Review
Estrogen receptor alpha phosphorylation and its functional impact in human breast cancer

https://doi.org/10.1016/j.mce.2015.01.016Get rights and content

Highlights

  • Identification and initial functional characterization of four novel ERα phosphorylation sites; S46/47, S282, S294, and S559 provides additional understanding of the regulation of ERα signaling.

  • Site specific ERα phosphorylation directs temporal recruitment of ERα and unique coactivator complexes to specific genes.

  • Stable disruption of ERα S118 and S167 phosphorylation in breast cancer cells elicit unique gene expression profiles that culminate in significant effects on breast cancer growth/morphology/migration/invasion.

  • The Src kinase signaling pathway impacts ERα phosphorylation to alter ERα function.

  • Light exposure at night leads to elevated ERK1/2, Src activity and ERα phosphorylation, concomitant with tamoxifen resistance in breast tumor models.

Abstract

Estrogen receptor α (ERα) is a member of the nuclear receptor superfamily of transcription factors that regulates cell proliferation, differentiation and homeostasis in various tissues. Sustained exposure to estrogen/estradiol (E2) increases the risk of breast, endometrial and ovarian cancers. ERα function is also regulated by phosphorylation through various kinase signaling pathways that will impact various ERα functions including chromatin interaction, coregulator recruitment and gene expression, as well impact breast tumor growth/morphology and breast cancer patient response to endocrine therapy. However, many of the previously characterized ERα phosphorylation sites do not fully explain the impact of receptor phosphorylation on ERα function. This review discusses work from our laboratory toward understanding a role of ERα site-specific phosphorylation in ERα function and breast cancer. The key findings discussed in this review are: (1) the effect of site specific ERα phosphorylation on temporal recruitment of ERα and unique coactivator complexes to specific genes; (2) the impact of stable disruption of ERα S118 and S167 phosphorylation in breast cancer cells on eliciting unique gene expression profiles that culminate in significant effects on breast cancer growth/morphology/migration/invasion; (3) the Src kinase signaling pathway that impacts ERα phosphorylation to alter ERα function; and (4) circadian disruption by light exposure at night leading to elevated ERK1/2 and Src kinase and phosphorylation of ERα, concomitant with tamoxifen resistance in breast tumor models. Results from these studies demonstrate that even changes to single ERα phosphorylation sites can have a profound impact on ERα function in breast cancer. Future work will extend beyond single site phosphorylation analysis toward identification of specific patterns/profiles of ERα phosphorylation under different physiological/pharmacological conditions to understand how common phosphorylation profiles in breast cancer program specific physiological endpoints such as growth, apoptosis, migration/invasion, and endocrine therapy response.

Introduction

Estrogen receptor alpha (ERα) is a member of the nuclear receptor superfamily of transcription factors whose activity is primarily regulated by binding of estrogen/estradiol (E2). E2 plays an indispensable role in growth, development, reproduction and maintenance of numerous physiological systems in mammals. Sustained exposure to E2 is a well-known stimulatory signal for breast cancer growth (Colditz, 1998, Hankinson et al, 2004). ERα is responsible for many of the effects of E2 on normal mammary and breast cancer tissue via its ligand induced transcriptional program (genomic actions) (McDonnell and Norris, 2002), as well as elicitation of rapid, cytoplasmic signaling cascades (non-genomic actions) (Levin, 2009).

ERα is a modular protein consisting of a number of functional domains including an N-terminal domain, two transcriptional activation functions (AF-1, AF-2), a centrally located DNA binding domain, hinge region, and a C-terminal ligand binding domain (Fig. 1). The C-terminal domain of ERα is highly structured upon ligand binding and this domain is highly conserved across many species. The N-terminal domain is less structured and poorly conserved across species, and is activated by both ligand-dependent and ligand-independent mechanisms. Though ERα function is strongly activated by ligand binding, ERα function is also regulated by posttranslational modifications (PTMs), most significantly by phosphorylation. ERα phosphorylation occurs at multiple sites, most located in the N-terminal domain, is regulated by ligand binding and by ligand-independent mechanisms such as peptide growth factor signaling (Lannigan, 2003, Murphy et al, 2011, Ward, Weigel, 2009). ERα phosphorylation sites contribute to regulation of multiple functional activities including hormone sensitivity, nuclear localization, DNA binding, protein/chromatin interactions, protein stability and gene transcription (Al-Dhaheri, Rowan, 2007, Duplessis et al, 2011, Huderson et al, 2012, Joel et al, 1995, Shah, Rowan, 2005, Williams et al, 2009).

In the absence of ligand, ERα like other steroid hormone receptors exists as a monomer bound to chaperone protein heat shock protein 90 (Hsp90). In the ‘classical genomic’ action of ERα, upon ligand binding ERα dissociates from heat shock proteins and binds either directly to estrogen response element (ERE) sequences in genes, or is tethered to promoters via interaction with other transcription factors [e.g. activation protein 1 (AP1); specificity protein 1 (SP1)], and recruits coregulators and the transcription machinery to induce transcription of ERα regulated genes. Dependent upon cell and promoter context, ERα regulates transcription through its two activation domains, AF-1 and AF-2. AF-2 is contained within the LBD and becomes active upon E2 binding. AF-1 however, is activated by phosphorylation of ERα at several sites, most notably serine 118 (S118) and serine 167 (S167), sites that are regulated by multiple signaling pathways.

This review will discuss evidence from the author’s laboratory on the role of ERα phosphorylation sites, particularly S118 and S167, on ERα function in breast cancer, including effects on chromatin interaction, gene expression, the impact on protein complexes at target promoters, breast cancer growth and endocrine therapy response.

Section snippets

ERα phosphorylation sites

There are fourteen serine residues in the NTD (10, 46, 47, 84, 91, 102, 104, 106, 118, 137, 154, 167, 173, and 178), of which serines 102, 104, 106, 118, and 167 have been identified as phosphorylation sites by numerous laboratories [for review, see Le Romancer et al. (2011)]. A number of these phosphorylation sites have been functionally characterized including serines 102 (S102), 104 (S104), 106 (S106), 118 (S118), and 167 (S167) in the AF-1 domain, serine 236 (S236) in the DNA binding

Phosphorylation of Ser118 exerts gene selective effects on promoter complex assembly and transcription

Gene-specific assembly of ERα and coregulator complexes at promoters may be dictated by unique DNA sequences acting as allosteric ligands that change receptor conformation thereby recruiting a distinct set of coregulators (Hall et al, 2002, Lefstin, Yamamoto, 1998, Tora et al, 1989). However, it is equally likely that conformational changes in ERα induced by phosphorylation could also dictate coregulator assembly. p160 steroid receptor coactivator (SRC) family members/nuclear receptor

Loss of ERα phosphorylation at S118 or S167 markedly impacts breast cancer growth, cellular morphology, and ERα signaling

To date, stable disruption of ERα phosphorylation sites in vivo has not been reported. Given the importance of S118 and S167 as surrogate markers for favorable outcome for tamoxifen adjuvant therapy in breast cancer, and as markers for a functional ERα signaling pathway, it was of interest to assess the impact of stable disruption of S118 and S167 phosphorylation on breast cancer growth, morphology and E2-regulated gene expression. Remarkably, MCF-7 breast cancer cells in which endogenous ERα

Src kinase promotes ERα interaction with promoters through phosphorylation at S167

A molecular link between Src activation and ERα function was demonstrated by Src-mediated changes in ERα phosphorylation at S118 (Feng et al., 2001). S167 phosphorylation is remarkable for the profound effect loss of S167 phosphorylation has on in vitro DNA binding (Arnold et al, 1995, Castano et al, 1997). Our laboratory was the first to report the connection between activated Src and ERα S167 phosphorylation. Src kinase activated PI3K/AKT and AKT that in turn, directly phosphorylated S167

Dim light exposure at night (dLEN) impacts the circadian clock and ERα phosphorylation

The circadian clock in peripheral tissues coordinates intrinsic timing in most organisms including humans, and controls various physiological, biochemical and behavioral processes that include sleep/wake cycles, body temperature, blood pressure, hormone production, digestive juice secretion, and the immune system [reviewed in Fu and Lee (2003)]. The master clock in the brain controls and coordinates the rhythmicity in various tissues according to the light input it receives. In the present 24/7

Summary

ERα phosphorylation serves as a cellular sensor of environmental cues that will modulate the gene expression program of ERα signaling in breast cancer. Even in the presence of saturating levels of E2, specific ERα phosphorylation sites regulate a distinct temporal recruitment of ERα to promoters, as well as direct recruitment of distinct coregulator complexes that are unique for each ERα regulated gene. We have directed our efforts toward study of single ERα phosphorylation sites to establish

References (80)

  • LeoC. et al.

    The SRC family of nuclear receptor coactivators

    Gene

    (2000)
  • LeoC. et al.

    Differential mechanisms of nuclear receptor regulation by receptor-associated coactivator 3

    J. Biol. Chem

    (2000)
  • MedunjaninS. et al.

    Glycogen synthase kinase-3 interacts with and phosphorylates estrogen receptor alpha and is involved in the regulation of receptor activity

    J. Biol. Chem

    (2005)
  • MegdalS.P. et al.

    Night work and breast cancer risk: a systematic review and meta-analysis

    Eur. J. Cancer

    (2005)
  • MukherjeeR. et al.

    Ligand and coactivator recruitment preferences of peroxisome proliferator activated receptor alpha

    J. Steroid Biochem. Mol. Biol

    (2002)
  • ParkK.J. et al.

    Formation of an IKKalpha-dependent transcription complex is required for estrogen receptor-mediated gene activation

    Mol. Cell

    (2005)
  • RigginsR.B. et al.

    Pathways to tamoxifen resistance

    Cancer Lett

    (2007)
  • RogatskyI. et al.

    Potentiation of human estrogen receptor alpha transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A-CDK2 complex

    J. Biol. Chem

    (1999)
  • ShahY.M. et al.

    Selective estrogen receptor modulator regulated proteins in endometrial cancer cells

    Mol. Cell. Endocrinol

    (2004)
  • SongR.X. et al.

    Estrogen rapid action via protein complex formation involving ERalpha and Src

    Trends Endocrinol. Metab

    (2005)
  • ToraL. et al.

    The human estrogen receptor has two independent nonacidic transcriptional activation functions

    Cell

    (1989)
  • YamnikR.L. et al.

    mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor alpha serine 167 phosphorylation

    FEBS Lett

    (2010)
  • Al-DhaheriM.H. et al.

    Application of phosphorylation site-specific antibodies to measure nuclear receptor signaling: characterization of novel phosphoantibodies for estrogen receptor alpha

    Nucl. Recept. Signal

    (2006)
  • Al-DhaheriM.H. et al.

    Protein kinase A exhibits selective modulation of estradiol-dependent transcription in breast cancer cells that is associated with decreased ligand binding, altered estrogen receptor alpha promoter interaction, and changes in receptor phosphorylation

    Mol. Endocrinol

    (2007)
  • AnbalaganM. et al.

    KX-01, a novel Src kinase inhibitor directed toward the peptide substrate site, synergizes with tamoxifen in estrogen receptor alpha positive breast cancer

    Breast Cancer Res. Treat

    (2012)
  • ArnoldS.F. et al.

    Serine 167 is the major estradiol-induced phosphorylation site on the human estrogen receptor

    Mol. Endocrinol

    (1994)
  • BunoneG. et al.

    Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation

    EMBO J.

    (1996)
  • CastanoE. et al.

    Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation

    Biochem. J.

    (1997)
  • ChenD. et al.

    Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera

    Oncogene

    (2002)
  • ChenM. et al.

    Phosphorylation of estrogen receptor alpha at serine 118 is correlated with breast cancer resistance to tamoxifen

    Oncol. Lett

    (2013)
  • ChengJ. et al.

    A functional serine 118 phosphorylation site in estrogen receptor-alpha is required for down-regulation of gene expression by 17beta-estradiol and 4-hydroxytamoxifen

    Endocrinology

    (2007)
  • ColditzG.A.

    Relationship between estrogen levels, use of hormone replacement therapy, and breast cancer

    J. Natl Cancer Inst

    (1998)
  • DasguptaS. et al.

    Nuclear receptor coactivators: master regulators of human health and disease

    Annu. Rev. Med

    (2014)
  • DauchyR.T. et al.

    Circadian and melatonin disruption by exposure to light at night drives intrinsic resistance to tamoxifen therapy in breast cancer

    Cancer Res

    (2014)
  • DesoukiM.M. et al.

    SRC kinase and mitogen-activated protein kinases in the progression from normal to malignant endometrium

    Clin. Cancer Res

    (2004)
  • DuplessisT.T. et al.

    Phosphorylation of estrogen receptor alpha at serine 118 directs recruitment of promoter complexes and gene-specific transcription

    Endocrinology

    (2011)
  • DutertreM. et al.

    Ligand-independent interactions of p160/steroid receptor coactivators and CREB-binding protein (CBP) with estrogen receptor-alpha: regulation by phosphorylation sites in the A/B region depends on other receptor domains

    Mol. Endocrinol

    (2003)
  • FaganD.H. et al.

    Crosstalk between IGF1R and estrogen receptor signaling in breast cancer

    J. Mammary Gland Biol. Neoplasia

    (2008)
  • FengW. et al.

    Potentiation of estrogen receptor activation function 1 (AF-1) by Src/JNK through a serine 118-independent pathway

    Mol. Endocrinol

    (2001)
  • FilardoE.J. et al.

    Estrogen action via the G protein-coupled receptor, GPR30: stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis

    Mol. Endocrinol

    (2002)
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