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti-androgens and anti-oestrogens. Similar ®ndings for oestradiol receptor a were observed in MCF-7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF-7 and T47D cells with SrcK ± abolishes steroid-stimulated S-phase entry. Data from transfected Cos cells con®rm and extend the ®ndings from these cells. Hormone-stimulated Src interaction with the androgen receptor and oestradiol receptor a or b is detected using glutathione S-transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor a and the Src SH3 domain with a proline-rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor a with Src and consequent activation of Src in intact Cos cells.
Analysis of Androgen Receptor Rapid Actions in Cellular Signaling Pathways: Receptor/Src Association
2011
Much evidence indicates that, with few exceptions, non-genomic actions of steroids are mediated by receptors universally known as nuclear receptors. Steroid receptors do not exhibit intrinsic tyrosine kinase activity. Nevertheless, they stimulate different signaling pathways in cytoplasm of target cells, including those dependent on Src, a cytoplasmic tyrosine kinase. Steroid-induced Src activation regulates cell cycle progression, survival, migration, and associated processes, such as cell growth and differentiation. Androgen stimulation of human prostate cancer-derived LNCaP cells triggers cell cycle progression and proliferation. The key event in this process is the association of androgen receptor (AR) with Src. This association triggers activation of the Src/Ras/Erk pathway and finally impacts cell cycle. Androgen stimulation of fibroblasts also induces AR/Src association, which triggers DNA synthesis. Prevention of this association by a receptor-derived peptide competing for AR interaction with Src specifically inhibits the androgen receptor-dependent proliferative effect in vitro and in vivo.
Epidermal Growth Factor Directs Sex-specific Steroid Signaling through Src Activation
Journal of Biological Chemistry, 2007
Estrogens and androgens exert many biological effects that do not require interactions of their receptors with chromosomal DNA. However, it has been a long-standing question how the sex steroid receptors provoke signal transduction outside the nucleus. Here we have shown that epidermal growth factor (EGF) directs sex-specific steroid signaling through Src activation. We have revealed that estrogen (E2)-induced Src activation takes place in, not only plasma, but also endomembranes. This was found ascribed to the existence of EGF and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor together with Src. EGFR, estrogen receptor, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast, the androgen 5␣-dihydrotestosterone-induced Src activation occurs only in the plasma membrane free from the interaction of EGFR with androgen receptor, irrespective of EGF. The cell growth occurred only moderately as a result. The spatial difference in Src activation between E2 and 5␣-dihydrotestosterone may be responsible for the different extent of observed cell growth.
Molecular and Cellular Endocrinology, 2010
Steroid receptors act as ligand-dependent transcriptional factors. It has been observed that in addition to responding to cognate hormones with transcription activation, once hormone bound they are also capable of rapid responses following association with signaling effectors in the extra nuclear compartment. This novel aspect of steroid hormone action could influence our view of the cross talk between growth factor and steroid receptors. Increasing evidence shows that in hormone-responsive cells, a cross talk occurs between growth factors (EGF, IGF-1) and steroid hormone receptors that reciprocally regulate their action. To date, this has mostly been explained by modulation of steroid receptor transcriptional activity through growth factor receptor signaling activation. However, it is now known that growth factors might also act on extra nuclear steroid receptors, activating them via a hormone-independent mechanism. On the other hand, extra nuclear steroid receptors can regulate growth factor receptor activity either directly interfering with their transduction pathways, or inducing autocrine growth factor secretion. Here we discuss findings indicating that EGF, like steroid hormones, induces association of steroid receptors with Src thereby activating pathways that can trigger cell proliferation and migration. Since mammary and prostate cancers respond to both steroid hormones and growth factors, this association might be a putative target for human cancer therapy. Findings from our laboratory supporting this view are discussed.
Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells
PLoS ONE, 2013
Background: Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR).
Biochemistry, 2001
SHP (short heterodimer partner) is an orphan nuclear receptor, first described for its interaction with nuclear receptors. This study explores a new way of inhibiting the androgen-signaling pathway. We demonstrated that SHP inhibited up to 97% of AR-induced activity. Characterization of AR/SHP interaction provided evidence of a clear ligand dependency. We also showed that the LXXI/LL motifs previously found on SHP mediated the interaction with the AR ligand-binding domain (AR-LBD), the motif responsible for the interaction being slightly different from that found with ER. The AR N-terminal domain (AR-NTD), in contrast to that of other nuclear receptors, accounts for most of the entire receptor transactivation potential. SHP also interacted with AR-NTD, thus stabilizing the interaction with AR. We demonstrated that SHP inhibited both AR-LBD and NTD-dependent transactivation, which evidenced for the first time a protein capable of inhibiting a steroid receptor amino-terminal-dependent transactivation. We further characterized the SHP mechanism of action by showing that SHP reversed AR coactivator-mediated activation. Conversely, FHL2 and TIF2 counteracted SHP-mediated inhibition of AR. SHP evidences a new way of inhibiting AR activity by competing with AR coactivators. This new type of inhibitor could dictate the activity of nuclear receptors, depending on the equilibrium between activators and inhibitors.
Crosstalk between EGFR and Extranuclear Steroid Receptors
Annals of the New York Academy of Sciences, 2006
Src activity, epidermal growth factor receptor (EGFR) phosphorylation on tyrosine, and the EGF-dependent signaling pathway activation. In these cells, the AR/ER/Src complex is required for the EGF action, as the growth factor effects are abolished upon receptor silencing by specific SiRNAs and steroid antagonists or Src inhibition by the kinase inhibitor PP2.
Effect of Small Molecules Modulating Androgen Receptor (SARMs) in Human Prostate Cancer Models
PLoS ONE, 2013
The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progressionrelated markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.