Journal of Mammary Gland Biology and Neoplasia

, Volume 14, Issue 1, pp 67–78

ER Re-expression and Re-sensitization to Endocrine Therapies in ER-negative Breast Cancers

Authors

  • Joeli A. Brinkman
    • University of Miami, Miller School of MedicineDepartment of Medicine, Sylvester Comprehensive Cancer Center
    • University of Miami, Miller School of MedicineDepartment of Medicine, Sylvester Comprehensive Cancer Center
Article

DOI: 10.1007/s10911-009-9113-0

Cite this article as:
Brinkman, J.A. & El-Ashry, D. J Mammary Gland Biol Neoplasia (2009) 14: 67. doi:10.1007/s10911-009-9113-0

Abstract

Breast cancer is the leading cause of cancer amongst women in the westernized world. The presence or absence of ERα in breast cancers is an important prognostic indicator. About 30–40% of breast cancers lack detectable ERα protein. ERα− breast cancers are resistant to endocrine therapies and have a worse prognosis than ERα+ breast cancers. Since expression of ERα is necessary for response to endocrine therapies, investigational studies are ongoing in order to understand the generation of the ERα− phenotype and develop interventions to restore ERα expression in ERα− breast cancers. DNA methylation and chromatin remodeling are two epigenetic mechanisms that have been linked with the lack of ERα expression and in these cases; demethylation of the ERα promoter or treatment with HDAC inhibitors shows promise in restoring ERα expression in ERα− breast cancers. Two additional potential mechanisms underlying generation of the ERα− phenotype involve E6-AP and Src, both of which have been shown to be elevated in ERα− breast cancer and can drive the proteasomal degradation of ERα. Recently, studies have demonstrated that upregulated growth factor signaling due to hyperactive MAPK activity significantly contributes to generation of the ERα− phenotype and that inhibition of MAPK activity can cause re-expression of the ERα and restore sensitivity to endocrine therapies. Given the challenges in treating ERα− breast cancer, understanding and manipulating the cellular mechanisms that effect expression of ERα are imperative in order to restore sensitivity to endocrine therapies and to design novel therapeutics for the treatment of ERα− breast cancers.

Keywords

Breast cancerEstrogen receptorEndocrine therapyMAPKEGFRHER2HDACE6-APSrc

Abbreviations

ERα

estrogen receptor alpha

ERE

estrogen response element

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

MAPK

mitogen-activated protein kinase

Her2

hairy-related 2

erbB2

v-erb-b2 erythroblastic leukemia viral oncogene homolog 2

TGF-α

transforming growth factor alpha

E2

estradiol

HDAC

histone deacetylase

DNMT

DNA methyltransferase

SAHA

Suberoylanilide hydroxamic acid

AZA

5-aza-2’-deoxycytide

TSA

Trichostatin A

PAK1

p21 protein (Cdc42/Rac)-activated kinase 1

AKT

serine/threonine protein kinase Akt

PR

progesterone receptor

Src

v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog

E6-AP

ubiquitin protein ligase E3A

HSP

heat shock protein

Pl3K

phosphoinositide 3-kinase

SHC

SH2 Containing Protein

PKC

Protein kinase C

ERK

Extracellular Signal Regulated Kinases

JNK

c-Jun N-terminal Kinase

Introduction

Among women in the westernized world, breast cancer is the leading cause of cancer and the second leading cause of cancer related deaths. The American Cancer Society estimates that in 2008, 182,460 new cases and 40,480 deaths in women will be reported in the USA alone [3]. Considering the current estimates of the incidence of breast cancer, a woman living in the USA today has approximately a one in eight chance of developing breast cancer during her lifetime [2]. Clinically, breast cancer presents as either estrogen receptor alpha positive (ERα+) or estrogen receptor alpha negative (ERα−) with 60–70% of all breast tumors expressing ERα. The presence or absence of ERα in breast tumors is an important prognostic indicator of the disease. The presence of ERα correlates with increased disease-free survival and an overall better prognosis compared to breast cancers that lack ERα, which are characterized by a more aggressive phenotype and a poor prognosis. Importantly, ERα+ breast cancers respond to endocrine therapies like tamoxifen whereas ERα− tumors are resistant to endocrine therapies [17, 22, 50, 60, 61]. Tamoxifen, the most common hormonal therapy is effective in both pre- and postmenopausal patients with ERα+ tumors. With longer courses of tamoxifen therapy, recurrence and survival benefits increase and have been demonstrated for at least 10 years after treatment [2]. However, 25–35% of all ERα+ tumors demonstrate de novo resistance or show no initial response to tamoxifen and even those that do initially respond can develop or acquire resistance to tamoxifen therapy [43]. Up to 50% of patients bearing ERα+ primary tumors lose ERα expression in recurrent tumors that develop after relapse and about one third of metastic tumors that demonstrate an initial response develop resistance to tamoxifen therapy and lose ERα expression [15, 43, 44, 68]. In these instances, loss of ERα expression precludes the use of tamoxifen as adjuvant therapy as tamoxifen has shown no demonstrative therapeutic effect in ERα− breast cancers [25, 36]. Therefore, in breast tumors in which ERα is not expressed initially or that subsequently lose expression of ERα, re-expression of the ERα receptor could allow for restoration of anti-estrogen sensitivity, and maintenance of ERα expression may provide a means of prolonging the response to tamoxifen and other endocrine therapies. Due to the fact that expression of ERα is necessary for response to endocrine therapies including tamoxifen, fulvestrant and the aromatase inhibitors, intensive investigational studies have been undertaken to understand the generation of the ERα− phenotype and develop interventions to restore ERα expression in ERα− breast cancers.

Estrogen Receptor Biology in Breast Cancer

In order to appreciate the generation of the ERα− phenotype in breast cancers, an understanding of the functioning of ER in normal breast development and in tumor progression is necessary. ER exists in both alpha and beta isoforms that are differentially expressed in various tissues throughout the body (reviewed in [23]). ERα is the ER isoform associated with mammary gland development and proliferation of normal and breast cancer cells, and is most associated with predicting response to tamoxifen. As such, the discussion here will focus on the biology of ERα. ERα is a 66 kDa nuclear hormone receptor that functions as a hormone dependant transcription factor reviewed in [9, 102, 106]. ERα contains two transcription activation functions, AF-1 and AF-2. AF-1 can be phosphorylated and activated in a ligand-independent manner following growth factor stimulation, while AF-2 is activated by ligand-stimulated changes in ERα conformation [21, 99]. In ligand dependent activation (depicted in Fig. 1), upon binding it’s ligand, estrogen, ERα becomes activated which causes a conformational change within the receptor that allows receptor dimerization and phoshorylation on a number of serine residues by various kinases. In its activated form, ERα dimers bind to estrogen response elements (ERE) which are located in the promoter region of estrogen responsive genes. The binding of activated ERα to the ERE leads to the recruitment of cofactors and chromatin remodeling factors that enhance transcription of estrogen regulated genes [40, 89, 100]. Subsequent translation leads to the synthesis of proteins which drive cell differentiation, growth, division, and survival all of which can promote breast cancer proliferation and progression. ERα can also be activated in a ligand independent fashion when it is phosphorylated in response to peptide growth factors like EGF, IGF-I or TGF-α that activate PKB and MAPK signaling pathways [5, 12, 41, 47].
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Figure 1

In the ligand-dependant pathway, the AF-2 transcriptional activation function of ER is stimulated by direct binding of estrogen to the ER. This releases the ER from an inhibitory complex with heat shock proteins (HSPs) and triggers conformational changes that allow ER to bind the responsive elements in the target gene promoters. Subsequently, the receptor-ligand complex binds to the estrogen response element (ERE) located in the target gene promoters and stimulates gene transcription. The ligand-independent or AF-1 transcriptional activation pathway is activated by growth factor stimulation through direct activation of the mitogenactivated protein kinase (MAPK/ERK) and phosphoinositide 3-kinase (Pl3K/AKT) pathway. Transcriptional activity is also affected by a number of regulatory cofactors including chromatinremodeling complexes, coactivators, and corepressors. Examples of ER coactivators include the cointegrators: CREB-Binding Protein (CBP) and p300. Corepressors like Nuclear Receptor Co-Repressor (NCoR) protein are associated with transcriptional silencing. It is the relative balance of receptors, coactivator, and corepressor proteins, which is a critical determinant of the ability of this pathway to initiate responses.

In the normal female breast, only a fraction of cells express ERα and this expression is largely restricted to the glandular epithelium [4, 6, 42, 52, 77]. The ERα+ cells in the glandular epithelium rarely proliferate but are seen in close proximity to the proliferating cells of the breast. This suggests that ERα-positive cells regulate growth of the surrounding epithelium through paracrine/juxtacrine mechanisms and the lack of proliferation in the ERα+ epithelium suggests a link between ERα expression and terminal differentiation in normal breast tissue. However, recent data from Cheng et al. demonstrates that ERα+ cells do proliferate in response to estradiol but lose receptor expression post treatment suggesting this is why ERα+/Ki67+ cells are normally not seen [14]. In the progression to breast cancer, cells in the breast increase their level of ERα expression and the relationship between ERα expression and lack of proliferation of ERα+ cells changes [1, 74, 90, 93]. Thus, while more DNA synthesis occurs in adjacent ERα− cells as is the case in normal breast tissue, ERα+ cells also proliferate in ERα+ breast cancers [18, 90].

Increased ERα expression is seen in the earliest stages of breast carcinoma. ERα expression increases in ductal hyperplasia and increases even more with progressing atypia, where up to 90% of cells in atypical ductal hyperplasia (ADH) and low grade carcinoma in situ (CIS) can demonstrate ERα+ staining [1, 82, 91]. However, as the lesions progress they begin to lose ERα expression with about 78% of high grade CIS lesions staining ERα+ [82]. As CIS progresses to invasive carcinoma, ERα expression continues to decrease [53, 96] such that 60–65% of DCIS cases are ERα+, about 55% of invasive carcinomas are ERα+, and approximately 50% to 70% of all breast cancers are ERα+ [6, 31, 36, 51, 105].

While the mechanisms underlying the positive regulation of ERα expression are largely unknown, mechanisms underlying the negative regulation of ERα expression in normal and cancerous tissue are more clearly defined. A common theme is that once its ligand, estrogen, binds to ERα, the receptor is downregulated through protein degradation of the receptor, reduced transcription of new receptor, and decreased mRNA stability [10, 11, 26, 27, 80, 81, 84]. Estrogen-induced down-regulation of existing ERα protein in the cell occurs through a ubiquitin-proteasome pathway [57, 67] and the conformational changes in the receptor that are associated with targeting for proteasomal degradation seem to be necessary for the transcriptional activity of the receptor itself, although the latter has not been uniformly demonstrated [30]. Ligand bound ERα represses its own transcription by simultaneous binding to multiple ERE half-sites that are located within the promoter region [84], and lastly, the activation of ERα causes destabilization of message via binding of 3′ UTR elements which decreases message half-life in breast cancer cells [64, 83].

Potential Mechanisms Underlying the ERα− Phenotype in Breast Cancer

Mechanisms for the lack of ERα expression in breast cancer have been investigated for last couple of decades with hypermethylation of the ERα promoter representing the first identified mechanism. However, hypermethylation of the ERα promoter has been observed in only a small minority of ERα− breast cancers [54]. Several studies have concluded that the loss of ER expression is rarely the result of mutations, deletions, loss of heterozygosity, or polymorphisms within the gene [7, 45, 48]. More recent efforts at understanding the generation of the ERα− phenotype have focused on the role of tyrosine kinase induced signaling pathways and the role of proteasomal degradation of ERα. About 30–40% of breast cancers lack detectable ERα protein and have a worse prognosis than ERα+ breast cancer. ERα− breast cancers are common in younger women, resistant to antiestrogens, and often resistant to chemotherapy. Triple negative breast cancers (negative for ERα, PR and Her2 amplification) are the most clinically aggressive: treatments are limited by frequent de novo or acquired resistance to standard chemotherapies. Given the challenge in treating ERα− breast cancer and its inherent poor prognosis, understanding and manipulating the cellular mechanisms that effect expression of ERα and thus generate the ERα− phenotype are imperative to being able to restore sensitivity to endocrine therapies and thus design novel therapies for ERα− breast cancer.

Epigenetic Regulation of ERα in Breast Cancers

DNA methylation and chromatin remodeling, two epigenetic mechanisms, have been linked with the lack of ERα expression in breast tumors. While being clustered throughout the genome, CpG islands are highly clustered within the promoter region of most genes. CpG islands are cytosine residues located 5′ of guanine residues that can be modified by DNA methyltransferase (DNMT) proteins to a methylated state which results in a lack of gene expression [33, 34, 54, 109]. The promoter region and the first exon of the ERα gene contains five CpG islands that are variously methylated, with methylation of two of these correlating strongly with the lack of ERα expression [54, 73]. However, hypermethylation of the ERα promoter has been observed in only a minority of ERα− breast cancers [54, 73], approximately 25% of breast cancers, while it is found in a majority of ER-negative breast cancer cell lines [109].

In addition to methylation of CpG islands, another layer of gene silencing can be added in the form of irregular chromatin remodeling. Inactive chromatin can form on methylated CpG islands due to the recruitment of HDAC family members that remove lysine residues from the H3 and H4 core histones. Deacylation of the core histones results in an overall positive charge on the lysine residues that when reacted with negatively charged DNA results in a highly compact nucleosome and reduced transcription due to the physical inability of the polymerase to access the DNA [107, 109]. Overall epigenetic mechanisms that cause transcriptional silencing of the ERα are believed to be due to a close interplay between DNA methylation and histone modifications.

Historically, studies have demonstrated that treatment with the general DNA methyltransferase inhibitor, 5-aza-2′deoxycytidine (AZA), leads to reactivation of functional ERα protein in ERα− human breast cancer cells [54, 73, 107]. Further investigation has led to the discovery that specific inhibition of a single DNA methyltransferase DNMT1, with antisense oligonucleotides is sufficient to cause re-expression of ERα in ERα− breast cancer cell lines [107], and investigations using a combination of the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (AZA), and the histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA) demonstrated that functional ERα mRNA and protein can be restored in ERα− breast cancer cell lines [109]. Further experimentation with combination therapies including the Scriptaid HDAC inhibitor and AZA demonstrated a 2,000–20,000-fold increase of ERα mRNA and re-expression of the progesterone receptor which is ERα dependent target gene product [49]. It has also been demonstrated that a combination of the AZA methyltransferase inhibitor and HDAC inhibitor, trichostatin A, can restore the response to endocrine therapy in ERα− tumors in a xenograft model in nude mice [29].

More recent studies have used the LBH589 clinically relevant HDAC inhibitor to examine the roles of histone deacytalases in the epigenetic silencing of the ERα gene [110]. In ERα− breast cancer cell lines, treatment with the LBH589 HDAC inhibitor restored ERα mRNA and protein expression without demethylation of the CpG island within the ER promoter. Treatment with LBH589 HDAC inhibitor released DNMT1, HDAC1, and the H3 lysine 9 (H3-K9) methyltransferase SUV39H 1 from the ERα promoter region. These changes resulted in formation of an active chromatin structure and indicate that HDAC inhibitors alone can restore expression of the silenced ER gene without the need to alter the DNA methylation state of the ERα promoter [110]. Additional studies utilizing the clinically applicable HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), in ERα− breast cancer cell lines have shown that not only does SAHA treatment result in a re-expression of ERα but an inhibition of EGFR expression by disruption of the EGFR mRNA stability [111]. As a result, treatment with SAHA caused a decrease in EGF-initiated signaling pathways including PAK1, p38MAPK and AKT. Overall, these findings indicate that HDAC inhibitors in general and particularly SAHA show promise in restoring ERα expression in ERα− breast cancers and clinical evaluation of these agents is underway.

MAPK Hyper-Activation as a Reversible Mechanism that Drives Generation of the ERα− Phenotype

Since ERα promoter methylation as a mechanism for ERα− breast cancer occurs in approximately 25% of ERα− breast cancers, there must be additional mechanisms involved in generating the ERα− phenotype. We have been investigating the role of upregulated growth factor signaling as a mechanism underlying ERα− breast cancer. A significant inverse relationship exists between expression of ERα and the overexpression of certain growth factor receptors, in particular those of the erbB family [35, 76, 85, 87, 94, 104]. Two such receptors, EGFR (EGFR/erbB1/Her1) and erbB2 (erbB2/neu/Her-2) are members of the receptor tyrosine kinase superfamily.

Once bound to extracellular growth factors like epidermal growth factor (EGF), TGF-α, amphiregulin, heparin-binding protein, β-cellulin, epiregulin, cripto-1, neuregulin and heregulin, the EGFR undergoes receptor dimerization, activation of its kinase function and phosphorylation on its C-terminal regulatory domain [32, 38, 103]. Among other downstream pathways that contribute to the maintenance or enhancement of the malignant state (depicted in Fig. 2), the phosphorylated EGFR can then subsequently activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathways [20, 97]. Incidentally ErbB-2/Her-2 does not have a cognate ligand, but rather can heterodimerize with liganded/activated EGFR or erbB-3 to form potent EGFR/erbB-2 or erbB-2/erbB-3 signaling heterodimers [71].
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Figure 2

Binding of extracellular growth factors like epidermal growth factor (EGF), heparin binding protein, or transforming growth factor α (TGF-α) to growth factor receptors of the tyrosine kinase superfamily causes phosphorylation and activation of the receptor kinase function. After binding ligand, growth factor receptors induce rapid phosphorylation of the adaptor proteins, Src and SH2 containing protein (SHC), resulting in a SHC–GRB2-SOS complex formation. This leads to the subsequent activation of phosphatidylInosiol-3-kinase (PI3K), serine-threonine kinase (Akt), Protein kinase C (PKC), Ras, Raf, and MAPKs, including extracellular signal regulated kinases (ERK-1/2), c-Jun N-terminal kinase (JNK), and p38. Once activated, they are then translocated to the nucleus and participate in gene transcription. Experimental evidence has also demonstrated that in some cases hyperactivation of the MAPK pathway can lead to elevated NFkB mediated transcriptional activity, most likely through induction of the heparinbinding EGF autocrine factor [58, 68, 74, 100].

In breast cancer, the overexpression of EGFR and/or overexpression/amplification of erbB-2/Her-2 are important prognostic indicators. Overexpression of EGFR occurs in approximately 50% of breast cancers and is inversely correlated with ERα [85], and EGFR+ tumors have a poor prognosis independent of ERα status [72, 86]. Double-label immunohistochemical detection of ERα and EGFR in breast tumor specimens and cell lines confirms the inverse correlation of expression [87, 88, 104]. Furthermore, in ERα+/EGFR+ tumors, individual tumor cells express high levels of ERα or EGFR, but not both. The EGFR+ cells in these tumors are associated with a higher growth rate than the ERα+/low EGFR cells [98]. Similarly, tumors that overexpress c-erbB-2 have a poorer prognosis and tend to be ERα-negative [76, 94]. It has been estimated that about 20% of erbB-2 overexpressing tumors are ERα+; importantly, these tend to have reduced ERα levels. Using phospho-erbB-2 specific antibodies, it was demonstrated that those erbB-2 overexpressing tumors that exhibited activation of erbB-2 were most likely to be ERα−/PR− [24] indicating that downstream signaling via this receptor is associated with the ERα− phenotype.

In support of the inverse relationship that is seen between the expression of EGFR and erbB-2 and ERα expression in breast cancer, several investigators have shown that stable transfection of growth factor signaling components like EGFR, erbB-2, heregulin, Ras, c-Raf, and MEK1 leads to both estrogen-independent growth and a reduction in ERα expression in ERα+ MCF-7 breast cancer cell lines [28, 46, 55, 62, 63, 78, 95]. We have demonstrated with MCF-7 cell line models that we generated via transfection of constitutively active c-erbB-2 [55], ligand-inducible EGFR [63], and constitutively active forms of c-Raf1 [28] and MEK1 [70], that hyperactivation of MAPK results in the down-regulation of ERα expression and inhibition of this hyperactive MAPK results in restoration of ERα expression, and this re-expressed ERα is functional [70].

Initially, we developed four different cell line models of hyperactive MAPK: two in which multiple signaling pathways including MAPK were hyperactivated by upstream tyrosine kinase receptor overexpression (constitutively active erbB-2 and ligand-activatable EGFR) and two in which there would be the clean activation of MAPK by its most immediate upstream activators (MEK-1 and Raf). Regardless of how MAPK was hyperactivated, that is in all four models, loss of ERα expression at both the protein and mRNA levels was observed. Additionally, transient expression of (ca)Raf or (ca)MEK also led to down-regulation of ERα expression [70]. When the hyperactive MAPK was abrogated with the pharmacologic MEK inhibitors PD 098059 or U0126 or genetically using dominant negative (dn) versions of ERK1 and ERK2 or siRNAs specific to ERK 1 and ERK2, ERα expression was restored in all four cell line models, again regardless of how the MAPK was being hyperactivated [39, 70]. We have more recently extended these studies to established ERα− breast cancer cell lines where again, inhibition of MAPK only results in re-expression of ERα [8]. These cell lines included lines which overexpressed either EGFR or erbB-2 or both, as well as a cell line that in addition to overexpression of EGFR also exhibited overexpression of RhoC leading to hyperactivation of NFkB. Collectively, these data indicate that it is the resultant hyperactivation of MAPK by overexpressed EGFR or erbB-2 that is responsible for down-regulation of ERα since abrogation of only MAPK activity via direct inhibition of MEK results in re-expression of ERα [8, 39, 70]. Further demonstration of the ability of MAPK inhibition to restore ERα expression in ERα− breast cancer using both ex vivo tissues and primary cultures from human ERα− breast tumors frequently results in re-expression of ER as well as restoration of response to antiestrogen treatment in the tumor cells and strongly supports the hypothesis that there are reversible mechanisms underlying ERα− breast cancer and these mechanisms can be targeted therapeutically [8]. Thus some ERα− breast cancer patients could benefit from a combined MAPK inhibition and hormonal therapy.

In compliment to these observations, molecular profiling of our four hyperactive MAPK cell lines identified a “MAPK signature” that is found in a large majority of human ERα− breast tumors [19]. This hyperactive MAPK signature consists of approximately 400 genes that were consistently up or down-regulated in each of the hyperactive MAPK cell lines. Genes down-regulated in this gene signature included not only ERα itself, as expected, but a large number of estrogen-induced genes. Similarly, the MAPK upregulated genes included estrogen down-regulated genes. Thus of the genes in common between both MAPK and ERα signaling, all were inversely regulated. In several independent profile data sets of human breast tumors, the MAPK signature could accurately distinguish between ERα+ and ERα− breast tumors [19]. Interestingly, while the MAPK signature could distinguish ERα− from ERα+ breast cancer, an estrogen regulated gene signature could not, suggesting that the MAPK gene signature represents genes involved in the actual difference in biology between ERα− and ERα+ breast cancers. And in fact, many of the genes in the MAPK signature are known to be involved in the aggressive behavior of ERα− breast cancer. The results of this study confirmed that increased MAPK activation causes loss of ERα expression and suggested that hyperactivation of MAPK contributes to the biology of the ERα− phenotype as well.

The mechanisms by which elevated MAPK signaling leads to ER down-regulation are still not completely understood. Since hyperactivation of the MAPK pathway is known to be able to phoshorylate ER at SER 118 resulting in ligand –independent activation of the receptor in vitro, a tempting hypothesis is that MAPK induced ligand-independent activation enhances proteasomal degradation of ER. However, both our earlier studies examining individual estrogen-induced gene expression [70], as well as the microarray data described above indicate that downregulation of ERα by MAPK hyperactivation is not due to ligand-independent activation of ERα.

We did discover an interaction between MAPK and NFkB in inducing the down-regulation of ERα expression. The NFkB transcription factor exists in the cytoplasm in the form of a complex with IkB. Cytokines, chemokines, and intracellular stress lead to the phosphorylation of IkB by IKK (IkB kinase), releasing NFkB, which can then translocate into the nucleus to modulate the transcription of target genes [92]. While MAPK does not directly phosphorylate IkB or activate IKK, there is evidence to show that hyperactivation of MAPK leads to elevated NFkB mediated transcriptional activity, in some cases through induction of an autocrine factor, most likely heparin-binding EGF [59, 69, 75, 101]. NFkB signaling plays important roles in both normal breast development and in breast cancer [13]. Elevated NFkB activity is associated with progression to hormone independent, ERα− breast cancer [66], and is implicated in enhanced cell survival and chemoresistance in cancer [37, 56]. Our four hyperactive MAPK cell line models all exhibited significantly increased activation of NFkB and this activation of NFkB was dependent on the hyperactive MAPK as inhibition of MAPK activity also completely abrogated the increased NFkB activity [39]. Specific inhibition of NFkB activity in these model cell lines resulted in partial restoration of ERα expression suggesting that at least one mechanism by which MAPK results in down-regulation of ERα expression involves NFkB [39]. Further studies to dissect the mechanism of MAPK hyperactivation contributing to generation of the ERα− phenotype and downregulation of ERα in breast cancers have implicated both a targeting of ERα for proteasomal degradation by MAPK as well as a transcriptional repression of new ERα mRNA by as of yet an unknown mechanism.

Taken together, the findings in these studies indicate that upregulated growth factor signaling due to hyperactive MAPK activity significantly contributes to generation of the aggressive ERα− phenotype in breast cancers and that inhibition of MAPK activity can cause re-expression of the ERα and restore sensitivity to endocrine therapies. Overall, these studies support the potential role of a combined MAPK inhibition/endocrine therapy in ER-negative breast cancer patients.

Additional Mechanisms that Downregulate ERα in ERα− Breast Cancers

An additional recently proposed mechanism for the downregulation of ERα in ERα− tumors is an increase in proteolysis. In the ligand dependent function of ERα mediated transcription, estrogen binding to the ERα rapidly stimulates ERα ubiquitylation and proteolysis [57, 67]. E6-AP is a coactivator of the transactvation function of steroid hormone receptors, including ERα and is also a component of the ubiquitin-proteasome pathway. E6-AP has been shown to act as an ubiquitin ligase for ERα and recently has been demonstrated to exhibit an inverse correlation of expression with ERα in breast cancer. Given that E6-AP in upregulated in ERα− breast cancers, it is possible that E6-AP may orchestrate the down-regulation of ERα protein and thus the be involved in the generation of the ERα− phenotype in a subset of ERα− breast cancers [79].

Chu and colleagues have recently demonstrated some ERα− breast tumors, while lacking expression of ERα protein, do in fact, still express ERα mRNA. In a series of a couple of hundred ERα− breast tumors, they demonstrated that while ERα+ tumors did have higher levels of ERα mRNA, ERα mRNA levels in ERα− breast tumors were highly variable and overlapping with those from ERα+ tumors. They have further found that crosstalk between the ligand bound ERα and the 60 kDa tyrosine kinase Src, appears to enhance the proteasomal degradation of ERα. Transfection of Src into MCF-7 cells resulted in decreased levels of ERα protein, but not mRNA and this effect could be prevented by a Src inhibitor. In addition, in the low ERα+ breast cancer cell lines, BT474 and MDA-MB-361, Src induction acted to increase the ERα proteolysis induced by estrogen. Src acted to drive ubiquitylation of ERα with subsequent proteasomal degredation [16]. Thus, it may be that in a subset of ERα− breast cancer with activated Src this is the underlying mechanism for their ERα− phenotype.

Summary and Conclusions

In this review, we have delineated several potential mechanisms underlying the ERα− phenotype in breast cancer. Each of these mechanisms has been demonstrated in cell lines, and sometimes in breast tumor specimens, to both drive the loss of ERα expression and more importantly, when inhibited, result in re-expression of ERα. Furthermore, with some of these mechanisms, it has been demonstrated that the restoration of ERα expression is sufficient to induce anti-estrogen responses in a subset of ERα− breast cancer cells.

The longest known mechanism for lack of ERα expression was methylation of the ERα promoter and previously, re-expression of ERα in established ERα− breast cancer cell lines had only been demonstrated via inhibition of DNA methylation or histone deacetylation in those cell lines in which the ERα promoter has been shown to be methylated [33, 73, 110]. The methylation of the ERα promoter is both a means of permanent repression secondary to some other down-regulating event as well as more recently appreciated, a transient event induced by other signaling pathways.

The down-regulation of ERα expression by hyperactive MAPK is a more direct mechanism and is dynamic and reversible. Inhibition of MAPK activity results in re-expression of ERα and similarly, down-regulation of ERα occurs again shortly after the return of MAPK activity. Our data indicates that in addition to hypermethylation of the ERα promoter, hyperactivation of MAPK resulting from overexpression of EGFR or erbB-2 can also be directly responsible for the lack of ERα expression in ERα− tumors. Importantly, this MAPK meditated down-regulation of ERα expression can be targeted to result in re-expression of ERα and in some cases, in restoration of anti-estrogen responses. We have demonstrated this in cell lines engineered to hyperactivate MAPK, in established ERα− breast cancer cell lines with overexpression of EGFR and/or erbB-2 and thus subsequent hyperactivation of MAPK, and in primary cultures from several ERα− tumors [8]. In support of this data, it has recently been shown that in a small study of ten ERα−/erbB-2+ patients treated for various lengths of time with Herceptin, three of these re-expressed ERα and went on to have durable responses to letrazole [65]. A more recent study by Massarweh et al. suggests this mechanism can also be exploited in ERα+/erbB-2+ tumors that lose ERα expression during treatment. They found that resistance to estrogen deprivation/fulvestrant in an ERα+/erbB-2+ MCF-7 xenograft model was accompanied by upregulation of MAPK activity and loss of ERα expression, and subsequent co-treatment with Iressa resulted in inhibition of MAPK activity and increased ERα expression [58]. Again, the re-expression of ERα was associated with restoration of anti-estrogen responses.

Both hypermethylation of the ERα promoter and down-regulation of ERα expression by hyperactive MAPK involve both lack of ERα protein expression as well as lack of ERα mRNA. While large studies of mRNA levels in ERα− breast cancer confirm that many ERα− breast tumors are indeed negative for both ERα protein and mRNA, it has been recently demonstrated that there are, in fact, some ERα− breast cancers that are ERα− for protein only and thus still express ERα mRNA [16]. Two potential mechanisms underlying this ERα− phenotype both involve increased proteolysis of ERα with no concomitant transcriptional repression of ERα mRNA. The first of these involves the ERα co-activator and ubiquitin ligase, E6-AP while the second mechanism involves Src. In both cases, expression/activation of E6-AP and Src are elevated in ERα− breast cancer compared to ERα+, and in both cases, each of these can drive the proteasomal degradation of ERα.

In order to translate these mechanisms into a clinical benefit for patients with ERα− breast cancer, regardless of the different potential mechanisms for down-regulating/restoring ERα expression, the re-expressed ERα must be not only functional upon re-expression, that is induce the regulation of estrogen-responsive genes, but must also be able to regulate growth in response to estrogen/anti-estrogens. In studies where demethylation of the ERα promoter or use of histone deacetylase inhibitors restored ERα expression, this ERα was functional in that it could regulate ERE-luciferase activity as well as the expression of specific estrogen regulated genes such as the progesterone receptor (PR) [108110]. And in both our previous studies using our hyperactive MAPK cell line models and primary cultures from ERα− breast tumors, we demonstrated that re-expression of ERα upon inhibition of MAPK also restored ERα’s transcriptional activity [39, 70]. Similarly, Chu et al have shown that Src inhibition can restore at least some estrogen-regulated gene expression. Importantly, for all three of these mechanisms, it has also been found that reversal of hypermethylation, or inhibition of MAPK or Src can restore anti-estrogen responses in various in vitro and in vivo models.

Together these data are suggestive of a number of important possibilities for the treatment of ERα− breast cancer (Fig. 3). First, approximately 25% of ERα− breast tumors exhibit methylation of the ERα promoter thus resulting in lack of ERα expression. Second, it is clear that in a large majority of ERα− breast tumors, hyperactivation of MAPK by upstream overexpressed/hyperactive EGFR or c-erbB-2 represses ERα expression and thus can be targeted to allow for re-expression of ERα. Similarly, a large number of ERα− breast tumors exhibit increased activation of Src, and upregulation of E6-AP expression, and thus these mechanisms could be targeted for re-expression of ERα. Finally, these data indicate that ERα status, rather than being solely positive or negative, is a dynamic process strongly impacted by the signaling environment of breast cancer cells.
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Figure 3

Subsets of ERα− breast cancer and possible therapeutics. Representative model of ERα− breast cancer subsets with potential mechanisms and therapies for each.

Interestingly, so far, each of these mechanisms has been studied in isolation as a linear pathway leading to the down-regulation of ERα expression. But in fact, these mechanisms could also be linked in several combinations and be working together in different subsets of ERα− breast cancer. For example, MAPK can stimulate DNMT expression and the transient methylation of CpG islands. Thus hyperactivation of MAPK could also be linking to hypermethylation of the ERα promoter. Similarly, both MAPK and Src are activated by tyrosine kinase signaling and thus could be interacting to drive both proteolysis of ERα protein and transcriptional repression of ERα mRNA. And finally, Src targeting of ERα for proteasomal degradation may involve the recruitment of E6-AP. Thus, while we and others have identified clear mechanisms underlying subsets of ERα− breast cancer, further investigation into the interactions between these different mechanisms is necessary to fully understand the generation of the ERα− phenotype and realize the ability to not only restore ERα expression but more importantly, functional ERα that can also restore anti-estrogen responses.

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© Springer Science+Business Media, LLC 2009