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Molecular Cancer Research 5, 1213-1221, November 1, 2007. doi: 10.1158/1541-7786.MCR-07-0150
© 2007 American Association for Cancer Research
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Signaling and Regulation

Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor {alpha} Tyrosine-Phosphorylated Peptide

Lilian Varricchio1, Antimo Migliaccio1, Gabriella Castoria1, Hiroshi Yamaguchi2, Antonietta de Falco1, Marina Di Domenico1, Pia Giovannelli1, William Farrar3, Ettore Appella2 and Ferdinando Auricchio1

1 Dipartimento di Patologia Generale, II Università di Napoli, Naples, Italy; 2 Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, Maryland; and 3 Science Applications International Corporation-Frederick, National Cancer Institute-Frederick, Frederick, Maryland

Requests for reprints: Ferdinando Auricchio, Department of General Pathology, II University of Naples, Via L. De Crecchio, 7, Naples 80138, Italy. Phone: 39-081-5665676; Fax: 39-081-291327. E-mail: ferdinando.auricchio{at}unina2.it


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
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 ER{alpha}. 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 peptide, in contrast with classic steroid antagonists, does not interfere in ER- or androgen receptor–dependent transcriptional activity. Nevertheless, it markedly inhibits the growth of MCF-7 cell xenografts induced in immunodepressed and estradiol-treated mice. The present report suggests that inhibition of association of steroid receptors with Src or other signaling effectors may have therapeutic applications for patients with ER-positive tumors. (Mol Cancer Res 2007;5(11):1213–21)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Breast tumor growth is mainly regulated by estrogens, and estrogen deprivation remains a key therapeutic approach (1). The essential function of estrogen in breast cancer led to the development of tamoxifen, an antagonist of the estrogen receptors and the most widely used compound in cancer hormone therapy. It reduces relapses of cancers expressing steroid receptors and significantly prevents cancer development (2). Two other drugs have recently been developed. Raloxifen, which belongs to a new generation of selective estrogen receptor modulators, is used in chemoprevention with reduced risk of endometrial cancer (3). Aromatase inhibitors are also prescribed for postmenopausal patients with estrogen receptor–positive cancers and in chemoprevention (1). They lower plasma estrogen levels synthesized from androgenic substrates.

Our understanding of the nongenomic action of steroid hormones is rapidly increasing and significantly contributing to our knowledge of the proliferative action of the sex-steroid hormones. Agonist-occupied estradiol receptor-{alpha} (ER{alpha}) or androgen receptor (AR) stimulates the Src/Shc/Ras/Erk pathway in human mammary cancer–derived MCF-7 cells (4-6). This stimulation requires association of the phosphorylated tyrosine residue in position 537 of hER{alpha} (6-8) with the SH2 domain of Src and a proline stretch of hAR with the SH3 domain of Src (6). This pathway is a part of a complex signaling network including phosphoinositide 3-kinase and various effectors of this kinase, such as Akt/PKB (9), Rac (10), and PKC{zeta} (11). Under proliferative conditions, estradiol activation of this network leads to increased expression of cyclin D1, nuclear exclusion of the CDK inhibitor p27, and stimulation of G1-S transition of MCF-7 cells (9, 11). The entire network is under the control of Src activity and ER{alpha}/AR/Src association. In human prostate cancer–derived LNCaP cells, which express ERß and AR, agonist occupancy of these receptors triggers a similar process. Androgens induce ERß/AR/Src association and activate the Src-dependent pathway, thereby stimulating G1-S transition. ERß probably interacts with Src through tyrosine 443, which is homologous with tyrosine 537 of ER{alpha} (12).

A new link between epidermal growth factor (EGF) signaling and extranuclear steroid receptors has been recently described (13). EGF induces ER/AR/Src association in MCF-7 and LNCaP cells. This association is required for DNA synthesis and cytoskeletal changes induced by EGF in these cells. It is noteworthy that in MCF-7 and LNCaP cells, ER{alpha} or ERß, respectively, is associated with AR under basal conditions (13). Therefore, challenging these cells with estradiol, androgen, or EGF induces the assembly of the mitogenic ER/AR/Src complex, and the simultaneous presence of either estradiol or androgen antagonists prevents complex assembly and G1-S transition.

Identification of this rapid mitogenic action of estradiol suggests the development of molecules to interfere with the signaling pathway stimulation by either estradiol, androgen, or EGF in human cancer cells expressing ER. Therefore, we synthesized a peptide of six residues derived from the sequence around the phosphotyrosine residue in position 537 of the human ER{alpha}. Such a peptide, at very low concentrations, inhibits ER/Src interaction, Src/Erk pathway, cyclin D1 expression, and DNA synthesis induced by estradiol, androgen, or EGF. In contrast to classic estradiol antagonists, the peptide does not affect the receptor-dependent transcriptional activity, and, unlike common signaling inhibitors, does not interfere with non–receptor-dependent signaling.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Phosphorylation of tyrosine 537 in hER{alpha} was initially reported by our group (7) and later found to be the only tyrosine phosphorylation present in ER{alpha} of MCF7-cells (8). This phosphorylation is required for a strong association of hER{alpha} with the Src-SH2 domain (6). LNCaP cells express ERß, which is highly homologous to ER{alpha} in the hormone-binding domain. Its tyrosine 443 corresponds to tyrosine 537 of ER{alpha}. Phosphorylation of this tyrosine could be involved in the interaction of ERß with the Src-SH2 domain (6). To inhibit these associations, we used a peptide mimicking the sequence of hER{alpha} around this tyrosine. Figure 1A shows the sequence of two six-amino-acid peptides (pY-pep and Y-pep), both derived from residues 536 to 541 of the hER{alpha}, which include tyrosine 537 and have a minimal size capable of binding to Src SH2 domain. One of the two peptides shown contains a phosphorylation at this tyrosine (Ac-LpY-DLLL-NH2: pY-pep), whereas the other does not (Ac-LY-DLLL-NH2: Y-pep). Both peptides are amino-terminal acetylated and carboxyl-terminal amidated to avoid cleavage by exopeptidases.


Figure 1
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  		FIGURE 1. Uptake of peptides mimicking the hER{alpha} 536-541 sequence and their action on steroid-dependent DNA synthesis. A. Sequences of the phosphorylated (pY-pep) and nonphosphorylated (Y-pep) peptides. The peptides are amino-terminal acetylated and carboxyl-terminal amidated. B. The uptake of the fluorescein-conjugated pY-pep in unfixed MCF-7 and LNCaP cells, respectively, at 4°C and 37°C. Similar images are detectable with the fluorescein-conjugated Y-pep. C. Effect of 10 µmol/L estradiol antagonist ICI 182,780, and 1 nmol/L pY-pep or Y-pep on BrdUrd incorporation in MCF-7 cells stimulated by 10 nmol/L estradiol. D. Effect of 10 µmol/L androgen antagonist, Casodex, and 1 nmol/L pY-pep or Y-pep on BrdUrd incorporation of LNCaP cells stimulated by 10 nmol/L R1881. BrdUrd incorporation was detected as described in Materials and Methods and analyzed by fluorescence microscopy. It was calculated by the following formula: percentage of BrdUrd-positive cells (number of BrdUrd-positive cells / number of total cells) x 100. For each experiment, data were derived from at least 500 scored cells. The results of three independent experiments have been averaged. Columns, mean; bars, SE. The significance was evaluated by t test (P ≤ 0.001).

 
The uptake of the carboxyfluoresceinated peptides by quiescent MCF-7 and LNCaP cells was analyzed by fluorescence microscopy of unfixed cells. At nanomolar concentration, after 30 min, the fluorescence of pY-pep was prevalently observed in the extranuclear compartment (Fig. 1B). Independent of temperature (4°C or 37°C), the peptide uptake by the two cell lines was similar, suggesting that it did not depend on energy. Similar results were observed in fixed cells as well as with fluorescein-labeled Y-pep.4 Cells were then maintained for 3 days in the absence of phenol red and in presence of charcoal-treated serum. Under these conditions, most of the bromodeoxyuridine (BrdUrd) incorporation depended on addition of estradiol (Fig. 1C) and the androgen R1881 to the medium (Fig. 1D). As expected, hormone action was completely inhibited by the estradiol antagonist ICI 182,780 or the androgen antagonist Casodex. Interestingly, addition of the pY-pep (1 nmol/L) to the cell medium inhibited estradiol- or androgen-induced DNA synthesis, comparable with the levels observed with each steroid antagonist. The peptide inhibitory effect on hormonal-stimulated MCF-7 and LNCaP cells was maximal at 0.1 and 1 nmol/L.4 In addition, substitution of pY-pep with an equal concentration of the nonphosphorylated peptide showed only a weak inhibitory effect (Fig. 1C). This weak inhibition suggests that tyrosine phosphorylation plays a major role in the observed inhibitory effect of pY-pep on hormone-induced DNA synthesis.

To determine cytotoxic effects by p-Y and Y peptide in MCF-7 cells, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used. This assay revealed that the number of viable cells after stimulation with 10 nmol/L estradiol in the presence of pY-pep or Y-pep was comparable with that of cells treated with hormone alone (85% and 87% of untreated control cells, respectively, versus 86%). The peptides had no cytotoxic activity even when added in the absence of estradiol. In fact, viable cells after treatment with pY-pep and Y-pep were 84% and 97% of control, respectively (not represented). The lack of cytotoxicity of the peptides is further supported by the finding that, under the conditions used for the analysis of BrdUrd incorporation, no reduction of Hoechst-stained nuclei in the presence of pY-pep or Y-pep was observed in MCF-7 or LNCaP, or the mammary cancer ER-negative MDA-MB 231 cells.4

We then determined whether the phosphorylated peptide inhibits the G1-S transition by preventing the mitogenic ER/Src or AR/Src association challenged by estradiol in MCF-7 cells and R1881 in LNCaP cells, respectively. Hormone treatment of MCF-7 and LNCaP cells triggered these associations (Fig. 2A and B, top and middle ), and steroid antagonists, ICI 182,780 or Casodex, abolished it. Remarkably, pY-pep exerted the same inhibitory action. In contrast, nonphosphorylated Y-pep did not affect the ER/Src association and slightly reduced the AR/Src complex assembly. The inhibition of association by pY-pep paralleled inhibition of Src activity (Fig. 2A and B, bottom), and, likewise, the inhibitory effect of Y-pep on Src activity was very weak. Analysis of the effect of pY-pep on ER{alpha}/Src association was then extended to in vitro conditions. In a pull-down assay, the [35S]methionine-labeled hER{alpha} interacted with the chimeric glutathione S-transferase–Src protein, whereas a point-mutated version, in which tyrosine 537 was substituted with phenylalanine (HEG537), did not interact with Src. These findings indicate that under these conditions phosphotyrosine is required for the ER/Src association. As observed in MCF-7 cells (Fig. 2A), the pY-pep blocked the direct interaction of the wild-type ER{alpha} with Src (Fig. 2C). Both results with the receptor mutant and with the peptide underline the importance of tyrosine phosphorylation in the association of the hormone receptor with Src. Because it is known that the Src/Erk pathway is activated by the steroid receptor/Src association (6), the pY-pep was also tested in this system. The data clearly show an inhibition of the Erk-2 activity in hormone-stimulated MCF-7 and LNCaP cells (Fig. 2D and E, top and middle). Here, again, the Y-pep does not show an activity.


Figure 2
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  		FIGURE 2. Effect of pY-pep and Y-pep on ER/Src and AR/Src association, Src-Erk pathway, and cyclin D1 expression in MCF-7 or LNCaP cells stimulated by estradiol or R1881. A. Top and middle, effect of 1 nmol/L pY-pep or Y-pep and 10 µmol/L ICI 187,780 on ER{alpha}/Src association induced by treatment of MCF-7 cells with 10 nmol/L estradiol (E2) for 5 min; bottom, effect of pY-pep or Y-pep on the activity of the immunoprecipitated Src assayed as enolase phosphorylation (en) under the same conditions of the association analysis. B. Top and middle, effect of 1 nmol/L pY-pep or Y-pep and 10 µmol/L Casodex on AR/Src association induced by treatment of LNCaP cells with 10 nmol/L R1881 for 5 min; bottom, effect of the two peptides under the same conditions of the association analysis on the activity of the immunoprecipitated Src assayed as phosphorylation of enolase. C. The inhibition by 100 nmol/L pY-pep of in vitro association of [35S]methionine-labeled hER{alpha} with glutathione S-transferase–Src (GST-src) in the presence of 10 nmol/L estradiol. The lack of association of HEG537 with glutathione S-transferase–Src is also shown. HEG537 is hER{alpha} whose tyrosine in position 537 has been replaced by the nonphosphorylable phenylalanine. D and E. Top and middle, the effect of 1 nmol/L pY-pep or Y-pep on Erk-2 activity immunoprecipitated from the indicated cells stimulated for 5 min with 10 nmol/L estradiol or R1881 for 5 min and assayed using myelin basic protein (MBP) as a substrate; bottom, the effect of the two peptides and 10 µmol/L PP2 (Calbiochem) on cyclin D1 expression in MCF-7 cells or LNCaP cells treated with 10 nmol/L estradiol or R1881 for 8 h.

 
Having shown that p-Y-pep inhibits the association between Src and ER{alpha} or AR as well as the Src/Erk activation, we next analyzed its effect on cyclin D1 expression induced by this association. We previously showed that the concerted Src/phosphoinositide 3-kinase activation by estradiol leads to increased transcription as well as expression of cyclin D1 in MCF-7 cells (9). These findings are now confirmed by the experiment in Fig. 2D (bottom), showing that estradiol increased cyclin D1 expression of MCF-7 cells, whereas the Src kinase inhibitor, PP2, abolished this hormonal effect. The phosphoinositide 3-kinase inhibitor (LY 294002) had a similar effect.4 Interestingly, pY-pep blocked the estradiol action, whereas Y-pep only slightly reduced the cyclin D1 expression. Comparable findings were observed in LNCaP cells treated with androgen (Fig. 2E, bottom): R1881 increased cyclin D1 expression and PP2 abolished it. It should be noted that the observed inhibitory effect of PP2 represents the first evidence of androgen up-regulation of cyclin D1 through signal transduction activation. Here, again, the phosphorylated peptide reduced the androgen-induced cyclin D1 expression to the basal level, whereas the nonphosphorylated peptide was without effect (Fig. 2E, bottom).

Altogether, the present experiments indicate that pY-pep inhibits receptor/Src association, Src-dependent cyclin D1 expression, and DNA synthesis stimulated by steroid hormones. In these experiments, the nonphosphorylated peptide showed weak effects, emphasizing the importance of the tyrosine phosphorylation for these interactions.

An additional peptide carrying the phosphomethyl phenylalanine instead of phosphotyrosine, which is refractory to the action of tyrosine phosphatases, has been analyzed for its effect on hormone action in MCF-7 cells. We find that 1 nmol/L phosphomethyl phenylalanine suppressed the estradiol-dependent ER{alpha}/Src association (Fig. 3A, inset ). When its action on DNA synthesis was analyzed at a concentration of 1 nmol/L under conditions identical to those of the experiments presented in Fig. 1C, an inhibitory effect on BrdUrd incorporation triggered by estradiol was observed (Fig. 3A). For comparison, the effect of 10 µmol/L ICI 182,780, which also inhibits ER/Src association and DNA synthesis (6), is shown in Fig. 3A.


Figure 3
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  		FIGURE 3. Inhibition of the phosphatase-resistant phosphomethyl phenylalanine peptide of estradiol action in MCF-7 cells, and effect of pY-pep or Y-pep on DNA synthesis induced by EGF in MCF-7 or LNCaP cells or ER-negative MDA-MB231 cells. A. One nanomolar phosphomethyl phenylalanine (pmp) or 10 µmol/L ICI 187,780 prevents the ER/Src association evaluated as ER coimmunoprecipitated with Src (inset) and inhibits the BrdUrd incorporation in MCF-7 cells stimulated by 10 nmol/L estradiol. B. The effect of 1 nmol/L pY-pep or Y-pep on AR/Src association in LNCaP cells and ER{alpha}/Src association in MCF-7 cells. Both cell lines were treated with 100 ng/mL EGF for 5 min. C to E. The effect of 10 µmol/L estradiol antagonist (ICI 182,780) and 1 nmol/L pY-pep or Y-pep on BrdUrd incorporation triggered by 100 ng/mL EGF in MCF-7, LNCaP, and MDA-MB231 cells, respectively. MDA-MB231 cells do not express ER. DNA synthesis was analyzed and calculated as described in the legend to Fig. 1. Data are derived from at least 1,000 scored cells, with mean values taken from at least three experiments. Columns, mean; bars, SE (P ≤ 0.005).

 
Recently, we have shown that EGF stimulates G1-S progression of MCF-7 or LNCaP cells through induction of the ER/AR/Src complex and that hER{alpha} tyrosine phosphorylation is crucial in promoting the receptor/Src assembly (13). Therefore, we have tested whether pY-pep inhibits the EGF-induced AR/Src and ER/Src association. LNCaP and MCF-7 cells were challenged with EGF in the absence or presence of either pY-pep or Y-pep. Cell lysates were immunoprecipitated with an anti-Src antibody and immunoblotted with anti-Src and anti-AR or anti-ER{alpha} antibodies. Figure 3B shows that EGF induced AR/Src and ER/Src associations that were blocked by pY-pep, whereas Y-pep had a weak inhibitory effect. Consistent with these findings and the observed inhibitory effect on receptor/Src association, pY-pep strongly reduced the effect of EGF on MCF-7 and LNCaP cell DNA synthesis, whereas the nonphosphorylated peptide actually showed a weak inhibitory effect (Fig. 3C and E). In addition, expression of the ER{alpha} is required for the pY-pep action. When an ER-negative mammary cancer–derived cell line (MDA-MB231) was used, neither ICI 182,780 nor the pY-pep inhibited the EGF-induced DNA synthesis of cells (Fig. 3D). Experiments with LNCaP and MCF-7 cells in Fig. 3 indicate that the reduction in EGF-mediated DNA synthesis by pY-pep is due to the inhibition of ER/Src complex assembly by the peptide.

In the previous experiments, we found that pY-pep and steroid antagonists had identical effects on hormone action. In contrast, a striking difference was detected when steroid receptor–dependent transcription was evaluated with a gene reporter assay. Figure 4A shows that the pY-pep as well as the Y-pep, unlike ICI 182,780, did not significantly affect the ability of ER{alpha} to transactivate an estradiol-responsive element in hormone-stimulated MCF-7 cells. Similar results were obtained in experiments with LNCaP cells treated with R1881 after transfection with an androgen-responsive element reporter gene. Again, the pY-pep did not affect the receptor-dependent transcription in contrast to the androgen antagonist (Fig. 4B). These results show that the phosphorylated peptide antagonizes the ER{alpha}-dependent signal transduction pathway activation without interfering in the activity of the receptor on gene transcription.


Figure 4
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  		FIGURE 4. The pY-pep does not affect the steroid receptor–dependent transcriptional activity, whereas it inhibits the growth of MCF-7 xenografts. MCF-7 (A) or LNCaP (B) cells were transfected with an estradiol-responsive element or ARE-reporter gene, respectively, then stimulated by 10 nmol/L of either estradiol or R1881 in the absence or presence of 10 µmol/L antagonist inhibitors or 1 nmol/L pY-pep or Y-pep. The luciferase activity was assayed, normalized using ß-galactosidase as an internal control, and expressed as arbitrary units of luciferase activity. The results are representative of three independent experiments. Columns, mean; bars, SE. The significance was evaluated by t test (P ≤ 0.005). C. MCF-7 cells were incubated for 60 min without or with 10 nmol/L estradiol alone or in the presence of the indicated compounds, then exposed for 90 min to 2 mmol/L H2O2 to induce apoptosis. Apoptotic and dead cells were counted as described in Materials and Methods. It was assumed as 100% the number of cells undergoing apoptosis or dead after 90-min treatment with H2O2 in the absence of hormone. Under these conditions, 39% of cells underwent apoptosis and 13% of cells died. D. MCF-7 cell xenografts were established in female nude mice as described in Materials and Methods. Mice were treated with either 200 µL of pY pep at 50 nmol/L ({bigtriangleup}), 100 nmol/L ({circ}), 250 nmol/L ({blacklozenge}), or with vehicle alone ({blacksquare}), and tumor mass was measured once weekly. The number of mice in each group is six. Points, means; bars, SE.

 
Estradiol exerts through a signaling network, including the Src-dependent pathway attenuation of apoptosis in different cell types (14, 15). Therefore, we addressed the question as to whether p-Y pep affects the hormone effect on apoptosis. To this end, MCF-7 cells were left untreated or treated with estradiol in the absence or in the presence of pY-pep or Y-pep; then, apoptosis was induced by exposing the cells to 2 mmol/L hydrogen peroxide for 90 min. Upon this treatment, ~39% of cells underwent apoptosis and 13% died (Fig. 4C). Estradiol reduced by ~31% the number of cells undergoing apoptosis and reduced by almost 70% the dead cells. Addition of pY-pep, but not Y-pep, significantly lowered the hormone protective effect. The pY-pep effect was similar to that of the pure antiestrogen ICI, which almost completely abolished the estradiol antiapoptotic action. Interestingly, no significant effect on cell apoptosis or death was observed when pY-pep was added in the absence of hormone.

Finally, the effect of pY-pep on in vivo MCF-7 cell growth was investigated. Human MCF-7 cell–derived tumor xenografts were established in female athymic nude mice and their growth was supported by estradiol cypionate (depo-estradiol injections, USP). Thereafter, three different concentrations of pY-pep were administered by subcutaneous injection every day to three randomly selected groups of six mice for 8 weeks. A fourth six-mouse group was used as a control and treated with vehicle alone for the same period. Tumor growth was followed by measuring tumor mass every week, as described in Materials and Methods. Figure 4D shows that treatment with the peptide strongly decreased or abolished the tumor growth in mice treated with the p-Y peptide. In contrast, an exponential increase of tumor mass could be observed in control group mice treated with vehicle alone. The peptide-treated mice did not show an overall decrease in weight when compared with untreated mice (not shown). Thus, the pY-pep was able to specifically inhibit tumor growth.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Breast cancer is a major cause of premature death, although a decline in mortality rate has been observed in recent years (ref. 16 and references therein). Adjuvant systemic treatments based on use of tamoxifen and its analogues as well as aromatase inhibitors are standard. Tamoxifen binds to the estrogen receptor and blocks the estrogen-induced proliferation of breast cancer. However, drug resistance and numerous side effects are frequently observed during tamoxifen treatment. Aromatase inhibitors inactivate or inhibit the enzyme-synthesizing estrogens from androgen compounds and strongly lower the estrogen plasma levels. However, resistance to these inhibitors is also frequently observed. In spite of these treatments, many endocrine-responsive breast cancers relapse and metastasize. Therefore, new cancer-specific drugs, particularly compounds targeting signaling effectors, are sought. We report here that a peptide mimic of the amino acid sequence surrounding the single phosphorylated tyrosine residue of ER{alpha} interferes in the association of the ER/AR complex with Src induced by estradiol or androgen. As expected on the basis of the inhibition of ER/Src association here described, which is required for Src-dependent signaling activation (6), the peptide prevents receptor-dependent Src/Erk signaling, cyclin D1 expression, the G1-S transition induced by estradiol or androgen, and DNA synthesis triggered by EGF in mammary and prostate cancer cells in vitro. Src-dependent pathway has been involved in the estradiol action not only on DNA synthesis stimulation but also on cell apoptosis attenuation (14, 15). Consistent with this knowledge, we now observe that pY-pep reduces the antiapoptotic effect of this hormone. The inhibitory effect on DNA synthesis and receptor association with Src in cancer cells in vitro is mostly, although not exclusively, dependent on the phosphorylation of the peptide. Our experience suggests that phosphorylation of Y-pep may occur during cell treatment. This possibility is supported by the observation that although 1 nmol/L Y-pep does not affect MCF-7 cell growth during the initial 7 days of culture, inhibition is detectable in the following days. In contrast, this late inhibitory action is not observed when a peptide containing phenylalanine, which cannot be phosphorylated, was used in place of tyrosine.4 The difference between Y-pep and the phenylalanine peptide supports the view that phosphorylation on tyrosine of Y-pep in cells may occur, thus increasing the inhibitory action of this peptide. Nonetheless, one of the concerns in using tyrosine-phosphorylated peptides is the possibility that these compounds under certain conditions could be dephosphorylated inside of whole cells. Hence, using of phosphatase-resistant derivatives could be preferable for long-term in vivo treatment. To this end, a modified pY-pep, with a phosphatase-resistant phosphomethyl phenylalanine in place of phosphotyrosine has been prepared and tested. Results show that this molecule works almost as efficiently as pY-pep.

Src-associated ER represents the specific target of the peptide, as shown by experiments in MDA-MB231 cell line. In these cells, which are AR positive and do not express ER (13), there is no inhibitory effect of the peptide on the Src-dependent DNA synthesis triggered by EGF. There is, however, peptide inhibition of the AR/Src association and DNA synthesis in LNCaP cells stimulated by R1881. This observation is not surprising, as in LNCaP and MCF-7 cells AR is associated under basal conditions with ER, the peptide target (13). Therefore, in these cells, the peptide interference in the ER/Src interaction also interferes in the AR/Src association required for the androgen or EGF signaling.

Remarkably, the peptide, either phosphorylated or unphosphorylated, does not modify the ER- or AR-dependent transcriptional activity, and therefore does not target the hormonal effects mediated by this activity. In addition, the finding that the receptor-dependent transcription action is refractory to pY-pep confirms that gene transcription and signal transduction action represent two independent functions of steroid receptors (10, 17). Notably, unlike the Src inhibitor AZD0530 proposed for breast cancer therapy (18), the peptide affects only the signaling effectors activated by ER, as shown by the lack of Src-dependent DNA synthesis inhibition in ER-negative and AR-positive MDA-MB231 cells stimulated by EGF. The inhibitory effect of pY-pep on tumor xenograft growth is remarkably strong, and it is probably the consequence of the action of the peptide not only on the steroid hormone action but also on the growth factor signaling. Such signaling requires ER/AR/Src association (13), which is prevented by the pY-pep. The specific action of the peptide, its strong inhibition of cell proliferation at very low inhibitory concentrations, and maintenance of mouse body weight suggest that the peptide might be useful in the treatment of breast and prostate cancers.

In conclusion, this report gives direct evidence that ER-dependent signaling activation by steroids or growth factors, which have a dominant role in breast cancer progression, triggers DNA synthesis and tumor growth. In the absence of ligand binding, there seems to be a nonfunctional association between the ER and AR. However, upon ligand binding, the conformation changes and a complex between ER/AR and Src is formed, resulting in the activation of the kinase. As shown in Fig. 5 , here we identified a peptide that binds the SH2 domain of Src and inhibits the growth of MCF7 tumor cells. Our work, therefore, provides evidence of the importance of the ER/AR/EGF signaling pathway in the progression of breast and prostate cancers. We also present a target for the development of a new approach to control cell proliferation in these cancers. In addition to Src, ER recruits different signaling effectors such as p85a, the regulatory subunit of phosphoinositide 3-kinase (9, 19), Shc (20), and MNAR (21). These effectors impinge on downstream pathways regulated by Src. Inhibitors of these interactions might exert effects similar to those of the inhibitory peptides here described.


Figure 5
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  		Figure 5. Model of extranuclear action of ER and AR and inhibitory action by the ER-derived peptide. A. In the absence of hormones, AR and ER are preassociated but do not interact with Src, which is inactive. B. Androgen, estrogen, or EGF promotes association of AR/ER complex with Src. Upon this interaction, the kinase achieves an active conformation. C. p-Y pep interacts with the SH2 domain of Src, hindering its association with the AR/ER complex and inhibiting its kinase activity.

 

   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture, Transfection, and Transactivation Assay
Human mammary cancer MCF-7 and MDA-MB231 cells as well as fast-growing human prostate cancer LNCaP cells were grown and made quiescent as reported (6, 17). For androgen- and estradiol-stimulated transcriptional analysis, LNCaP and MCF-7 cells were made quiescent and then transfected by Superfect (Qiagen) with 4 µg of purified plasmids, either estradiol-responsive element–luc or androgen-responsive element–luc 3416. The 3416 construct, containing four copies of the wild-type slp-HRE2 (5'-TGG-TCAgccAGTTCT-3'), was cloned in the NheI site in pTK-TATA-Luc (22). Twenty-four hours later, transfected cells were left unstimulated or stimulated for 12 h with the indicated compounds. Lysates were prepared and the luciferase activity was measured using a luciferase assay system (Promega). The results were corrected using CH110-expressed ß-galactosidase activity (Amersham Biosciences).

DNA Synthesis Analysis
For BrdUrd incorporation analysis, quiescent cells on coverslips were left unstimulated or stimulated for 24 h with the indicated compounds. After a 6-h pulse with 100 µmol/L BrdUrd (Sigma), BrdUrd incorporation was analyzed as described (17) using Alexa Fluor 594–conjugated mouse monoclonal anti-BrdUrd antibody (Molecular Probes). The coverslips were finally stained with Hoechst 33258, inverted, and mounted in Mowiol (Calbiochem). The fields were analyzed with a DMLB fluorescent microscope (Leica) equipped with x40 and x63 objectives.

Uptake Analysis of Labeled Peptide
For this analysis, exponentially growing cells were dissociated with a nonenzymatic cell dissociation medium (Sigma). Cells (2.5 x 105) were plated on 30-mm plates on a glass coverslips and then made quiescent. The medium was discarded, and the cells were washed with NaCl/Pi (pH 7.3). NaCl/Pi was discarded and the cell monolayer was incubated with the SH2-binder peptide conjugated to 5-(6)-carboxylfluorescein succinimidyl ester (Molecular Probes). Fluorescein-conjugated peptide was dissolved in Opti-MEM and added (at 1 nmol/L) at 37°C or 4°C for 30 min to the cell medium. For direct detection of fluorescein-labeled peptide, the cells were washed thrice in NaCl/Pi at room temperature before being processed in Vectashield mounting medium (Vector Laboratories). The distribution of fluorescence was analyzed by a DMIRB fluorescent microscope (Leica) equipped with x40 and x63 objectives. Images were generated using an IM1000 software (Leica).

Human Tumor Xenografts
Human MCF-7–derived tumor xenografts were established in female athymic Ncr-nu/nu nude mice (National Cancer Institute-Frederick) as described (23, 24). To support the growth of the estrogen-dependent MCF-7 tumor, the depo-estradiol (estradiol cypioniate injection, USP) was administered at a dose of 3 mg/kg (i.m.) once weekly. Animals were treated with different doses of PY peptide (Ac-LpY-DLLL-NH2) or control vehicle once daily (subcutaneously) for a total of 8 weeks. Each group consisted of six animals. The tumors were measured in two dimensions by caliper and the animals were weighed twice weekly. Tumor volume is calculated as a2 x b x 0.5, where a is the width and b is the length of the tumor. Animal experimentation was reviewed and approved by the Animal Research Committee of the NIH (Bethesda, MD).

Production of Recombinant Protein
The glutathione S-transferase–Src fusion protein construct was transformed in Escherichia coli JM109 cells. Fusion protein was extracted as described (25) with minor modifications. Briefly, the cells were resuspended in one-hundredth volume and extract was obtained by three cycle of freezing-thawing in lysis buffer [PBS, 1%, Triton X-100 (pH 7.4), containing the protease inhibitors leupeptin, antipain and pepstatin, LAP, at 10 µg/mL, and phenylmethylsulfonyl fluoride]. Lysozyme at 5 mg/mL was added to the lysis buffer and lysates were clarified at 13,000 rpm. Fusion protein was then purified on glutathione-agarose beads (1:1 in PBS containing 1% Triton X-100, 10 µg/mL LAP, and 1 mmol/L phenylmethylsulfonyl fluoride); the matrix with the adsorbed fusion protein was used for protein-protein interaction assay.

Protein-Protein Interaction Assay
Coupled in vitro transcription/translation reactions were used to produce [35S]methionine-labeled estrogen receptors {alpha} (HEG0 and HEG537F) in rabbit reticulocyte lysate (Promega). For protein-protein interaction assay, the matrix with adsorbed fusion proteins was incubated with in vitro synthesized proteins for 1 h at room temperature by gentle shaking in PBS (containing 0.2% Triton X-100, 10 µg/mL LAP, and 1 mmol/L phenylmethylsulfonyl fluoride) in the presence of hormone, in absence or in the presence of p-Y peptide. Beads were then washed thrice in the same buffer and proteins were eluted with Laemmli sample buffer. They were finally resolved by SDS-PAGE, and protein bands were revealed by autoradiography.

Lysates, Immunoprecipitation, and Kinase Assays
Lysates were prepared as previously described (5), and protein concentration was measured with a Bio-Rad protein assay kit (Bio-Rad). Equal amounts of cell lysates (2 mg protein/mL) were used for immunoprecipitation of Src and Erk-2 (5). Src activity was assayed using enolase as a substrate and Erk-2 activity was detected using myelin basic protein as a substrate (5).

Electrophoresis and Immunoblotting
The electrophoresis and immunoblotting procedures were done as described elsewhere (26). Src was revealed using the mouse monoclonal anti-Src antibody (clone 327; Calbiochem). Erk-2 was detected using the rabbit polyclonal anti-Erk-2 antibody (C-19; Santa Cruz Biotechnology). Cyclin D1 was revealed using the mouse monoclonal antibody (AM20; Zymed). ER{alpha} was immunoblotted using the rabbit polyclonal anti-ER (HC-20; Santa Cruz Biotechnology) antibody. AR was immunoblotted using rabbit polyclonal antibodies (N-20; Santa Cruz Biotechnology). Immunoreactive proteins were revealed with the enhanced chemiluminescence detection system (Amersham Biosciences).

Flow Cytometric Analysis
Cells were treated with 10 nmol/L estradiol alone or in the presence of the indicated compounds for 1 h and then exposed to 2 mmol/L H2O2 for 90 min. Cells undergoing apoptosis were detected with the use of double staining with Annexin V–FITC and propidium iodide in the dark according to the manufacturer's instructions. Briefly, cells attached to plastic dishes were harvested by 0.25% trypsin and washed twice with cold PBS. The cell pellets were suspended in 1x binding buffer [10 mmol/L HEPES/NaOH (pH 7.4), 140 mmol/L NaCl, 2.5 mmol/L CaCl2] at a concentration of 1 x 106 cells/mL. Then, the cells were incubated with Annexin V–FITC and propidium iodide for 15 min (22-25°C) in dark. The stained cells were immediately analyzed by flow cytometry (FACSCalibur BD). Annexin V–FITC selectively passed through the plasma membranes of apoptotic cells and stained them with green fluorescence. Apoptosis was considered to have taken place in cells positive for Annexin V–FITC and negative for propidium iodide. All data were analyzed with Cell Quest software (BD). Each measurement was carried out at least in triplicate.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assays
A tetrazolium-based assay was used to determine cytotoxicity. Briefly, ~5 x 104 cells were seeded in 96-well plates and cultured as for BrdUrd assay. Then, 10 nmol/L estradiol alone or 1 nmol/L pY-pep or Y-pep in the absence and in the presence of 10 nmol/L estradiol were added. Cell viability was examined at 24 h after treatment. Before testing, 10 µL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium labeling reagent (5 mg/mL in PBS) were added, and the cells were incubated for 4 h at 37°C. Then, 100 µL DMSO were added, and the plate was incubated for a further 15 min at room temperature to dissolve formazan crystals. The absorbance was measured at a wavelength of 570 nm. Cell viability was calculated as the ratio between treated and untreated cells.

Statistical Methods
Where indicated, the statistical significance of the results was evaluated by paired t test. P values are reported in the legends to the figures.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank F. Claessens (Department of Molecular Cell Biology, University of Leuven, Leuven, Belgium) for the 3416 ARE-Luc construct, L. Altucci for the estradiol-responsive element–luc construct, Zeneca (Italy) for the antiestrogen ICI 182,780 and Casodex, and Flavia Vitale for technical assistance.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: Associazione Italiana per la Ricerca sul Cancro, Ministero dell'Università e della Ricerca Scientifica (Cofin 2006), and Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: L. Varricchio and A. Migliaccio contributed equally to this work.

4 Unpublished data. Back

Received 4/ 3/07; revised 6/14/07; accepted 7/23/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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M. Lombardi, G. Castoria, A. Migliaccio, M. V. Barone, R. Di Stasio, A. Ciociola, D. Bottero, H. Yamaguchi, E. Appella, and F. Auricchio
Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells
J. Cell Biol., July 28, 2008; 182(2): 327 - 340.
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