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Cell proliferation regulated by estradiol receptor: Therapeutic implications
Steroids 75 (2010) 524–527
Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Cell proliferation regulated by estradiol receptor: Therapeutic implications Gabriella Castoria, Antimo Migliaccio, Pia Giovannelli, Ferdinando Auricchio ∗ Dipartimento di Patologia Generale, II Università di Napoli, Via L. De Crecchio 7, 80138 Naples, Italy
a r t i c l e
i n f o
Article history: Available online 30 October 2009 Keywords: Estradiol receptor Non-genomic action Nuclear export Specific interfering peptides Breast cancer therapy
a b s t r a c t Estrogen receptor (ER) is a ligand-regulated transcription factor that controls human breast cancer cell proliferation. About 60–70% of human breast cancers express ER. In spite of major progress in the therapy of human breast cancer, many patients become resistant to pharmacologic treatments and develop metastatic breast tumors. Several mechanisms have been proposed to explain tumor progression and resistance to the therapies. However, the causes of hormone-dependent breast tumor progression as well as therapy resistance are still debated. An increasing body of evidence from our and other laboratories shows that in breast cancer cells, in addition to its classical transcriptional action, ER stimulates proliferative and anti-apoptotic signaling pathways in response to either ligand binding or growth factors. This discovery has led to the synthesis of new compounds specifically interfering in the rapid responses mediated by ER. It also suggests that the modalities currently in use for breast cancer treatment need to be reconsidered. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Breast cancer is very common in developed countries and the status of estradiol receptor (ER) is a prognostic marker in this tumor. Implication of ER in breast cancer progression is corroborated by the finding that about 60–70% of human breast cancers are ERalpha-positive. Most patients with ERalpha-positive breast cancer are treated with estradiol antagonists or aromatase inhibitors. Although these therapies improve the outcome of breast cancer, many patients become resistant to the treatment and develop metastatic breast tumors. Several mechanisms have been proposed to explain the causes of resistance to endocrine therapy [1]. These include expression of ER variants and ligand-independent activation of ER, as well as over-expression and activation of EGF-R and tyrosine kinases, including Src [2]. Estradiol controls proliferation and survival of breast cancer cells. This activity has until now been attributed to regulation of gene transcription [3]. In addition to well-known nuclear functions, ER (alpha or beta) participates in extra-nuclear and membrane-mediated signaling events. This non-genomic action has been linked to rapid responses elicited by estradiol and involves activation of Src, mitogen-activated protein kinase (MAPK),
Abbreviations: CRM1, chromosome region maintenance 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ER, estradiol receptor; FKHR, forkhead in rhabdomyosarcoma; MAPK, mitogen-activated protein kinase; NES, nuclear export signal; PI3-K, phosphatidylinositol-3-kinase; PKC, protein kinase C. ∗ Corresponding author. Tel.: +39 0815665676; fax: +39 081291327. E-mail address: [email protected] (F. Auricchio). 0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2009.10.007
phosphatidylinositol-3-kinase (PI3-K), protein kinase C (PKC) and etherotrimeric G-proteins in cytoplasm or membrane of target cells [4]. In addition, cytoplasmic cross talk between EGF and extranuclear ER has been investigated in breast cancer cells [5]. Thus, important biological responses such as DNA synthesis, cell growth and processes related to ER cytoplasm re-localization occur upon activation of these signaling effectors [6–11]. In this review, we will highlight the role of the signaling network regulated by ER in breast cancer cells. Our findings are integrated with further data we obtained using peptides specifically interfering in this network. 2. Genomic and non-genomic models of estradiol action In mammals, estradiol influences cell proliferation, inflammatory response, cardiovascular health, immunity, bone integrity, cognition and behaviour. Regulation of these effects may be mediated by a complex interface between signaling cascade activation and control of gene expression. ER (alpha or beta) in the cell nucleus regulates gene expression, whereas classical ER localized in the extra-nuclear compartment of target cells activates signal transduction pathways [4]. Genomic effects of estradiol usually occur via ligand-dependent binding of receptors to target gene promoters [3]. However, the resulting fluctuation in mRNAs and the proteins they encode takes place within hours or even days following hormonal exposure. In contrast, estradiol activation of signal transducing pathways occurs within seconds or minutes and is independent of RNA or protein synthesis. To date, all the members of the sex steroid hormone family exhibit non-genomic actions in reproductive as well as non-reproductive cells. These actions range
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from activation of Src, MAPKs, adenylyl cyclase and PI3-K to rises in intracellular calcium concentrations [12]. We previously focused on the analysis of interplay between estradiol-induced signaling activation and cell cycle progression in target cells. We identified and characterized ER/Src interaction that intersects PI3-K signaling in breast cancer cells [7,9,13–15]. This network controls G1/S progression in cultured MCF-7 cells [6–8], cell growth in mouse models of tumorigenesis [11] and even ER cytoplasm re-localization in MCF-7 cells [9,10]. This model of estradiol action points to the proliferative role of rapid hormonal action and the associated extra-nuclear localization of ER, thus linking signaling pathway activation by estradiol with sub-cellular ER localization.
3. The role of Src and PI3-K in non-genomic action of estradiol Some years ago we observed that estradiol rapidly (within seconds) and transiently triggers Src activation as well as protein tyrosine phosphorylation in breast cancer-derived MCF-7 cells [16]. Evidence collected in our laboratory also pointed to the role of classical ER in mediating this response [13,16]. We subsequently showed that ERalpha directly interacts through its tyrosine 537 with the SH2 domain of Src. Activation of the Src-dependent pathway follows this association, with dramatic effects on nuclear events leading to cell proliferation [6,13,15]. A similar mechanism also takes place upon EGF stimulation of breast cancer MCF-7 cells [5]. EGF, like estradiol, induces ER/Src association, thereby activating the Src-dependent pathway. This activation triggers both DNA synthesis and motility of breast cancer MCF-7 cells [5]. Thus, ER/Src association represents a novel and promising target in therapy of breast cancer, since these tumors respond to both steroid hormones and growth factors. Estradiol activation of Src occurs simultaneously with activation of PI3-K in MCF-7 cells, where it triggers the assembly of a multi-molecular complex made up of ER, Src, the regulatory subunit of PI3-K, p85 alpha, and other signaling effectors [7,8]. Estradiolactivated PI3-K triggers AKT and atypical PKC zeta activation. Once activated, AKT up-regulates cyclin D1 [7], while PKC zeta fosters p27 nuclear release by recruiting Ras to the ER/Src/PI3-K complex and inducing Erk-2 nuclear translocation [8]. This interplay between signaling effectors and cell cycle regulators controls cell cycle progression in MCF-7 cells. Altogether, our findings suggested that interference in the ER/Src interaction inhibits estradiol-activated cell cycle progression in cultured cells and most likely impairs cell growth in vivo. Data on estradiol activation of extra-nuclear signaling pathways point to the extra-nuclear localization of ER. In addition, it has been reported that ERalpha (or beta) exists as a monomer to caveolae rafts at cell surface, and that ligand addition induces receptor dimerization and recruitment of signaling effectors in different cell types [17]. Altogether, these findings show that ER extra-nuclear localization can orchestrate receptor-mediated signaling activation and biological responses in target cells. It has been recently reported that estradiol activation of PI3K/AKT pathway controls the nuclear export of ERalpha in MCF-7 cells. The nuclear export sequence has been mapped within the COOH-terminus of ER and it has been shown that an ERalpha mutant (NES-ERalpha mutant), unable to interact with CRM1, does not exit nuclei and impairs S-phase entry in MCF-7 cells [9]. Thus, estradiol activation of this signaling pathway has been linked with ER nuclear export and cell cycle progression in breast cancer cells. FKHR is a member of the FOXO transcriptional factor family which targets different factors, including cyclin D1 [18]. Since FKHR nuclear export depends on its phosphorylation by AKT [19]
Fig. 1. Model of ERalpha nuclear export. Estradiol stimulates the PI3-K/AKT pathway, which triggers FKHR phosphorylation and the associated export of FKHR/ER. This export results in cyclin D1 up-regulation and DNA synthesis stimulation.
and estradiol activation of the PI3-K/AKT pathway positively controls S-phase entry in MCF-7 cells [7], we speculated that FKHR might play a role in this network. Thus, we observed that the estradiol-activated PI3-K/AKT pathway leads to phosphorylation of FKHR at Ser 256 in MCF-7 cells. In addition, over-expression of the FKHR triple mutant (FKHR-AAA), which cannot be phosphorylated by AKT, inhibits estradiol-induced S-phase entry in MCF-7 cells [9]. This FKHR mutant is permanently localized in nuclei, thereby inducing G1 arrest in cells [20]. We observed that over-expression of this mutant sequesters ERalpha in the nuclear compartment of estradiol-treated MCF-7 cells and that, in turn, over-expression of NES-ERalpha mutant retains wild type FKHR in the nuclei. Thus, estradiol simultaneously regulates ER and FKHR nuclear export. From this and other experimental approaches, we concluded that FKHR phosphorylation induced by estradiol in MCF-7 breast cancer cells is required for its association with receptor as well as the export of the FKHR/ER complex with the consequent release of FKHR-mediated cell cycle inhibition. Thus, in ER-positive breast cancer cells, FKHR moves from nucleus to cytoplasm via a receptordependent mechanism [9]. FKHR nuclear export is regulated by PI3-K/AKT pathway, which through FKHR export induces cell cycle progression up-regulating cyclin D1 and down-regulating p27. In addition, FOXO factors down-regulate cyclin D1 and up-regulate p27 expression through a transcriptional mechanism [21–23]. In both cases, cell cycle arrest follows. Recent findings from our laboratory suggest that estradiolinduced FKHR phosphorylation controls cyclin D1 expression in MCF-7 cells (unpublished data). This, together with previous results on induction of cyclin D1 transcription by estradiol-activated PI3K/AKT pathway in MCF-7 cells [7], led us to propose the model presented in Fig. 1. According to this model, estradiol-stimulated PI3-K/AKT pathway induces FKHR phosphorylation and positively controls the nuclear export of FKHR/ERalpha complex. In this way, the inhibitory action of FKHR on cyclin D1 transcription is removed and breast cancer cells enter S-phase. These findings also imply that interference in ER nuclear export blocks cell cycle progression by inhibiting cytoplasmic release of FKHR in breast cancer cells. 4. An ER-derived peptide specifically interferes in ER/Src association The observation that ERalpha phosphotyrosine 537 is required for the association of estradiol receptor alpha with the Src-SH2 domain [15] led us to design and synthesize a small peptide derived from ERalpha sequence surrounding Tyr537 [11]. This six-amino
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Table 1 pY-peptide: Ac-Leu-pTyr-Asp-Leu-Leu-Leu-NH2. Rapidly penetrates across cell membrane and diffuses through the extra nuclear compartment of MCF-7 cells by a temperature-independent mechanism Is active at very low concentration (1 nM) in MCF-7 cells In contrast to classic estradiol antagonists, does not affect receptor-dependent transcriptional activity
acid peptide (536–541; Table 1) was used in parallel with the non-phosphorylated form of the same peptide to evaluate the role of tyrosine phosphorylation in the peptide [24]. The peptides were amino-terminal acetylated and carboxyl-terminal amidated to reduce cleavage by exopeptidases. In short-term experiments (up to 24 hours) the non-phosphorylated peptide had a much weaker inhibitory effect than that of the phosphorylated peptide, suggesting a dominant role of tyrosine phosphorylation. In longterm experiments, this difference disappeared. Use of the peptide with the same sequence, except for substitution of tyrosine with the non-phosphorylatable phenylalanine derivative, restores the difference, indicating that in long-term experiments the tyrosine residue of peptide undergoes phosphorylation. When MCF-7 cells were treated with estradiol or EGF, DNA synthesis was stimulated. This stimulation was abolished by the antiestrogen ICI 182780 as well as by 1 nM phosphorylated peptide. The phospho-peptide effect follows inhibition of ERalpha association with Src and all the events following this association, including Src and Erk activation as well as cyclin D1 up-regulation in MCF-7 cells [11]. The peptide does not affect EGF-induced DNA synthesis in human mammary cancerderived MDA-MB231 cells, which do not express ER, thus indicating that the peptide specifically targets ER [11]. In MCF-7 cells, the survival effect induced by estradiol is inhibited by the phosphopeptide, widening the potential interest for the applicative aspects of this peptide [11]. Lastly, the specificity of the Src-dependent pathway on proliferative inhibitory action is clearly illustrated by the absence of inhibition by the phospho-peptide on estradiolinduced ERE-dependent transcriptional activity in MCF-7 cells [11]. Finally, the peptide inhibits tumor growth in MCF-7 cell xenografts established in immunodepressed mice in a dose-dependent fashion. Table 1 summarizes the properties of the peptide. 5. An ER-derived peptide specifically interferes in ER nuclear export
Table 2 Tat-peptide: Ac-EFVCLKSIILLNS-AAA-RKKRRQRRR-NH2. Penetrates across cell membrane and rapidly reaches the nuclear compartment of MCF-7 cells Specifically retains ERalpha in the nuclear compartment of MCF-7 cells Specifically inhibits estradiol-induced S-phase entry in MCF-7 cells
analysis revealed that the carboxyfluorescein-conjugated peptide translocated across the plasma membrane and, within 30 min, accumulated in nuclei of MCF-7 cells [9]. This peptide displaced the estradiol-induced in vitro interaction between recombinant CRM1 and ERalpha, and sequestered the receptor in nuclei of 60 min estradiol-treated breast cancer MCF-7 cells [9]. The Tat-conjugated peptide did not affect p27 or p53 nuclear export [9], indicating that it specifically interferes in ERalpha localization. Interestingly, the Tat-conjugated peptide markedly reduced estradiol-induced DNA synthesis, whereas it did not interfere in serum-induced DNA synthesis in MCF-7 cells, thus indicating that it specifically interferes in estradiol action. Table 2 illustrates the properties of the Tat-peptide. 6. Conclusions Evidence collected in the last few years reveals a crucial role of non-genomic estradiol action in breast cancer cell proliferation. It also identifies a new role for ER nuclear export in DNA synthesis in breast cancer cells. It is plausible that alterations in nuclear trafficking of steroid receptors contribute to human proliferative diseases. Therefore, a detailed analysis of nuclear transport pathways and regulation of this process is expected to provide new insights into how sub-cellular compartmentalization contributes to cell cycle regulation in both normal and patho-physiological states. Lastly, our findings support the notion that specific targeting of interaction between ERalpha and signaling effectors (e.g. Src; 11) or nuclear export receptor (e.g. CRM1; 9) antagonizes the proliferative rate of breast cancer cells. Src tyrosine kinase is frequently deregulated [2] and ERalpha is mislocalized in human breast cancers [27]. In this respect, the strategy of interfering in ER/Src association or trapping ER in the nuclear compartment may make an important contribution to the development of novel therapeutic approaches for human breast cancers. Conflict of interest
Our findings on the regulation of ERalpha nucleo-cytoplasm shuttling supported the conclusion that the 444–456 sequence of ER contains a putative NES. This sequence shows homology with the conserved leucine-rich and REV-like NES of p53 and, remarkably, is conserved in other steroid receptors, such as ERbeta, PgR, AR, GR and MR, thus suggesting that this sequence is responsible for CRM1-dependent nuclear export of most steroid receptors [9,25]. Mutations in the core of this sequence impair estradiolinduced nuclear export of full-length ERalpha in MCF-7 cells [9]. Since the NES-ERalpha mutant we prepared fails to mediate estradiol-induced S-phase entry, we reasoned that targeting of NES-ER sequence by synthetic peptides inhibits estradiol-induced DNA synthesis by trapping ER/FKHR complex in nuclei of target cells. Small cationic peptides (also called protein transduction domains, PTDs) derived from nucleic acid- binding proteins, such as HIV-Tat protein, deliver a myriad of peptides into animal models and represent a biological and potentially therapeutic tool for targeting proteins in the cells [26]. To this end, we synthesized a Tat-conjugated peptide construct corresponding to the residues 444–456 of ERalpha (Table 2). Confocal microscopy
The authors declare that they do not have competing financial interests. Acknowledgements The work here presented was supported by grants from Associazione Italiana per la Ricerca sul Cancro (National and Regional Grants) and Regione Campania (Legge 5). References [1] Gururaj AE, Rayala SK, Vadlamudi RK, Kumar R. Novel mechanisms of resistance to endocrine therapy: genomic and nongenomic considerations. Clin Cancer Res 2006;12:1001s–7s. [2] Biscardi JS, Ishizawar RC, Silva CM, Parsons SJ. Tyrosine kinase signalling in breast cancer: epidermal growth factor receptor and c-Src interactions in breast cancer. Breast Cancer Res 2000;2:203–10. [3] Beato M, Herrlich P, Schütz G. Steroid hormone receptors: many actors in search of a plot. Cell 1995;83:851–7. [4] Migliaccio A, Castoria G, Auricchio F. Src-dependent signalling pathway regulation by sex-steroid hormones: therapeutic implications. Int J Biochem Cell Biol 2007;39:1343–8.
G. Castoria et al. / Steroids 75 (2010) 524–527 [5] Migliaccio A, Di Domenico M, Castoria G, Nanayakkara M, Lombardi M, de Falco A, et al. Steroid receptor regulation of EGF signaling through Src in breast and prostate cancer cells: steroid antagonist action. Cancer Res 2005;65:10585–93. [6] Castoria G, Barone MV, Di Domenico M, Bilancio A, Ametrano D, Migliaccio A, et al. Non-transcriptional action of estrogen and progestin triggers DNA synthesis. EMBO J 1999;18:2500–10. [7] Castoria G, Migliaccio A, Bilancio A, Di Domenico M, de Falco A, Lombardi M, et al. PI3-kinase in concert with Src promotes the S-phase entry of estradiolstimulated MCF-7 cells. EMBO J 2001;20:6050–9. [8] Castoria G, Migliaccio A, Di Domenico M, Lombardi M, de Falco A, Varricchio L, et al. Role of atypical PKC in estradiol-triggered G1/S progression of MCF-7 cells. Mol Cell Biol 2004;24:7643–53. [9] Lombardi M, Castoria G, Migliaccio A, Barone MV, Di Stasio R, Ciociola A, et al. Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells. J Cell Biol 2008;182:327–40. [10] Le Romancer M, Treilleux I, Leconte N, Robein-Lespinasse Y, Sentis S, Bouchekioua-Bouzaghou K, et al. Regulation of estrogen rapid signaling through arginine methylation by PRMT1. Mol Cell 2008;31:212–21. [11] Varricchio L, Migliaccio A, Castoria G, Yamaguchi H, de Falco A, Di Domenico M, et al. Inhibition of estradiol receptor/Src association and cell growth by an estradiol receptor {alpha} tyrosine-phosphorylated peptide. Mol Cancer Res 2007;11:1213–21. [12] Castoria G, Migliaccio A, D’Amato L, Di Stasio R, Ciociola A, Lombardi M, et al. Integrating signals between cAMP and MAPK pathways in breast cancer. Front Biosci 2008;13:1318–27. [13] Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, et al. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J 1996;15:1292–300. [14] Migliaccio A, Piccolo D, Castoria G, Di Domenico M, Bilancio A, Lombardi M, et al. Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor. EMBO J 1998;17:2008–18. [15] Migliaccio A, Castoria G, Di Domenico M, de Falco A, Bilancio A, Lombardi M, et al. Steroid-induced androgen receptor-oestradiol receptor beta Src complex triggers prostate cancer cell proliferation. EMBO J 2000;19:5406–17.
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[16] Migliaccio A, Pagano M, Auricchio F. Immediate and transient stimulation of protein tyrosine phosphorylation by estradiol in MCF-7 cells. Oncogene 1993;8:2183–91. [17] Hammes SR, Levin ER. Extranuclear steroid receptors: nature and actions. Endocr Rev 2007;28:726–41. [18] Birkenkamp KU, Coffer PJ. Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors. Biochem Soc Trans 2003;31:292–7. [19] Biggs 3rd WH, Meisenhelder J, Hunter T, Cavenee WK, Arden KC. Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci USA 1999;96:7421–6. [20] Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, Sellers WR. Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Biol 2000;20:8969–82. [21] Medema RH, Kops GJ, Bos JL, Burgering BM. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1 . Nature 2000;404:782–7. [22] Schmidt M, de Mattos SF, van der Horst A, Klompmaker R, Kops GJPL, Lam EWF, et al. Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D1. Mol Cell Biol 2002;22:7842–52. [23] Ramaswamy S, Nakamura N, Sansal I, Bergeron L, Sellers WR. A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell 2002;2:81–91. [24] Auricchio F, Migliaccio A, Castoria G. Sex-steroid hormones and EGF signalling in breast and prostate cancer cells: targeting the association of Src with steroid receptors. Steroids 2008;73:880–4. [25] Castoria G, Migliaccio A, Auricchio F. Signaling-dependent nuclear export of estradiol receptor controls cell cycle progression in breast cancer cells. Mol Cell Endocrinol 2009;308:26–31. [26] Joliot A, Prochiantz A. Transduction peptides: from technology to physiology. Nat Cell Biol 2004;6:189–96. [27] Kumar R, Wang RA, Mazumdar A, Talukder AH, Mandal M, Yang Z, et al. A naturally occurring MTA1 variant sequesters oestrogen receptor-alpha in the cytoplasm. Nature 2002;418:654–7.
Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Cell proliferation regulated by estradiol receptor: Therapeutic implications Gabriella Castoria, Antimo Migliaccio, Pia Giovannelli, Ferdinando Auricchio ∗ Dipartimento di Patologia Generale, II Università di Napoli, Via L. De Crecchio 7, 80138 Naples, Italy
a r t i c l e
i n f o
Article history: Available online 30 October 2009 Keywords: Estradiol receptor Non-genomic action Nuclear export Specific interfering peptides Breast cancer therapy
a b s t r a c t Estrogen receptor (ER) is a ligand-regulated transcription factor that controls human breast cancer cell proliferation. About 60–70% of human breast cancers express ER. In spite of major progress in the therapy of human breast cancer, many patients become resistant to pharmacologic treatments and develop metastatic breast tumors. Several mechanisms have been proposed to explain tumor progression and resistance to the therapies. However, the causes of hormone-dependent breast tumor progression as well as therapy resistance are still debated. An increasing body of evidence from our and other laboratories shows that in breast cancer cells, in addition to its classical transcriptional action, ER stimulates proliferative and anti-apoptotic signaling pathways in response to either ligand binding or growth factors. This discovery has led to the synthesis of new compounds specifically interfering in the rapid responses mediated by ER. It also suggests that the modalities currently in use for breast cancer treatment need to be reconsidered. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Breast cancer is very common in developed countries and the status of estradiol receptor (ER) is a prognostic marker in this tumor. Implication of ER in breast cancer progression is corroborated by the finding that about 60–70% of human breast cancers are ERalpha-positive. Most patients with ERalpha-positive breast cancer are treated with estradiol antagonists or aromatase inhibitors. Although these therapies improve the outcome of breast cancer, many patients become resistant to the treatment and develop metastatic breast tumors. Several mechanisms have been proposed to explain the causes of resistance to endocrine therapy [1]. These include expression of ER variants and ligand-independent activation of ER, as well as over-expression and activation of EGF-R and tyrosine kinases, including Src [2]. Estradiol controls proliferation and survival of breast cancer cells. This activity has until now been attributed to regulation of gene transcription [3]. In addition to well-known nuclear functions, ER (alpha or beta) participates in extra-nuclear and membrane-mediated signaling events. This non-genomic action has been linked to rapid responses elicited by estradiol and involves activation of Src, mitogen-activated protein kinase (MAPK),
Abbreviations: CRM1, chromosome region maintenance 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ER, estradiol receptor; FKHR, forkhead in rhabdomyosarcoma; MAPK, mitogen-activated protein kinase; NES, nuclear export signal; PI3-K, phosphatidylinositol-3-kinase; PKC, protein kinase C. ∗ Corresponding author. Tel.: +39 0815665676; fax: +39 081291327. E-mail address: [email protected] (F. Auricchio). 0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2009.10.007
phosphatidylinositol-3-kinase (PI3-K), protein kinase C (PKC) and etherotrimeric G-proteins in cytoplasm or membrane of target cells [4]. In addition, cytoplasmic cross talk between EGF and extranuclear ER has been investigated in breast cancer cells [5]. Thus, important biological responses such as DNA synthesis, cell growth and processes related to ER cytoplasm re-localization occur upon activation of these signaling effectors [6–11]. In this review, we will highlight the role of the signaling network regulated by ER in breast cancer cells. Our findings are integrated with further data we obtained using peptides specifically interfering in this network. 2. Genomic and non-genomic models of estradiol action In mammals, estradiol influences cell proliferation, inflammatory response, cardiovascular health, immunity, bone integrity, cognition and behaviour. Regulation of these effects may be mediated by a complex interface between signaling cascade activation and control of gene expression. ER (alpha or beta) in the cell nucleus regulates gene expression, whereas classical ER localized in the extra-nuclear compartment of target cells activates signal transduction pathways [4]. Genomic effects of estradiol usually occur via ligand-dependent binding of receptors to target gene promoters [3]. However, the resulting fluctuation in mRNAs and the proteins they encode takes place within hours or even days following hormonal exposure. In contrast, estradiol activation of signal transducing pathways occurs within seconds or minutes and is independent of RNA or protein synthesis. To date, all the members of the sex steroid hormone family exhibit non-genomic actions in reproductive as well as non-reproductive cells. These actions range
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from activation of Src, MAPKs, adenylyl cyclase and PI3-K to rises in intracellular calcium concentrations [12]. We previously focused on the analysis of interplay between estradiol-induced signaling activation and cell cycle progression in target cells. We identified and characterized ER/Src interaction that intersects PI3-K signaling in breast cancer cells [7,9,13–15]. This network controls G1/S progression in cultured MCF-7 cells [6–8], cell growth in mouse models of tumorigenesis [11] and even ER cytoplasm re-localization in MCF-7 cells [9,10]. This model of estradiol action points to the proliferative role of rapid hormonal action and the associated extra-nuclear localization of ER, thus linking signaling pathway activation by estradiol with sub-cellular ER localization.
3. The role of Src and PI3-K in non-genomic action of estradiol Some years ago we observed that estradiol rapidly (within seconds) and transiently triggers Src activation as well as protein tyrosine phosphorylation in breast cancer-derived MCF-7 cells [16]. Evidence collected in our laboratory also pointed to the role of classical ER in mediating this response [13,16]. We subsequently showed that ERalpha directly interacts through its tyrosine 537 with the SH2 domain of Src. Activation of the Src-dependent pathway follows this association, with dramatic effects on nuclear events leading to cell proliferation [6,13,15]. A similar mechanism also takes place upon EGF stimulation of breast cancer MCF-7 cells [5]. EGF, like estradiol, induces ER/Src association, thereby activating the Src-dependent pathway. This activation triggers both DNA synthesis and motility of breast cancer MCF-7 cells [5]. Thus, ER/Src association represents a novel and promising target in therapy of breast cancer, since these tumors respond to both steroid hormones and growth factors. Estradiol activation of Src occurs simultaneously with activation of PI3-K in MCF-7 cells, where it triggers the assembly of a multi-molecular complex made up of ER, Src, the regulatory subunit of PI3-K, p85 alpha, and other signaling effectors [7,8]. Estradiolactivated PI3-K triggers AKT and atypical PKC zeta activation. Once activated, AKT up-regulates cyclin D1 [7], while PKC zeta fosters p27 nuclear release by recruiting Ras to the ER/Src/PI3-K complex and inducing Erk-2 nuclear translocation [8]. This interplay between signaling effectors and cell cycle regulators controls cell cycle progression in MCF-7 cells. Altogether, our findings suggested that interference in the ER/Src interaction inhibits estradiol-activated cell cycle progression in cultured cells and most likely impairs cell growth in vivo. Data on estradiol activation of extra-nuclear signaling pathways point to the extra-nuclear localization of ER. In addition, it has been reported that ERalpha (or beta) exists as a monomer to caveolae rafts at cell surface, and that ligand addition induces receptor dimerization and recruitment of signaling effectors in different cell types [17]. Altogether, these findings show that ER extra-nuclear localization can orchestrate receptor-mediated signaling activation and biological responses in target cells. It has been recently reported that estradiol activation of PI3K/AKT pathway controls the nuclear export of ERalpha in MCF-7 cells. The nuclear export sequence has been mapped within the COOH-terminus of ER and it has been shown that an ERalpha mutant (NES-ERalpha mutant), unable to interact with CRM1, does not exit nuclei and impairs S-phase entry in MCF-7 cells [9]. Thus, estradiol activation of this signaling pathway has been linked with ER nuclear export and cell cycle progression in breast cancer cells. FKHR is a member of the FOXO transcriptional factor family which targets different factors, including cyclin D1 [18]. Since FKHR nuclear export depends on its phosphorylation by AKT [19]
Fig. 1. Model of ERalpha nuclear export. Estradiol stimulates the PI3-K/AKT pathway, which triggers FKHR phosphorylation and the associated export of FKHR/ER. This export results in cyclin D1 up-regulation and DNA synthesis stimulation.
and estradiol activation of the PI3-K/AKT pathway positively controls S-phase entry in MCF-7 cells [7], we speculated that FKHR might play a role in this network. Thus, we observed that the estradiol-activated PI3-K/AKT pathway leads to phosphorylation of FKHR at Ser 256 in MCF-7 cells. In addition, over-expression of the FKHR triple mutant (FKHR-AAA), which cannot be phosphorylated by AKT, inhibits estradiol-induced S-phase entry in MCF-7 cells [9]. This FKHR mutant is permanently localized in nuclei, thereby inducing G1 arrest in cells [20]. We observed that over-expression of this mutant sequesters ERalpha in the nuclear compartment of estradiol-treated MCF-7 cells and that, in turn, over-expression of NES-ERalpha mutant retains wild type FKHR in the nuclei. Thus, estradiol simultaneously regulates ER and FKHR nuclear export. From this and other experimental approaches, we concluded that FKHR phosphorylation induced by estradiol in MCF-7 breast cancer cells is required for its association with receptor as well as the export of the FKHR/ER complex with the consequent release of FKHR-mediated cell cycle inhibition. Thus, in ER-positive breast cancer cells, FKHR moves from nucleus to cytoplasm via a receptordependent mechanism [9]. FKHR nuclear export is regulated by PI3-K/AKT pathway, which through FKHR export induces cell cycle progression up-regulating cyclin D1 and down-regulating p27. In addition, FOXO factors down-regulate cyclin D1 and up-regulate p27 expression through a transcriptional mechanism [21–23]. In both cases, cell cycle arrest follows. Recent findings from our laboratory suggest that estradiolinduced FKHR phosphorylation controls cyclin D1 expression in MCF-7 cells (unpublished data). This, together with previous results on induction of cyclin D1 transcription by estradiol-activated PI3K/AKT pathway in MCF-7 cells [7], led us to propose the model presented in Fig. 1. According to this model, estradiol-stimulated PI3-K/AKT pathway induces FKHR phosphorylation and positively controls the nuclear export of FKHR/ERalpha complex. In this way, the inhibitory action of FKHR on cyclin D1 transcription is removed and breast cancer cells enter S-phase. These findings also imply that interference in ER nuclear export blocks cell cycle progression by inhibiting cytoplasmic release of FKHR in breast cancer cells. 4. An ER-derived peptide specifically interferes in ER/Src association The observation that ERalpha phosphotyrosine 537 is required for the association of estradiol receptor alpha with the Src-SH2 domain [15] led us to design and synthesize a small peptide derived from ERalpha sequence surrounding Tyr537 [11]. This six-amino
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Table 1 pY-peptide: Ac-Leu-pTyr-Asp-Leu-Leu-Leu-NH2. Rapidly penetrates across cell membrane and diffuses through the extra nuclear compartment of MCF-7 cells by a temperature-independent mechanism Is active at very low concentration (1 nM) in MCF-7 cells In contrast to classic estradiol antagonists, does not affect receptor-dependent transcriptional activity
acid peptide (536–541; Table 1) was used in parallel with the non-phosphorylated form of the same peptide to evaluate the role of tyrosine phosphorylation in the peptide [24]. The peptides were amino-terminal acetylated and carboxyl-terminal amidated to reduce cleavage by exopeptidases. In short-term experiments (up to 24 hours) the non-phosphorylated peptide had a much weaker inhibitory effect than that of the phosphorylated peptide, suggesting a dominant role of tyrosine phosphorylation. In longterm experiments, this difference disappeared. Use of the peptide with the same sequence, except for substitution of tyrosine with the non-phosphorylatable phenylalanine derivative, restores the difference, indicating that in long-term experiments the tyrosine residue of peptide undergoes phosphorylation. When MCF-7 cells were treated with estradiol or EGF, DNA synthesis was stimulated. This stimulation was abolished by the antiestrogen ICI 182780 as well as by 1 nM phosphorylated peptide. The phospho-peptide effect follows inhibition of ERalpha association with Src and all the events following this association, including Src and Erk activation as well as cyclin D1 up-regulation in MCF-7 cells [11]. The peptide does not affect EGF-induced DNA synthesis in human mammary cancerderived MDA-MB231 cells, which do not express ER, thus indicating that the peptide specifically targets ER [11]. In MCF-7 cells, the survival effect induced by estradiol is inhibited by the phosphopeptide, widening the potential interest for the applicative aspects of this peptide [11]. Lastly, the specificity of the Src-dependent pathway on proliferative inhibitory action is clearly illustrated by the absence of inhibition by the phospho-peptide on estradiolinduced ERE-dependent transcriptional activity in MCF-7 cells [11]. Finally, the peptide inhibits tumor growth in MCF-7 cell xenografts established in immunodepressed mice in a dose-dependent fashion. Table 1 summarizes the properties of the peptide. 5. An ER-derived peptide specifically interferes in ER nuclear export
Table 2 Tat-peptide: Ac-EFVCLKSIILLNS-AAA-RKKRRQRRR-NH2. Penetrates across cell membrane and rapidly reaches the nuclear compartment of MCF-7 cells Specifically retains ERalpha in the nuclear compartment of MCF-7 cells Specifically inhibits estradiol-induced S-phase entry in MCF-7 cells
analysis revealed that the carboxyfluorescein-conjugated peptide translocated across the plasma membrane and, within 30 min, accumulated in nuclei of MCF-7 cells [9]. This peptide displaced the estradiol-induced in vitro interaction between recombinant CRM1 and ERalpha, and sequestered the receptor in nuclei of 60 min estradiol-treated breast cancer MCF-7 cells [9]. The Tat-conjugated peptide did not affect p27 or p53 nuclear export [9], indicating that it specifically interferes in ERalpha localization. Interestingly, the Tat-conjugated peptide markedly reduced estradiol-induced DNA synthesis, whereas it did not interfere in serum-induced DNA synthesis in MCF-7 cells, thus indicating that it specifically interferes in estradiol action. Table 2 illustrates the properties of the Tat-peptide. 6. Conclusions Evidence collected in the last few years reveals a crucial role of non-genomic estradiol action in breast cancer cell proliferation. It also identifies a new role for ER nuclear export in DNA synthesis in breast cancer cells. It is plausible that alterations in nuclear trafficking of steroid receptors contribute to human proliferative diseases. Therefore, a detailed analysis of nuclear transport pathways and regulation of this process is expected to provide new insights into how sub-cellular compartmentalization contributes to cell cycle regulation in both normal and patho-physiological states. Lastly, our findings support the notion that specific targeting of interaction between ERalpha and signaling effectors (e.g. Src; 11) or nuclear export receptor (e.g. CRM1; 9) antagonizes the proliferative rate of breast cancer cells. Src tyrosine kinase is frequently deregulated [2] and ERalpha is mislocalized in human breast cancers [27]. In this respect, the strategy of interfering in ER/Src association or trapping ER in the nuclear compartment may make an important contribution to the development of novel therapeutic approaches for human breast cancers. Conflict of interest
Our findings on the regulation of ERalpha nucleo-cytoplasm shuttling supported the conclusion that the 444–456 sequence of ER contains a putative NES. This sequence shows homology with the conserved leucine-rich and REV-like NES of p53 and, remarkably, is conserved in other steroid receptors, such as ERbeta, PgR, AR, GR and MR, thus suggesting that this sequence is responsible for CRM1-dependent nuclear export of most steroid receptors [9,25]. Mutations in the core of this sequence impair estradiolinduced nuclear export of full-length ERalpha in MCF-7 cells [9]. Since the NES-ERalpha mutant we prepared fails to mediate estradiol-induced S-phase entry, we reasoned that targeting of NES-ER sequence by synthetic peptides inhibits estradiol-induced DNA synthesis by trapping ER/FKHR complex in nuclei of target cells. Small cationic peptides (also called protein transduction domains, PTDs) derived from nucleic acid- binding proteins, such as HIV-Tat protein, deliver a myriad of peptides into animal models and represent a biological and potentially therapeutic tool for targeting proteins in the cells [26]. To this end, we synthesized a Tat-conjugated peptide construct corresponding to the residues 444–456 of ERalpha (Table 2). Confocal microscopy
The authors declare that they do not have competing financial interests. Acknowledgements The work here presented was supported by grants from Associazione Italiana per la Ricerca sul Cancro (National and Regional Grants) and Regione Campania (Legge 5). References [1] Gururaj AE, Rayala SK, Vadlamudi RK, Kumar R. Novel mechanisms of resistance to endocrine therapy: genomic and nongenomic considerations. Clin Cancer Res 2006;12:1001s–7s. [2] Biscardi JS, Ishizawar RC, Silva CM, Parsons SJ. Tyrosine kinase signalling in breast cancer: epidermal growth factor receptor and c-Src interactions in breast cancer. Breast Cancer Res 2000;2:203–10. [3] Beato M, Herrlich P, Schütz G. Steroid hormone receptors: many actors in search of a plot. Cell 1995;83:851–7. [4] Migliaccio A, Castoria G, Auricchio F. Src-dependent signalling pathway regulation by sex-steroid hormones: therapeutic implications. Int J Biochem Cell Biol 2007;39:1343–8.
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