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in breast cancer
Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22903, USA
(Requests for offprints should be addressed to R X-D Song who is now at Division of Endocrinology, University of Virginia Health Science Center, Charlottesville, Virginia 22903, USA; Email: rs5wf{at}virginia.edu)
This paper was presented at the 2nd Tenovus/AstraZeneca Workshop, Cardiff (2006). AstraZeneca supported the meeting and the Welsh School of Pharmacy, Cardiff University has supported the publication of these proceedings.
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Abstract |
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 Top Abstract IntroductionIdentity of membrane E2-binding...Summary and conclusionsReferences |
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(ER). Present investigative emphasis focuses on the additional importance of ER residing in or near the plasma membrane. A small fraction of ER is associated with the cell membrane and mediates the rapid effects of E2. Unlike classical growth factor receptors, such as IGF-1R and EGFR, ER has no transmembrane and kinase domains and is known to initiate E2 rapid signals by forming protein/protein complexes with many signaling molecules. Our recent studies demonstrate that the IGF-1R is involved in tethering ER to the plasma membrane, in activating the EGFR, and in the initiation of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling. The formation of a multi-protein complex containing these receptors as well as adaptor proteins is a critical step in this process. A full understanding of the mechanisms underlying these relationships with the ultimate aim of abrogating specific steps, should lead to more targeted strategies for treatment of hormone-dependent breast cancer.
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Introduction |
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TopAbstractIntroductionIdentity of membrane E2-binding...Summary and conclusionsReferences |
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Autocrine mechanisms mediated by the ER can take place in the nucleus of the cell or via extranuclear events. For the past two decades, major investigative emphasis in breast cancer research has focused upon transcriptional events resulting from the binding of E2 to ERs (ER and ERß) in the nucleus of the cell. Specifically, the liganded receptor interacts with canonical or non-canonical estrogen response elements in the promoter region of E2 responsive genes, leading to gene transactivation. Other nuclear actions of E2 involve the tethering of the ER to nuclear transcription factors, such as NF-Y and Sp-1, which also lead to gene transactivation (Wang et al. 1999). These nuclear events usually require hours or days for maximal gene activation. More recently, multiple studies demonstrated very rapid ER-mediated actions, which occur in the region of the cell membrane and mitochondria. This evolution in understanding of the ER function has led to a new terminology, which distinguishes the nuclear (genomic) and extranuclear (non-genomic) actions of E2. An alternative but parallel nomenclature describes nuclear-initiated steroid signaling and membrane-initiated steroid signaling (Nemere et al. 2003).
A series of E2-induced extranuclear events has been described in benign as well as in malignant cells of various origins. These effects occur from seconds to minutes after administration of E2 and involve rapid activation of many signaling molecules (Cheskis 2004, Shupnik 2004, Levin 2005), such as (i) the insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR); (ii) p21ras and Raf; (iii) mitogen-activated protein kinase (MAPK) and Akt; (iv) protein kinase C; (v) intracellular calcium transients; (vi) nitric oxide and prolactin secretion; and (vii) Maxi-K channels. While the majority of studies describing extranuclear events focus on biochemical changes, convincing biologic evidence from many systems has accumulated. Within 2 min, vasodilatation in association with nitrous oxide formation occurs in response to physiologic concentrations of E2 in isolated femoral arterial wall (Chambliss et al. 2005, Guo et al. 2005). These effects can be blocked by inhibitors of MAPK and by anti-estrogens. E2 also rapidly stimulates the secretion of prolactin in isolated pituitary cells (Watters et al. 2000). In cancer cells, E2 causes a rapid increase in dynamic membrane structures that can be abrogated with an anti-estrogen (Song et al. 2002).
A recent focus of ER research has been precisely to define the mechanisms underlying extranuclear actions. Substantial attention has been directed toward proving the hypothesis that kinases activated in the membrane are involved in later nuclear transcriptional events. According to the recent model of O’Malley (2005) ER on the membrane initially activates cytoplasmic kinases, which in turn phosphorylate and activate coactivator proteins in the cytoplasm. These coactivators then travel to the nucleus and modulate ER-mediated transcriptional events. This integrated view of extranuclear ER signaling suggests that events occurring initially at the level of the plasma membrane later modulate more classical transcriptional events by crosstalk mechanisms.
Our recent studies have examined the protein–protein complexes, which occur when E2 binds to its receptor in breast cancer cells. Special emphasis is given to the formation of large protein complexes that activate various kinases and mediate downstream effects. We hypothesize that the formation of this ER-centered large protein complex plays a critical role in initiation of E2 rapid action in breast cancer cells. Activation of many signaling pathways by E2 is regulated by the formation of such complexes and could lead to the activation of MAPK and Akt and translocation of ER to the region of the plasma membrane. In terms of ER-centered protein complex formation, c-Src tyrosine kinase, Shc, PELP-1, modulator of nongenomic activity of estrogen receptor (MNAR), phosphoinositide 3-kinase (PI3K), caveolins, and G-proteins have all been reported to serve as components of large complexes of interacting proteins (Song et al. 2005). Through the mediation of these molecules, E2 activates the Shc/MAPK and PI3K/Akt pathways, which are likely to be the major effectors of cell proliferation and cell survival.
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Identity of membrane E2-binding proteins |
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TopAbstractIntroductionIdentity of membrane E2-binding...Summary and conclusionsReferences |
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(Razandi et al. 1999); (ii) a truncated form of the ER with a molecular weight of 46 kDa (Li et al. 2003); (iii) a unique estrogen membrane protein called ER-X, whose ligand affinity differs from that of the classical ER but is recognized by antibodies directed against ER ligand-binding domain (Toran-Allerand et al. 2002); (iv) sex steroid-binding protein acting in concert with a membrane protein megalin (Catalano et al. 1997, Hammes et al. 2005); (v) G-protein-coupled receptor 30 (Thomas et al. 2005); and (vi) an unknown protein present in non-ER, non-ERß expressing, chinese hamster ovary (CHO) and COS-7 cells (Nethrapalli et al. 2005). While these various E2-binding proteins exist in specific systems, accumulating evidence supports the classical full length ER as the membrane estradiol-binding protein. This has been demonstrated in several types of cells, including breast epithelial cells, osteoblasts, endothelial cells, and vascular smooth muscle cells. Using multiple antibodies against ER, Watson & Gametchu (1999) demonstrated that the classical ER is expressed near the region of the cell membrane in MCF-7 cells. Using confocal microscopic methods, our group demonstrated translocation of full length ER into or near the plasma membrane in response to E2. A proof of principle experiment demonstrated that transfection of ER-negative breast cancer cells with ER resulted in 5% of ER located in the plasma membrane and the remainder predominantly in the nucleus (Razandi et al. 1999).
Biochemical studies also demonstrated full length ER protein in isolated plasma membranes as detected by mass spectrometry analysis (Pedram et al. 2006). Functional studies demonstrated that the rapid biochemical and biological effects of E2 seen in wild-type mice were abolished in ERKO (ER knockout) and BERKO (ERß knockout) animals and that these animals have no demonstrable ERs in isolated plasma membrane preparations (Razandi et al. 2004). Additional evidence supporting the classical ER in the membrane was obtained from studies in which this receptor was knocked down by a selective siRNA (Song et al. 2004). In this study, E2 activated MAPK in a matter of minutes but abrogation of ER with a selective siRNA abolished this effect in human breast cancer MCF-7 cells.
Additional studies demonstrated that E2 stimulates MAPK activation only in ER transfected cells but not in those non-transfected (Razandi et al. 2004). Studies with ER transfected COS-1 cells demonstrated that only the membrane localized receptor could activate MAPK (Zhang et al. 2002). Harrington et al. demonstrated rapid activation of MAPK using membrane impermeable dendrimers (Harrington et al. 2005). Taken together, these data provide strong evidence that ER can localize to the region of the cell membrane and initiate kinase-mediated events. However, a critical overview of this field would suggest that truncated ER, G-protein-coupled receptors, or other proteins may also be involved in rapid estrogen-initiated effects in some cell types and under certain circumstances (Filardo et al. 2000, Chambliss & Shaul 2002, Li et al. 2003).
Mechanism of membrane localization of ER
Even though ER has no transmembrane domain and is not intrinsically a membrane protein, several laboratories have demonstrated that extranuclear actions of E2 require ER translocation in or near the plasma membrane (Lu et al. 2004, Song et al. 2004). Whether ER membrane translocation and its activation on the cell membrane are sequential or an independent event is not presently known. We also do not understand the biological role of cytosol ER in E2 rapid action, although effects on the mitochondria have been reported (Chen & Yager 2004).
Our working model suggests that translocation of the ER to the membrane involves Shc as a transporter. In response to E2, Shc is phosphorylated and binds to ER. At the same time, the Shc binding sites on the IGF-1R are phosphorylated, allowing Shc to bind to the IGF-1R. This is analogous to Shc acting as a bus, picking up ER and then delivering ER to the Shc binding site on IGF-1R. ER can then be tethered to the membrane through Shc interaction with IGF-1R (Razandi et al. 2002, Song et al. 2004). Substantial proof for our working model has emanated from immunoprecipitation/western blot experiments, from the use of siRNA and dominant negative constructs, from confocal microscopy, and from biochemical studies of kinase activation (Song et al. 2002, 2004). We demonstrated that E2 induces formation of a ternary complex consisting of ER, Shc, and IGF-1R in the cell membrane of MCF-7 cells. We also showed that E2 causes phosphorylation of the IGF-1R and of Shc. Elimination of Shc either by siRNA expression or by dominant negative constructs abrogates estrogen-induced MAPK activation (Fig. 1
). More importantly, knockdown of Shc with a specific siRNA prevents E2-induced ER translocation to the plasma membrane (Song et al. 2004). However, this knockdown strategy does not eliminate estrogen-induced membrane ruffling, a morphologic change of the plasma membrane, which is associated with cell mobility. Our results suggest that pathways not involving Shc might mediate E2-induced cell mobility changes. Recently, the adapter protein p130Cas was reported to associate with ER and c-Src in T47D breast cancer cells (Cabodi et al. 2004). As p130Cas is known to interact with focal adhesion kinase, it is conceivable that estrogen-induced cell morphologic changes might be mediated by p130Cas independently of Shc and IGF-1R.
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into the plasma membrane of EAhy926 human aortic endothelial hybridoma cells, by interacting with amino acid 185–253 of N-terminal ER (Lu et al. 2004). The protein complex of striatin and ERleads to the targeting of ERon the cell membrane. Notably, our preliminary data suggest that E2 also increased the binding of the ER to the EGFR (Song et al. 2003). Furthermore, palmitoylation of ER has been reported to enable the association of a truncated ER with the plasma membrane (Acconcia et al. 2005). Therefore, it is possible that Shc transports ER to the membrane and that ER palmitoylation facilitates the persistent residence of this receptor in the membrane. Data from Levin et al. (Razandi et al. 2002) suggest that caveolin is also involved in the reversible shuttling of ER from the cytosol to the membrane. Role of the IGF-1R and EGFR in E2 rapid action
IGF-1R is a key receptor involved in the growth of breast cancer cells and its expression is always positively correlated with ER expression in breast cancer. Estrogens appear to favor synergistic interactions with the IGF-1 system, leading to the expression of several components, such as IGF-1R, IRS1, and IGF-1 (Mauro et al. 2001). These upregulated proteins act locally in autocrine loops to mediate the biologic effects of estrogen. A close relationship between ER and IGF-1R has been confirmed by the fact that uterine cells do not respond to estrogen if the IGF-IR pathway is blocked and that IGF-1 also loses its effect on gene transactivation and cell proliferation in ER knockout cells (Klotz et al. 2002). A direct interaction between two pathways also occurs at the receptor level whereby estrogen can induce IGF-1R phosphorylation in uterine epithelia cells, ER transfected COS-7 cells, and MCF-7 breast cancer cells (Richards et al. 1996, Kahlert et al. 2000). These data indicate that the IGF-1R-mediated signaling pathway is important in estrogen action. Interestingly, the expression of EGFR correlates inversely with the expression of ER. However, a variety of evidence shows that both receptors are functional in the regulation of breast cancer growth regardless of which one is dominantly expressed (Klijn et al. 1992, van Agthoven et al. 1994). For example, EGFR is functionally involved in estrogen-induced MAPK activation in MCF-7 cells, a cell line with high expression of IGF-1R and ER but low EGFR.
Both EGF and E2 also crosstalk at other levels. For example, administration of the anti-estrogen ICI 182780 reduces the response to EGF in the mammary gland (Ankrapp et al. 1998). Studies in ER-knockout mice demonstrated that EGF could not induce DNA synthesis in the uterus even in the presence of wild-type levels of EGF and EGFR (Curtis et al. 1996, Ankrapp et al. 1998). These results indicate that there is a true requirement for the presence of ER for EGF-mediated biological functions, at least in some cell types. These and other data suggest that ER serves as a nodal point, which allows interactions between the ER and growth factor pathways.
Mechanism of ER-mediated activation of the MAPK and Akt pathways
Both MAPK and Akt play important roles in estrogen-induced cancer cell growth. Using a fusion protein consisting of an oncogenic form of human Raf and the ER HBD domain, 
Raf:ER, Samuels et al. in 1994 observed that quiescent 3T3 cells transfected with Raf:ER responded to estrogen and showed rapid, sustained activation of MAPK (Samuels & McMahon 1994). Migliaccio et al.(1996) then first demonstrated, in endogenous ER expressing MCF-7 breast cancer cells that estrogen triggers rapid activation of MAPK. These findings were further confirmed in many other cell types, including osteoblasts (Endoh et al. 1997), rat brain cells (Bi et al. 2000) and human colon carcinoma-derived Caco-2 cells (Di Domenico et al. 1996). E2 also rapidly induces the activation of the PI3K/Akt pathway in endothelium cells (Koga et al. 2004), cardiomyocytes (Patten et al. 2004), rat PC-12 neuronal cells (Alexaki et al. 2004) and MCF-7 cells (Ahmad et al. 1999). In the presence of E2, ER interacts with the regulatory subunit of PI3K, p85, thus triggering activation of the catalytic subunit p110 of PI3K, leading to the downstream kinase Akt/PKB activation (Castoria et al. 2001). Activation of Akt mediates many of the downstream cellular effects of PI3K, including phosphorylation and inactivation of Bad to prevent Bad-mediated cell apoptosis as well as rapid activation of the endothelial isoform of eNOS (Chambliss & Shaul 2002).
Our studies demonstrated in MCF-7 breast cancer cells that E2 stimulation of MAPK occurs within 5 min. Later, MAPK phosphorylates and activates the transcription factor, Elk-1. The mechanism for E2-induced MAPK and Akt activation in breast cancer cells is now focused on the involvement of classical growth factor receptors, such as IGF-1R and EGFR (Kahlert et al. 2000, Levin 2003). E2 co-opts both IGF-1R and EGFR signaling pathways and utilizes their downstream common adapter proteins to transduce signals, leading to the activation of MAPK and Akt (Fig. 2). These steps occurred within the time frame that E2 stimulated the activation of MAPK and Akt. Knockdown of Shc blocked E2-induced MAPK activation, which was also confirmed by the expression of a dominant negative Shc in MCF-7 cells (Song et al. 2002). E2-induced activation of Akt could be attenuated by administration of the IGF-1R tyrosine kinase inhibitor AG1024. These experiments demonstrated a mechanistic link between the membrane growth factor receptors and their downstream kinase cascades in rapid E2 action.
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interacting molecules
Prior to MAPK and Akt activation, ER needs to interact with many signaling molecules to form a protein complex. It is now known that c-Src activation in the ER-centered complex is a key step in initiating many downstream signaling pathways in E2 action (Fig. 3). However, many other signaling molecules in the complex also play an important role in initiating and stabilizing the complex.
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has no intrinsic kinase domain and therefore is not capable of phosphorylating other proteins. Accordingly, E2 must stimulate the activity of a kinase, which serves this function. A key kinase candidate is c-Src tyrosine kinase, which has previously been identified to physically interact with ER. Our working model (Fig. 3
) suggests that E2 activates c-Src, which in turn phosphorylates Shc as well as the IGF-1R (Peterson et al. 1996). The c-Src then would serve as a crucial molecule to facilitate other protein/protein interactions involved in estrogen rapid action.
We have introduced the term, ‘activating particle’ for this multi-protein complex containing ER, c-Src, and other proteins and suggested that this ‘activating particle’ is a key component involved in estrogen rapid action. This name implies that a complex of proteins is necessary to activate c-Src and that this complex is a discrete molecular entity, which is required to initiate membrane receptor-mediated growth factor-induced signals. The involvement of the above proteins with ER and c-Src raises many questions regarding estrogen-induced signal initiation mechanisms which now require investigation. While our working model (Fig. 3) suggests that the EGFR and IGF-1R act downstream of c-Src activation, this remains to be precisely established (Song et al. 2004).
Role of p85 of PI3K
The PI3K molecule is composed of an 85Kd regulatory domain (p85) and a p110 catalytic domain. p85 is an adapter protein known to be involved in estrogen rapid action by forming a protein complex with ER and c-Src (Castoria et al. 2001). PI3K plays an important role in cell survival and in prevention of cell death (Vanhaesebroeck & Waterfield 1999). Activation of growth factor receptors, such as IGF-1R and EGFR on the cell membrane will recruit p85 to the receptors, leading to the activation of the p110 catalytic subunit (Yamamoto et al. 1992, Altschuler et al. 1994). The p85 as a substrate of IGF-1R and EGFR not only transduces IGF-1R signals to activate Akt, but also forms a protein complex with ER and c-Src (Fig. 3), leading to estrogen-induced c-Src activation. In this complex, c-Src SH2 interacts with pY-537 of ER and c-Src SH3 with a polyproline sequence in the linker region of p85 (Renzoni et al. 1996, Barletta et al. 2004).
Role of Shc, G-protein, GPCR, and caveolins
The adapter protein Shc, the G-proteins (Gs and Gi), the G-protein-coupled receptor 30 (GPR30), and caveolin-1 have all been involved in estrogen-induced MAPK activation by association with ER (Migliaccio et al. 1996, Kousteni et al. 2001, Song et al. 2002, Razandi et al. 2003, Revankar et al. 2005). Among these proteins, Shc was demonstrated to directly interact with ER in MCF-7 cells (Fig. 3), in which the SH2 domain of Shc physically interacts with ER A/B region (Song et al. 2002). Shc has no intrinsic kinase domain. It transduces signals based on protein–protein interactions through three functional domains: a PTB domain, a region rich in proline, and glycine residues called the collagen-homology domain, and a carboxy-terminal SH2 domain (Pelicci et al. 1996).
Both G-proteins and caveolin-1 might also be potential candidates in the formation of the ‘activating particle’ mediating the rapid effects of E2 since they have been reported to be in the complex with ER (Wyckoff et al. 2001, Razandi et al. 2002). However, so far there is no evidence showing that they directly interact with ER in a cell-free system. Presently the physiological significance of this protein–protein interaction is unclear. GPR30, a G-protein-coupled receptor (GPCR), was recently reported to specifically localize to the endoplasmic reticulum and bind to estrogen (Revankar et al. 2005).
ER-initiated sequential interactions among receptor components
Recent studies in non-breast cancer models have suggested a linear pathway whereby the IGF-R signals through the EGFR to activate MAPK. This process involves the sequential phosphorylation of the IGF-1R, activation of the matrix metalloproteinases (MMPs) 2 and 9, shedding of the extracellular domains of HB-EGF, binding of HB-EGF to the EGFR, and enhanced EGFR tyrosine kinase activity (Fig. 2). Meantime another report demonstrated that IGF-1R-induced EGFR phosphorylation is c-SRC dependent in IGF-II action (Knowlden et al. 2005). IGF-1R is not the only receptor to act upstream of the EGFR in this way. The receptors for angiotensin I, thrombin, and ß adrenergic and cholinergic ligands, human growth hormone, and prolactin can all act in a similar fashion (Hackel et al. 1999, Luttrell et al. 1999, Zwick et al. 1999). Accordingly, the EGFR has been considered a nodal point upon which converges many cytokine- and hormone-induced signals, which can lead to MAPK activation. Our present studies examine the hypothesis that the E2/ER complex can similarly engage the IGF-1R pathway and sequentially active metaloproteinases, and the EGFR. The biologic consequences of this putative pathway could involve the mediation of E2-induced cell growth and protection from cell death.
We have examined the effect of E2 on the phosphorylation of the IGF-1R, EGFR, and MAPK. Our preliminary data were presented in Endo2006, Boston (Song & Santen 2006) and show the following evidence. AG1024, a specific IGF-1R phosphorylation inhibitor blocked E2-induced MAPK phosphorylation as did AG1478, an EGFR phosphorylation inhibitor. In order to focus on the upstream/downstream relationships of IGF-1R and EGFR on E2-induced MAPK activation in MCF-7 cells, we utilized a strategy, which bypassed the ER and stimulated both receptors directly with their cognate ligands. Our preliminary data show that IGF-1, but not EGF, increased IGF-1R phosphorylation in a dose-dependent manner. Unlike IGF-1R, the phosphorylation status of EGFR was increased not only by EGFR, but also by IGF-1, indicating that IGF-1 is an upstream molecule that mediates EGFR tyrosine kinase activation. Matrix metaloproteinases are involved in this process since a MMP2/9 inhibitor blocked the effect of IGF-1 to activate the EGFR.
We then focused on the ability of IGF-1 and EGF to activate MAPK. IGF-1R and EGFR were specifically knocked down with a selective siRNA and the resultant effects on MAPK examined. Using a non-specific siRNA as control, IGF-1 and EGF produced in a 9- and 12-fold increase of MAPK phosphorylation respectively. The siRNA against IGF-1R significantly blocked IGF-1-stimulated, but not EGF-stimulated, MAPK phosphorylation. Together, the results indicate that EGFR not only plays a role in maintaining the basal level of MAPK activation, but also one of the molecules is affected by the upstream actions of IGF-1R in MCF-7 cells.
After biochemical demonstration of the involvement of EGFR in IGF-1-induced MAPK activation, we wished to assess the biological consequences of both receptors on E2 action in MCF-7 cell and examined both cell proliferation and the apoptosis in response to E2. For this purpose, both siRNA and tyrosine kinase inhibitor strategies were employed. First, we examined proliferation. Knockdown of either IGF-1R or EGFR significantly decreased the numbers of cells growing in the absence of E2. Compared with vehicle treatment, knockdown of IGF-1R significantly blocked E2 or IGF-1, but not EGF, induced stimulation of cell number, suggesting that ligand-induced EGFR activation is a downstream event in E2 or IGF-1 action. At the same time, knockdown of EGFR with siRNA significantly retarded the stimulatory effect induced by E2, IGF-1, and EGF supporting that EGFR is a common converging point mediating E2 and IGF-1R action. Together, these results reinforce the hypothesis that EGFR activation is a downstream signal involved in E2 and IGF-1 action.
Next, we examined apoptosis. We determined whether blockade of IGF-1R or EGFR could reverse the effects of E2 or growth factors on protection against cell death. Approximately a twofold increase of apoptosis was observed in both AG1024- and AG1478-treated groups. Treatment of cells with E2, IGF-1, and EGF not only significantly decreased cell death rate compared with vehicle-treated control, but also in general protected cell apoptosis induced by both AG1024 and AG1478. Together, the data indicate that both IGF-1R and EGFR play a role in the effect of E2 in protecting against cell death and that IGF-1R might exert a stronger anti-apoptotic effect than on proliferation.
ER involved non-genomic action in anti-hormonal therapy
Exposure to hormonal therapy can cause breast cancer cells to adapt and change their properties. We postulate that long-term exposure to tamoxifen might enhance the utilization of the EGFR pathways. Our experimental model was to expose MCF-7 cells to tamoxifen for a period of 3 months to 2 years until they became resistant to tamoxifen (TAM-R). Our published data show that TAM-R cells were more sensitive to the stimulatory effect of EGF (Fan et al. in press). Wild-type control MCF-7 cells did not respond to 0.1 ng/ml EGF with MAPK phosphorylation, whereas the TAM-R cells responded robustly. In addition, the rapid, extranuclear, MAPK response to E2 was also increased in TAM-R cells, which is in agreement with enhanced sensitivity to EGF-stimulated MAPK activation. TAM-R cells became more sensitive to growth inhibition by small molecule inhibitors that block kinase activity of EGFR or MEK. These results suggest that MCF-7 cells become more dependent on the EGFR/MAPK pathway for growth after long-term exposure to tamoxifen.
As a possible explanation for our findings, we questioned whether or not ER bound more effectively to the EGFR in the TAM-R cells. These studies demonstrated an enhanced interaction between ER and the EGFR in the TAM-R cells. However, the levels of ER and EGFR were not increased in TAM-R cells. This result suggested that an enhanced interaction between EGFR and ER might result from redistribution of ER. In support of this possibility, confocal microscopy studies demonstrated a marked enhancement of cytoplasmic ER content in the TAM-R cells. To confirm that long-term tamoxifen treatment increased the pool of cytoplasmic ER, fractions of the plasma membrane, nucleus, and cytoplasm were prepared from TAM-R and control cells. In the control cells, ER was recovered from both cytoplasmic and nuclear fractions with higher levels in the nucleus. In TAM-R cells, ER was detectable in the membrane fraction. The level of ER in cytoplasm was increased, whereas the level of nuclear ER was reduced compared with the control cells.
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Summary and conclusions |
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TopAbstractIntroductionIdentity of membrane E2-binding...Summary and conclusionsReferences |
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action to enhance cell proliferation and diminish apoptosis. The formation of large protein/protein complexes, which involve the IGF-1R, the EGFR, c-Src, Shc, and ER are required for extranuclear ER actions. Depending upon the cell type and context, additional proteins in this complex can include PELP-1 (MNAR), striatin, caveolin, P130Cas, and certain G-protein-coupled receptors. Complex interactions occur between the extranuclear signaling pathways and the nuclear transcriptional events. Our studies suggest that E2, when bound to ER, can co-opt classical growth factor signaling pathways to activate MAPK and PI3K. Our working model emphasizes a sequential process involving several steps. Initially E2 binds to ER and induces the phosphorylation of Shc and subsequent binding to ER. Shc acts as a ‘bus’ to transport ER to Shc binding sites on the IGF-1R, which in turn is phosphorylated in response to E2. After formation of this complex, metaloproteinases 2 and 9 are activated, which release HB-EGF from its membrane tethering. HB-EGF serves as a ligand to activate the EGFR, which in turn enhances the activity of the MAPK and PI3K pathways. With exposure to anti-hormonal therapies, such as tamoxifen, breast cancer cells can adapt by upregulation of growth factor pathways. Our data demonstrate that long-term tamoxifen exposure results in an increase in the relative amounts of ER in extranuclear sites of the cell, a process that can be abrogated by the c-Src inhibitor PP2. With more ER outside the nucleus, the processes that utilize the IGF-1R and EGFR are enhanced. These adaptive mechanisms provide specific targets, such as blockade of the IGF-1R, EGFR, c-Src, and Shc, which could potentially abrogate development of resistance to anti-hormonal therapy (Knowlden et al. 2005). |
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