Antimo Migliaccio
email: antimo.migliaccio@unina2.it

Gabriella Castoria

Pia Giovannelli

Ferdinando Auricchio

CROSS TALK BETWEEN EPIDERMAL GROWTH FACTOR (EGF) RECEPTOR AND EXTRANUCLEAR STEROID RECEPTORS IN CELL LINES

Keywords: EGF, EGF-R, steroid receptors, cross talk, Src-dependent signaling pathway

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, extranuclear 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.

A c c e p t e d M a n u s c r i p t

THE COMPLEX NETWORK OF EXTRA NUCLEAR INTERACTIONS OF STEROID RECEPTORS: THE EXAMPLE OF ESTROGEN RECEPTOR ALPHA

Estrogen receptor/s (ERs) interact with a multitude of proteins inside as well as outside the nuclear compartment. Inside the nucleus, these receptors interact with hundreds of proteins that in different ways regulate ER transcriptional action [START_REF] O'malley | A life-long search for the molecular pathways of steroid hormone action[END_REF]. Outside the nucleus protein/protein interactions shape the signaling pathway followed by signal transduction from membrane to cell interior [START_REF] Pawson | Dynamic control of signaling by modular adaptor proteins[END_REF]. Recent work from different laboratories shows that ERs located outside the nucleus activate different signal transduction pathways with multiple effects, such as cell growth and survival [START_REF] Kousteni | Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity[END_REF], cell migration [START_REF] Giretti | Extra-nuclear signalling of estrogen receptor to breast cancer cytoskeletal remodelling, migration and invasion[END_REF], and vasorelaxation [START_REF] Simoncini | Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase[END_REF]. How can ERs, which unlike growth factor receptors are soluble and lack intrinsic enzyme activity, exert these stimulatory effects? The answer to this question is provided by a growing number of observations that once occupied by estradiol this receptor interacts with different signaling proteins and these interactions trigger signaling activity (for reviews, see [START_REF] Cato | Rapid actions of steroid receptors in cellular signaling pathways[END_REF][START_REF] Levin | Plasma membrane estrogen receptors[END_REF]. Such associations are facilitated by adaptor proteins and post-transcriptional modifications of the receptor. Signaling effectors interacting with the hormone-occupied receptor include the non-receptor-associated tyrosine kinase Src [START_REF] Migliaccio | Steroid-induced androgen receptor-oestradiol receptor-beta Src complex triggers prostate cancer cell proliferation[END_REF], the p85alpha regulatory subunit of PI3K [START_REF] Simoncini | Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase[END_REF][START_REF] Castoria | PI3-kinase in concert with Src promotes the S-phase entry of estradiol-stimulated MCF-7 cells[END_REF], the adaptor protein SHC [START_REF] Song | The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane[END_REF], the focal adhesion kinase FAK [START_REF] Le Romancer | Regulation of estrogen rapid signaling through arginine methylation by PRMT1[END_REF] and membrane receptor tyrosine kinases, such as EGF-R and erb-B2 [START_REF] Fox | Novel actions of estrogen to promote proliferation: Integration of cytoplasmic and nuclear pathways[END_REF]. Interestingly, in the case of ERalpha/Src association the interacting sequences have been identified. Direct ERalpha/Src association triggered by estradiol occurs "in vitro". This suggests that ligand modification of receptor triggers its affinity for Src, most likely through allosteric modification. In addition an adaptor protein, p130 Cas, is involved in association of ERalpha with Src [START_REF] Cabodi | p130Cas interacts with estrogen receptor alpha and modulates non-genomic estrogen signaling in breast cancer cells[END_REF]. Receptor modifications also play a key role in ER/Src association. ERalpha phosphorylation at tyrosine 537 is required for the direct interaction of ER with the Src-SH2 domain triggered by physiological concentration (10 nM) of estradiol [START_REF] Migliaccio | Steroid-induced androgen receptor-oestradiol receptor-beta Src complex triggers prostate cancer cell proliferation[END_REF]. In addition, it has recently been shown that ER arginine methylation regulates ER association with Src, p85 and FAK (Le Romacer at al., 2008). In conclusion, signal transduction pathway stimulation by estradiol requires ligand activation of the receptor, receptor modifications and adaptor proteins.

EGF DIRECTS CROSS TALK BETWEEN EGF-R AND EXTRA NUCLEAR STEROID RECEPTORS

Based on the knowledge that EGF-R shares many signaling effectors with the ER-activated pathway, we will present evidence that ER and androgen receptor (AR) not only participate in a large signalosome built around the EGF-R family kinases (IGFR, erb-B2, etc), but are required for EGF action in cells expressing ERs and AR.

A c c e p t e d M a n u s c r i p t 4 ERalpha and beta associate and cross talk with soluble receptors, AR and progesterone receptor B (PR-B) [START_REF] Migliaccio | Steroid-induced androgen receptor-oestradiol receptor-beta Src complex triggers prostate cancer cell proliferation[END_REF][START_REF] Migliaccio | Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor[END_REF] as well as membrane receptors [START_REF] Levin | Integration of the Extra nuclear and Nuclear Actions of Estrogen[END_REF]. Here we will focus on cross talk between EGF-R and extra nuclear ERalpha or beta in human mammary and prostate cancer-derived cells respectively. Many findings imply that ERs play a role in EGF signaling. Initial evidence comes from the observation that the estradiol antagonist Tamoxifen impairs EGF-induced DNA synthesis in breast cancer-derived MCF-7 cells [START_REF] Vignon | Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens[END_REF]. It was later shown that EGF and IGF-1 lose the ability to induce proliferative markers in uterine epithelial cells of ER-knockout mouse [START_REF] Hewitt | Lessons in estrogen biology from knockout and transgenic animals[END_REF]; see also Table I). On the other hand, EGF is known to be involved in hormone-independent steroid receptor signaling in human cancer cells. EGF-R-related tyrosine kinase Erb-B2, was shown to be gene-amplified in 20-25% [START_REF] Slamon | Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer[END_REF] of ER-positive breast cancers, and erb-B2 overexpression can cause Tamoxifen-stimulated growth as a mechanism of de novo resistance [START_REF] Shou | Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer[END_REF][START_REF] Chung | Resistance to tamoxifen-induced apoptosis is associated with direct interaction between Her2/neu and cell membrane estrogen receptor in breast cancer[END_REF]. This EGF-induced ER escape from hormone regulation could be explained by evidence that EGF triggers ligand-independent ER transcriptional activity through phosphorylation of specific critical residue(s) of nuclear estrogen receptor promoting interaction with its co-activator SRC1 [START_REF] Schiff | Breast Cancer Endocrine Resistance: How Growth Factor Signaling and Estrogen Receptor Coregulators Modulate Response[END_REF]. EGF stimulates through MAPK-dependent signaling ERalpha serine phosphorylation on 118 [START_REF] Kato | Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase[END_REF] and 154 residues [START_REF] Britton | A novel serine phosphorylation site detected in the N-terminal domain of estrogen receptor isolated from human breast cancer cells[END_REF], likely enhances 305 serine phosphorylation [START_REF] Rayala | P21-activated kinase1 regulation of estrogen receptor-activation involves serine 305 activation linked with serine 118 phosphorylation[END_REF], and induces 167 serine phosphorylation through a PI3-K dependent pathway [START_REF] Campbell | Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance[END_REF]. It has also been shown that EGF activated MAPK pathway can phosphorylate the ER coactivator AIB1 and nuclear co-repressor NCoR (Font de [START_REF] Font De Mora | AIB1 is a conduit for kinase-mediated growth factor signaling to the estrogen receptor[END_REF]. Therefore, such phosphorylations triggered by growth factors lead to ligand-independent activation of ER transcriptional activity. This evidence seemed to point to a model where growth factors drive ER-regulated nuclear signaling through non-genomic pathways, thereby suggesting the existence of a unidirectional signaling flow from membrane to nuclear receptors (Figure 1). In turn, it was also observed that in breast cancer cells hormone-occupied ERalpha [START_REF] Razandi | Proximal events in signaling by plasma membrane estrogen receptors[END_REF], or a G protein-coupled receptor homologue [START_REF] Filardo | Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF[END_REF], promote EGF-R transactivation and downstream signaling through c-Src-mediated activation of the MMP cascade and subsequent release of membrane-associated heparin binding EGF (HB-EGF) (Fig. 2A).

We analyzed the impact of extra-nuclear ERalpha and beta on EGF signaling on the basis of our previous findings on these receptors. In MCF-7 cells, estradiol triggers association of ERalpha with Src and the regulatory subunit of PI3-K, p85alpha. This association activates a complex signaling network that stimulates G1-S transition through up-regulation of cyclin D1 and p27 nuclear release [START_REF] Castoria | PI3-kinase in concert with Src promotes the S-phase entry of estradiol-stimulated MCF-7 cells[END_REF]2004). In MCF-7 cells, we also found that a small amount of ERalpha and AR (less than 10% of the total receptors) is associated under basal conditions. Therefore, either estradiol or androgen triggers association of both receptors with Src in these cells [START_REF] Migliaccio | Steroid receptor regulation of EGF signaling through Src in breast and prostate cancer cells: steroid antagonist action[END_REF]. These findings could also explain the inhibition by antiandrogens of estradiol-stimulated Src activation as well as the inhibition by antiestrogens of androgen-stimulated Src activity. Therefore, we asked whether EGF acts in a similar way to estradiol or androgen in inducing both the A c c e p t e d M a n u s c r i p t assembly of multi-molecular complexes including ERalpha, AR and Src, and DNA synthesis. To evaluate this hypothesis, DNA synthesis was stimulated in quiescent MCF-7 cells with EGF; this stimulation was ER-and AR-dependent as it was suppressed by pure estradiol or androgen antagonists (ICI 182,780 or Casodex, respectively). Inhibition of EGF-induced DNA synthesis was also observed by ERalpha or AR silencing. Again, the Src inhibitor PP2 had a dramatic inhibitory effect on DNA synthesis elicited by EGF. Interestingly, ICI 182,780, Casodex and PP2 also abolished cytoskeletal changes induced by EGF. These findings suggest that in MCF-7 cells, EGF signaling depends entirely on ERalpha, AR and Src. This view was confirmed by the finding that, like estradiol and androgen, EGF induces rapid ERalpha/AR/Src association in these cells. A similar association was also induced by EGF in Cos cells ectopically expressing hERalpha and hAR [START_REF] Migliaccio | Steroid receptor regulation of EGF signaling through Src in breast and prostate cancer cells: steroid antagonist action[END_REF]. In contrast, no association occurred when wild type hERalpha was substituted with mutant hERalpha containing a phenylalanine instead of tyrosine 537, which is the only phosphorylated tyrosine residue of ERalpha in MCF-7 cells [START_REF] Arnold | Phosphorylation of the human estrogen receptor on tyrosine 537 in vivo and by src family tyrosine kinases in vitro[END_REF]. Phosphotyrosine is required for interaction of ERalpha with the Src-SH2 domain in cells stimulated by estradiol [START_REF] Migliaccio | Steroid-induced androgen receptor-oestradiol receptor-beta Src complex triggers prostate cancer cell proliferation[END_REF]. In MCF-7 cells, tyrosine phosphorylation of ERalpha is induced by EGF and is abolished by the inhibitor of EGF-R tyrosine kinase, Iressa, or Herceptin, the antibody directed against the external domain of erb-B2 [START_REF] Migliaccio | Steroid receptor regulation of EGF signaling through Src in breast and prostate cancer cells: steroid antagonist action[END_REF]. From these data, we concluded that EGF treatment of MCF-7 cells through the EGF-R/erb-B2 complex induces phosphorylation on tyrosine 537 of AR-associated ERalpha [START_REF] Migliaccio | Steroid receptor regulation of EGF signaling through Src in breast and prostate cancer cells: steroid antagonist action[END_REF]. Once hormone occupied, AR interacts in turn with the Src-SH3 domain [START_REF] Migliaccio | Steroid-induced androgen receptor-oestradiol receptor-beta Src complex triggers prostate cancer cell proliferation[END_REF]. Association between these proteins is triggered by EGF-induced ERalpha tyrosine phosphorylation as shown by co-immunoprecipitation of EGF-R, ERalpha, AR and Src. This complex triggers activation of Src and the Src-dependent pathway, which controls DNA synthesis and cell migration. Surprisingly, in addition to activating the Src/Ras/Erk pathway, the ERalpha/AR/Src signaling complex directs Src activity associated with ERalpha/AR on EGF-R. In fact, ICI 182,780, Casodex, or silencing of ERalpha, AR or the dominant negative form of Src strongly reduce EGF-R tyrosine phosphorylation. Experiments in Cos cells, which do not express AR or ER, confirm that ERalpha and AR up-regulate EGF-R tyrosine phosphorylation [START_REF] Migliaccio | Steroid receptor regulation of EGF signaling through Src in breast and prostate cancer cells: steroid antagonist action[END_REF]. In fact, in these cells, ectopically expressed hERalpha and AR significantly increase EGF-R phosphorylation triggered by EGF. Steroid receptors also play a key role in EGF action in a different hormone-dependent environment such as prostate cancer cells. LNCaP cells derive from human prostate cancer and are largely used to study androgen action. In addition to AR they express ERbeta, whose role in prostate cancer is debated (Imanov et al., 2004). A small percentage of the two receptors is associated under basal conditions and stimulation of LNCaP cells with estradiol or androgen triggers proliferation following AR/ERbeta/Src association and consequent Src-dependent pathway activation [START_REF] Migliaccio | Steroid-induced androgen receptor-oestradiol receptor-beta Src complex triggers prostate cancer cell proliferation[END_REF]. EGF stimulation of LNCaP cells also induces ERbeta/AR/Src association, Src activation, DNA synthesis and actin architectural changes. All these effects are A c c e p t e d M a n u s c r i p t inhibited by ICI 182,780 or Casodex. On this basis we concluded that, as in MCF-7 cells, EGF signaling also depends on extra nuclear steroid receptors in LNCaP cells [START_REF] Migliaccio | Steroid-induced androgen receptor-oestradiol receptor-beta Src complex triggers prostate cancer cell proliferation[END_REF]. These findings raise the question of whether other proteins also direct Src activity to EGF-R phosphorylation in cells not expressing ER and/or AR. Analysis of this aspect might reveal new EGF-signaling regulatory proteins. However, from the evidence described above and many other findings not described here it can be firmly established that a dynamic bi-directional interplay exists between EGF-R and steroid receptor signaling. Two different models of how this occurs can be envisaged and are briefly presented in Fig. 2. According to the model derived from [START_REF] Razandi | Proximal events in signaling by plasma membrane estrogen receptors[END_REF] and shown in panel A, estradiol-bound ER activates EGFR activity by inducing metalloproteinase activation (MMP-9 and -2), which, in turn, causes HB-EGF secretion. HB-EGF transactivates EGFR to signal to ERK. MMP activation is mediated by Src kinase. In this model Src activation is mediated by G protein isoforms located at membranes. The mechanism of activation of these G proteins by ER needs to be further investigated. Interestingly, this model suggests that under some circumstances (e.g. in ER-negative cancer) other G protein-coupled receptors, such as the orphan receptor GPR 30 [START_REF] Filardo | Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF[END_REF][START_REF] Vivacqua | G protein-coupled receptor 30 expression is up-regulated by EGF and TGF alpha in estrogen receptor alpha-positive cancer cells[END_REF], might substitute ER in activating the EGFR-dependent pathway. Panel B in the figure represents a model derived from our work and shows cross phosphorylation between two tyrosine kinases, EGFR and Src associated with ER. Ligand-activated EGFR phosphorylates ERalpha on tyrosine 537, inducing ER-Src association and kinase activation. Activated Src phoshorylates EGFR, amplifying receptor activity and inducing signal transduction activation. These two models show some substantial differences, and further analysis is required to assess whether they co-exist or work under different cell settings. Nevertheless, both of them clearly suggest that EGFR as well as ERalpha potentiate each other's activity and point to a key role of Src in cooperation between these receptors.

EGF action on DNA synthesis is inhibited by interfering in steroid receptor-Src association through steroid receptor-derived peptides.

The observation that in mammary and prostate cancer-derived cells estradiol and androgen or EGF can participate in the same transducesome to exert their proliferative and migratory action profoundly modifies our current view on the interplay between hormones and growth factors, and may have important therapeutic implications. Both steroid hormones and growth factors control progression of these cancers. Therefore, ERalpha/AR/Src association is a very promising target for specific approaches to mammary and prostate cancer therapy. The ERalpha phosphotyrosine 537, and probably the homologous ERbeta phosphotyrosine 443, is required for association of estradiol receptor alpha or beta with the Src-SH2 domain, whereas the 377-386 proline-rich sequence of AR interacts with the Src-SH3 domain [START_REF] Migliaccio | Steroid-induced androgen receptor-oestradiol receptor-beta Src complex triggers prostate cancer cell proliferation[END_REF]. We therefore synthesized small peptides derived from these sequences of the two receptors [START_REF] Varricchio | Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor {alpha} Tyrosine-Phosphorylated Peptide[END_REF]Migliaccio et al., 2007). The peptides were expected to compete for Src association with the receptors, thereby A c c e p t e d M a n u s c r i p t 7 inhibiting DNA synthesis induced by 10 nM estradiol or R1881 treatment. The peptide derived from the sequence surrounding ERalpha phosphotyrosine 537 is a six-amino acid peptide (536-541), and was used in parallel with the non-phosphorylated form of the same peptide to evaluate the role of tyrosine phosphorylation in the Src-peptide interaction [START_REF] Varricchio | Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor {alpha} Tyrosine-Phosphorylated Peptide[END_REF]. In short-term experiments (up to 24 hours) the non-phosphorylated peptide had a much weaker inhibitory effect than that of the phosphorylated peptide, implying a dominant role of tyrosine phosphorylation. In long-term experiments, this difference disappeared. Use of the peptide with the same sequence, except for substitution of tyrosine with the non-phosphorylatable phenylalanine, restores the difference, suggesting that in long-term experiments the tyrosine residue of peptide undergoes phosphorylation. The peptides were amino-terminal acetylated and carboxyl-terminal amidated to reduce cleavage by exopeptidases. When MCF-7 cells or LNCaP cells were treated with 100 ng/mL EGF, DNA synthesis was stimulated. Such a stimulation was abolished by ICI 182,780 and Casodex, as well as by nanomolar concentrations of the phosphorylated peptide [START_REF] Varricchio | Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor {alpha} Tyrosine-Phosphorylated Peptide[END_REF] (Fig. 3, Panels A and B) This peptide also prevents DNA synthesis induced in the same cells by estradiol or androgen, indicating that EGF and steroid hormones share the mechanism triggering S phase in estradiol-or androgen-stimulated cells. The phospho-peptide effect follows inhibition of ERalpha and AR association with Src, Src and Erk activation and induction of cyclin D1 expression in MCF-7 and LNCaP cells [START_REF] Varricchio | Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor {alpha} Tyrosine-Phosphorylated Peptide[END_REF]. The peptide has no effect on EGF-induced DNA synthesis in human mammary cancer-derived MDA-MB231 cells which express AR but not ER, indicating that the peptide specifically targets ER. The specificity of the proliferative inhibitory action is notably illustrated by the absence of inhibition by the phospho-peptide of estradiol-induced ERE-dependent transcriptional activity in MCF-7 cells. In these cells, protection from apoptosis induced by estradiol is inhibited by the phospho-peptide, widening the potential interest for the applicative aspects of this peptide [START_REF] Varricchio | Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor {alpha} Tyrosine-Phosphorylated Peptide[END_REF]. Following a similar approach, we synthesized a peptide derived from the proline-rich 377-386 AR sequence interacting with the Src-SH3 domain. A peptide with the same amino acid composition but with a scrambled sequence was used in parallel experiments. This peptide shows a much weaker inhibitory effect than that of the 377-486 peptide, indicating that sequence rather than proline content plays a major role [START_REF] Migliaccio | Inhibition of the SH3 domain-mediated binding of Src to the androgen receptor and its effect on tumor growth[END_REF]. In LNCaP and MCF-7 cells, EGFinduced DNA synthesis was abolished by the peptide (Fig. 3, Panel C), which also inhibits S-phase entry induced by estradiol or androgen. Inhibition of the hormonal effect follows failure of AR/Src association, Src-dependent pathway activation and cyclin D1 expression. Furthermore, the peptide does not affect serum-induced DNA synthesis in LNCaP or MCF-7 cells, nor does it affect DNA synthesis or cytoskeletal changes triggered by EGF-activated Src in AR-negative prostate cancer DU145 cells [START_REF] Migliaccio | Inhibition of the SH3 domain-mediated binding of Src to the androgen receptor and its effect on tumor growth[END_REF]. This implies that the peptide specifically targets AR, without interfering in intrinsic Src activity. The peptide also prevents S-phase entry induced by androgen or EGF in AR-positive NIH3T3 fibroblasts [START_REF] Castoria | Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action[END_REF], thus reinforcing the view that AR is the target of the proline-rich peptide. In contrast with classic androgen antagonists, the peptide does not affect ARE-dependent transcriptional activity in LNCaP cells stimulated with androgen A c c e p t e d M a n u s c r i p t 8 [START_REF] Migliaccio | Inhibition of the SH3 domain-mediated binding of Src to the androgen receptor and its effect on tumor growth[END_REF]. In addition to interfering with steroid/Src interaction, the phosphorylated and proline-rich peptides share some interesting properties: i) they rapidly penetrate across cell membrane, and within 30 min diffuse through the extra nuclear compartment of MCF-7 and LNCaP cells by a temperature-independent mechanism; ii) like anti-proliferative agents, they are active at very low concentration (1 nM) in MCF-7 and LNCaP cells; iii) unlike steroid antagonists, they do not affect receptor-dependent transcriptional activity; iv) unlike Src inhibitors, they do not interfere in intrinsic Src activity. These promising properties led us to investigate the activity of these peptides in MCF-7 and LNCaP cell xenografts established in immunodepressed mice. Xenografts of MCF-7 cells were grown in ovariectomized and estrogen-stimulated mice intraperitoneally injected with the phosphorylated peptide. A dose-dependent inhibition of tumor growth was observed up to total suppression [START_REF] Varricchio | Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor {alpha} Tyrosine-Phosphorylated Peptide[END_REF]. In addition, xenografts of LNCaP cells were grown in intact male mice, which were treated with the proline-rich peptide. Here again, the peptide suppressed tumor growth. Immunohistochemistry of LNCaP xenografts showed a decrease in proliferative marker Ki67 and an increase in apoptotic cells in mice treated with the peptide, while no changes in body weight was detected [START_REF] Migliaccio | Inhibition of the SH3 domain-mediated binding of Src to the androgen receptor and its effect on tumor growth[END_REF]. Therefore, use of these molecules provides some potential advantages over conventional hormone antagonists and EGF inhibitors. They act downstream of ligand-receptor interaction and could even be effective in cancer cells bearing mutated receptors unable to bind hormones or growth factors. Furthermore, they are extremely selective in inhibiting only pathways potentially involved in deregulated growth without interfering with other effects of EGF or steroid hormones. Figure 3, panel D, summarizes the action of these molecules.

CONCLUSIONS

From findings by our and other laboratories it can be reasonably concluded that a continuous cross talk occurs between EGF-R family receptors and ER and AR. This cross talk does not only mean that growth factors can affect steroid-regulated gene expression and vice-versa, but it also exists at extra nuclear level, where EGF-R and steroid receptors can reciprocally and rapidly regulate activation of their transduction pathways. In particular, the observation that EGF-R requires steroid receptors in hormone-dependent cells raises some intriguing questions. Why, for example, does EGF-R need ER and/or AR expression to act on DNA synthesis in mammary and prostate cancer cells, whereas it is able to stimulate growth in many other cell types that do not express steroid receptors? It could be argued that the dependence of EGF-R signaling on steroid receptors represents a mechanism by which hormone-dependent cells recognize a hierarchy among signals regulating their functions, where the steroid hormones have a dominant role. In such a view it could be inferred that, when extra nuclear steroid receptors are down regulated or translocated to the nucleus, i.e. upon hormone stimulation, cell responsiveness to EGF goes to be also reduced. On the other hand it is conceivable that in different cell lines, devoid of ER or AR, other proteins such A c c e p t e d M a n u s c r i p t as estrogen-related receptor family members, which are almost ubiquitously expressed, might substitute steroid receptors in this regulatory role. However, as previously shown, the observation of a direct cross talk between EGF and steroid receptors provides novel selective weapons in the treatment of hormone-dependent and independent cancers. It also worth noting that, in addition to associating with Src, activated ER and AR interact with other signaling effectors, which participate in a complex network controlling tumor progression. Once domains of steroid receptors involved in the interaction with these effectors have been revealed, it will become feasible to design other molecules (peptides or peptide derivatives) to inhibit growth and metastasis of tumors expressing steroid receptors. These custom-made drugs could be used in combination with the peptides described in the present article to improve the efficacy of tumor therapy. The increasing number of non-classical hormoneresponsive normal and cancer cells expressing steroid receptors, such as fibroblasts or colon cancer-derived cell lines [START_REF] Castoria | Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action[END_REF][START_REF] Di Domenico | Estradiol activation of human colon carcinoma-derived Caco-2 cell growth[END_REF], suggests that this therapeutic approach might be tried in a wide variety of human cancers. In A, MCF-7 cells were stimulated with 100 ng/mL EGF alone or in presence of 10 µM ICI 182,780 or 1 nM of a six aa. peptide corresponding to the sequence 536-541 of ERalpha, including the phosphotyrosine 537 (PY pep), or a peptide with the same sequence but with unphosphorylated tyrosine. (Y pep). For comparison, cells were also stimulated with 10 nM 17 estradiol. BrdU incorporation was measured after 24 hrs of treatment. In B, LNCaP cells were treated, as MCF-7 cells, with EGF alone, in the presence of PY pep, or Y pep. In these cells the EGF effect on DNA synthesis was compared with that of 10 nM of synthetic androgen R1881. In C, LNCaP cells were incubated with 100 ng/mL EGF alone, in the presence of 1 µM ICI 182,780, 1 nM of a peptide corresponding to the proline-rich sequence 377-386 of AR, (pro-pep), or a 1 nM of peptide containing the same amino acid composition but with a scrambled sequence (c-pep).

In D, a model of inhibitory action of receptor-derived peptides on EGF signaling is shown. In untreated cells EGF stimulates ER phosphorylation by EGFR, inducing the assembly of a ternary complex between ER, AR which is pre-associated with ER and Src. This induces Src activation, which phosphorylates EGFR activating the signaling cascade. The competitor peptides Y-Pep or pro-peptide, inhibit the formation of this complex by competing for binding of ER and AR to SH2 and SH3 domains of Src, respectively. In this way Src kinase fails to be activated and to phosphorylate EGFR. Therefore, the signaling activity is abolished.

Fig. 1 .

 1 Fig. 1. A model of EGF activation of steroid receptors located in the nucleus.

Fig. 2 .

 2 Fig. 2. Two models of ER-mediated EGF-R pathway activation. The model proposed by Razandi et al. of E2-dependent ER-mediated transactivation of EGF-R is shown in A. Through a G-protein dependent mechanism, estradiol bound ER stimulates Src activity, which in turn activates metalloproteinases 2 and 9, increasing HB-EGF release. HB-EGF binds to EGF-R and activates its signaling. The model reported by Migliaccio et al. of EGF-dependent, ER-Src mediated EGF-R activation is shown in B. EGF-activated EGF-R phosphorylates ERalpha on tyrosine 537, inducing ER-Src association and kinase activation. Activated Src phoshorylates EGF-R, amplifying receptor activity and its downstream targets inducing signal transduction activation.

Fig. 3 .

 3 Fig. 3. Effect of peptides inhibiting ER/AR/Src association on EGF-induced DNA synthesis.In A, MCF-7 cells were stimulated with 100 ng/mL EGF alone or in presence of 10 µM ICI 182,780 or 1 nM of a six aa. peptide corresponding to the sequence 536-541 of ERalpha, including the phosphotyrosine 537 (PY pep), or a peptide with the same sequence but with unphosphorylated tyrosine. (Y pep). For comparison, cells were also stimulated with 10 nM 17 estradiol. BrdU incorporation was measured after 24 hrs of treatment. In B, LNCaP cells were treated, as MCF-7 cells, with EGF alone, in the presence of PY pep, or Y pep. In these cells the EGF effect on DNA synthesis was compared with that of 10 nM of synthetic androgen R1881. In C, LNCaP cells were incubated with 100 ng/mL EGF alone, in the presence of 1 µM ICI 182,780, 1 nM of a peptide corresponding to the proline-rich sequence 377-386 of AR, (pro-pep), or a 1 nM of peptide containing the same amino acid composition but with a scrambled sequence (c-pep).In D, a model of inhibitory action of receptor-derived peptides on EGF signaling is shown. In untreated cells EGF stimulates ER phosphorylation by EGFR, inducing the assembly of a ternary complex between ER, AR which is pre-associated with ER and Src. This induces Src activation, which phosphorylates EGFR activating the signaling cascade. The competitor peptides Y-Pep or pro-peptide, inhibit the formation of this complex by competing for binding of ER and AR to SH2 and SH3 domains of Src, respectively. In this way Src kinase fails to be activated and to phosphorylate EGFR. Therefore, the signaling activity is abolished.

  Fig.1

ACKNOWLEDGEMENTS

This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (National and Regional Grants to F.A. and A.M.).

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Piotr T Chruściel

Gregory J Galloway

UNIQUENESS OF STATIC BLACK-HOLES WITHOUT ANALYTICITY by

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Introduction

One of the hypotheses in the current theory of uniqueness of static vacuum black holes is that of analyticity. This is used to exclude null Killing orbits, equivalently to prove non-existence of non-embedded degenerate prehorizons covering a closed set, within the domain of outer-communications; see [START_REF] Chruściel | On uniqueness of stationary black holes[END_REF] for the details. The aim of this note is to show that analyticity is not needed to exclude such prehorizons, and therefore can be removed from the set of hypotheses of the classification theorems in the static case.

More generally, such prehorizons need to, and have been, excluded in dimension n + 1 with n -1 commuting Killing vectors [START_REF]On higher dimensional black holes with abelian isometry group[END_REF] without assuming analyticity. Our analysis here provides an alternative, simpler, approach to this issue for any stationary solutions satisfying the null energy condition, without the need to invoke more Killing vectors. (Note, however, that for solutions that are not static, all n -1 Killing vectors are used to prove that existence of a null Killing orbit implies existence of a prehorizon.)

In this work we consider asymptotically flat, or Kaluza-Klein (KK) asymptotically flat (in the sense of [START_REF] Chruściel | On the topology of Kaluza-Klein black holes[END_REF]) spacetimes, and show that (for definitions, see below and [START_REF] Chruściel | On uniqueness of stationary black holes[END_REF]):

Theorem 1.1. -I + -regular stationary domains of outer communication M ext satisfying the null energy condition do not contain prehorizons, the union of which is closed within M ext .

The reader is referred to [START_REF] Alexakis | Hawking's local rigidity theorem without analyticity[END_REF] and references therein for progress towards removing the hypothesis of analyticity in the general stationary case.

M ext ∂S S M ext E + Figure 1.
The hypersurface S from the definition of I + -regularity.

The time of flight argument

For the convenience of the reader we recall some definitions from [START_REF]On higher dimensional black holes with abelian isometry group[END_REF][START_REF] Chruściel | On uniqueness of stationary black holes[END_REF]:

Definition 2.1.
-Let (M , g) be a space-time containing an asymptotically flat, or KK-asymptotically flat end S ext , and let K be a stationary Killing vector field on M . We will say that (M , g, K) is I + -regular if K is complete, if the domain of outer communications M ext is globally hyperbolic, and if M ext contains a spacelike, connected, acausal hypersurface S ⊃ S ext , the closure S of which is a topological manifold with boundary, consisting of the union of a compact set and of a finite number of asymptotic ends, such that the boundary ∂S := S \ S is a topological manifold satisfying

(2.1) ∂S ⊂ E + := ∂ M ext ∩ I + (M ext ) ,
with ∂S meeting every generator of E + precisely once. (See Figure 1.)

The definition appears to capture the essential ingredients required for a successful classification of vacuum [START_REF] Chruściel | On uniqueness of stationary black holes[END_REF] or electro-vacuum [START_REF]On the classification of stationary electro-vacuum black holes[END_REF] black holes. Whether or not the definition is optimal from this point of view remains to be seen. In any case, one of its consequences is the structure theorem [START_REF]On higher dimensional black holes with abelian isometry group[END_REF][START_REF] Chruściel | On uniqueness of stationary black holes[END_REF], which in essence goes back to [START_REF]On the topology of stationary black holes[END_REF]Lemma 2], and which represents M ext globally as R × S , with the Killing vector tangent to the R factor.

Another notion that is essential for the current work is:

Definition 2.2.
-Let K be a Killing vector and set

(2.2) N [K] := {p ∈ M | g(K, K)| p = 0 , K| p = 0} .
Every connected, not necessarily embedded, null hypersurface N 0 ⊂ N [K] to which K is tangent will be called a Killing prehorizon.

It follows from [5, Corollary 3.3 and Lemma 5.14] that in vacuum I + -regular space-times which are static, or four-dimensional stationary and axisymmetric, or (n+1)-dimensional with n-1 commuting Killing vectors, the set covered by Killing prehorizons associated with a Killing vector field K is closed within M ext . This remains true for electrovacuum space-times in dimension 3 + 1.

For further purposes it is convenient to introduce Definition 2.3. -Let (M , g) be a space-time with a complete Killing vector field K, and let Ω ⊂ M . We shall say that a closed set K ⊂ Ω is an invariant quasihorizon in Ω if K is a union of pairwise disjoint null (not necessarily embedded) hypersurfaces, called leaves. We further assume that the leaves of K are invariant under the flow of K, and that every null geodesic maximally extended within Ω and initially tangent to a leaf of K remains on K .

From what has been said, it follows that Proposition 2.4. -In static, or stationary axi-symmetric, I + -regular vacuum space-times, the union of Killing prehorizons forms a (possibly empty) spatially bounded quasi-horizon in M ext .

Here "spatially bounded" means that it does not extend infinitely far out in the end {0} × S ext . In cases of interest the Killing vector flow acts as a translation along the R factor, so in fact one has a t-independent bound on the extent on each slice {t} × S ext .

Theorem 1.1 follows now from:

Theorem 2.5. -Consider an asymptotically flat, or KK-asymptotically flat, globally hyperbolic domain of outer communications M ext , satisfying the null energy condition, diffeomorphic to R × S , with the Killing vector, tangent to the R factor, approaching a time translation in the asymptotic region. Then there are no invariant quasi-horizons in M ext .

Proof of Theorem 2.5: Let R ∈ R be large enough so that the constant-time spheres lying on the timelike hypersurface

T := R × {| x| = R}
are both past and future inner trapped, as defined in [START_REF] Chruściel | On the topology of Kaluza-Klein black holes[END_REF]. Without loss of generality we can assume that K does not intersect the region {| x| ≥ R}; indeed, K cannot be tangent to the null leaves of K in the asymptotically flat region, where it is timelike. Let C denote the following class of causal curves:

C := {γ | γ : [0, 1]
→ M is a causal curve which starts and ends at T , and meets

K 0 := K ∩ ({0} × S ) } .
The time of flight τ γ of γ is defined as

τ γ := t(γ(1)) -t(γ(0)) ,
where t is the time-function associated with the decomposition M = R × S . We write S τ for t -1 (τ ) ≡ {τ } × S . Let τ denote the infimum of τ γ over γ ∈ C . We wish to show that if K 0 is non-empty, then τ is attained on a smooth null geodesic γ, with a) initial and end points on T , b) meeting S 0 at K 0 , c) meeting T normally to the level sets of t.

In order to construct γ, let γ i ∈ C be any sequence of causal curves such that τ γi → τ . Let γ be any causal curve in C , then 0 > t(γ i (0)) ≥ -τ γ and 0 < t(γ i (1)) ≤ τ γ for i large enough. Hence for i large enough all the γ i (0)'s belong to the compact set [-τ γ , 0] × {| x| = R}; similarly the γ i (1)'s belong to the compact set [0, τ γ ] × {| x| = R}. By global hyperbolicity there exists an accumulation curve γ of the γ i 's which is a C 0 causal curve.

Since K 0 is closed in M ext , γ meets K 0 at some point p. It is standard that γ ∩ {t < 0} is a smooth null geodesic, since otherwise p would be timelike related to γ(0), which would imply existence of a curve in C with time of flight less than τ . Similarly, γ ∩ {t > 0} is a smooth null geodesic.

Next, in a similar fashion (see [15, Lemma 50, p. 298]), γ meets T t(γ(0)) and T t(γ(1)) orthogonally, where T τ := S τ ∩ T .

We claim that γ is also smooth at p. To see that, let K denote that leaf of K that passes through p. Then the portion of γ that lies to the causal past of p must meet K transversally: Otherwise γ ∩ J -(p) would coincide with that portion of the null Killing orbit of K through p that lies to the past of p, but those never reach T , since K is spatially bounded. Similarly the portion of γ that lies to the causal future of p must meet K transversally. Suppose that the two geodesic segments forming γ do not join smoothly at p. Then there exist arbitrary small deformations of γ which produce a timelike curve with the same end points as γ, and hence the same time of flight. By transversality, and because there exists a small neighbourhood V of p in which the connected component of K ∩ V passing through p forms a null embedded hypersurface, any such deformation, say γ, will meet K at some point p. Let φ t denote the flow of K, then

γ := φ -t( p) (γ)
is a timelike curve in C which has the same time of flight as γ. Since γ is timelike, it can be deformed to a causal curve with shorter time of flight. This contradicts the definition of τ , and hence proves a), b) and c).

Let τ * = t(γ(0)). We claim that d) γ minimizes the time of flight amongst all nearby differentiable causal curves from T τ * to T . Indeed, by transversality of γ to K , there exists a neighbourhood U of γ in the space of differentiable curves such that every curve γ in this neighbourhood intersects K . Suppose, then, that there exists a causal curve γ ∈ U which starts at T τ * , ends at T , and has time of flight smaller than τ . Then γ intersects K at some p. But then φ -t(p) (γ) is in C and has time of flight smaller than τ , which contradicts the definition of τ , whence d) holds.

This provides a contradiction to K being non-empty, as there are no causal curves with the property d) by [6, Proposition 3.3].

Non-rotating horizons and maximal hypersurfaces

In this section we provide an alternative simple argument to exclude prehorizons within the domain of outer communication, which applies to four-dimensional static vacuum space-times.

Let (M , g) be an asymptotically flat, I + -regular, vacuum space-time with a hypersurface orthogonal Killing vector K. By [START_REF] Chruściel | On non-existence of static vacuum black holes with degenerate components of the event horizon[END_REF] all components of the future event horizon E + are non-degenerate. We can therefore carry-out the construction of [START_REF] Rácz | Global extensions of space-times describing asymptotic final states of black holes[END_REF] if necessary to obtain that ∂ M ext is the union of bifurcate Killing horizons. By [START_REF] Chruściel | Maximal hypersurfaces in stationary asymptotically flat space-times[END_REF], M ext contains a maximal Cauchy hypersurface S . By [START_REF] Sudarsky | Extrema of mass, stationarity and staticity, and solutions to the Einstein-Yang-Mills equations[END_REF] (compare the argument at the end of [5, Section 7.2]), S is totally geodesic. Decomposing K as K = N n + Y , where n is the field of future-directed unit normals to S , and where Y is tangent to S , one finds from the Killing vector equations that

D i Y j + D j Y i = -2N K ij .
But the right-hand-side vanishes, thus Y is a Killing vector of the metric γ induced on S by g. Now, Y is asymptotic to zero as one recedes to infinity in M ext , hence Y = 0 by usual arguments, (see, e.g., the proof of [7, Proposition 2.1]). Since K has no zeros within M ext we conclude that N has no zeros on S . Alternatively, N satisfies the equation

(3.1) ∆N = K ij K ij N ,
vanishes on ∂S , and is asymptotic to one as one recedes to infinity along the asymptotically flat region, and thus has no zeros by the strong maximum principle. Whatever the argument, K = N n is timelike everywhere on M ext , and there are no prehorizons within M ext .

The above argument applies verbatim to higher dimensional vacuum metrics, as well as to four-dimensional electrovacuum metrics, for configurations where all horizons are non-degenerate. A proof of existence of maximal hypersurfaces with sufficiently controlled asymptotic behaviour near the degenerate horizons would extend this argument to the general case. In any case, the proof based on the time of flight covers more general situations.

Conclusions

Recall that a manifold S is said to be of positive energy type if there are no asymptotically flat complete Riemannian metrics on S with nonnegative scalar curvature and vanishing mass except perhaps for a flat one. This property has been proved so far for all n-dimensional manifolds S obtained by removing a finite number of points from a compact manifold of dimension 3 ≤ n ≤ 7 [START_REF] Schoen | Variational theory for the total scalar curvature functional for Riemannian metrics and related topics[END_REF], or under the hypothesis that S is a spin manifold of any dimension n ≥ 3, and is expected to be true in general [START_REF] Christ | Singular minimal hypersurfaces and scalar curvature[END_REF][START_REF] Lohkamp | The higher dimensional positive mass theorem I[END_REF].

Using results already established elsewhere [START_REF] Chruściel | The classification of static vacuum space-times containing an asymptotically flat spacelike hypersurface with compact interior[END_REF][START_REF] Chruściel | On uniqueness of stationary black holes[END_REF][START_REF] Chruściel | The classification of static electro-vacuum space-times containing an asymptotically flat spacelike hypersurface with compact interior[END_REF][START_REF] Costa | On black hole uniqueness theorems[END_REF][START_REF]On the classification of stationary electro-vacuum black holes[END_REF] together with Theorem 1.1 one has: Theorem 4.1. -Let (M , g) be a vacuum n + 1 dimensional space-time, n ≥ 3, containing a spacelike, connected, acausal hypersurface S , such that S is a topological manifold with boundary, consisting of the union of a compact set and of a finite number of asymptotically flat ends. Suppose that there exists on M a complete static Killing vector K, that M ext is globally hyperbolic, and that ∂S ⊂ M \ M ext . Let S denote the manifold obtained by doubling S across the non-degenerate components of its boundary and compactifying, in the doubled manifold, all asymptotically flat regions but one to a point. If S is of positive energy type, then M ext is isometric to the domain of outer communications of a Schwarzschild space-time.

Theorem 4 . 2 .

 42 -Under the remaining hypotheses of Theorem 4.1 with n = 3, suppose instead that (M , g) is electrovacuum with the Maxwell field invariant under the flow of K. Then M ext is isometric to the domain of outer communications of a Reissner-Nordström or a standard Majumdar-Papapetrou space-time.

Acknowledgements: PTC is grateful to the University of Miami for hospitality and support during part of work on this paper.

Supported in part by the Polish Ministry of Science and Higher Education grant Nr N N201 372736. Supported in part by NSF grant DMS-0708048.