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Departments of Medicine, Microbiology (C.M.S.), Physiology (M.A.S.) and the Cancer Center, University of Virginia Health System, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Corinne M. Silva, Department of Medicine, PO Box 800578, University of Virginia Health System, Charlottesville, Virginia 22908. E-mail: cms3e{at}virginia.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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STEROID AND GROWTH FACTOR SIGNALING PATHWAYS |
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TOPABSTRACTINTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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or ERß), members of the nuclear receptor family of transcription factors. The ER proteins have similar structure in their central DNA-binding (98% identity) and C-terminal ligand-binding domains (54%); but less than 15% homology in their N termini, which may allow unique protein-protein interactions. These two proteins have both overlapping and divergent tissue expression and biological activities, particularly in response to selective ER modulators (SERMS) and on specific promoters (5). Knockout mouse models have demonstrated that ER is the major regulator of estrogen-induced development of the mammary gland (6). Although the ER knockout mouse has the component structures necessary for mammary gland development (including a rudimentary ductal tree), it does not undergo the pre- or postpubertal stages of growth of these epithelial ducts through the stromal fat pad (7). In contrast, the ERß knockout has the normal ductal structure of the mammary gland at 4–5 months and also develops the lobuloalveolar structures necessary for lactation (8). Although both ER and ERß are expressed in normal mammary tissue, ER is overexpressed in breast tumors compared with normal tissue and is the predominant ER form in tumors and cell lines (9). Because the role of ERß remains less well defined and is the subject of a number of recent publications (10, 11), we will focus here on the ER. However, it may be noted that the majority of ER-positive breast tumors contain both ER and ERß, as well as the PR (12, 13).
The classical genomic or nuclear effects of steroids occur through ligand-induced receptor dimers binding to DNA. Ligand binding alters the conformation of the receptor, allowing the binding of coactivator and other regulatory proteins that alter chromatin function, or communicate with the transcriptional machinery. The ER dimer binds either directly to DNA at an estrogen response element or tethers to other bound transcription factors, thereby altering the transcription of estrogen-sensitive genes (14). The ER also exerts nongenomic effects that are initiated in the cytosolic/membrane compartment (15). Although most ER is in the nucleus, a population resides in the cytoplasm and/or membrane, available for cross talk with other cytoplasmic/membrane-associated signaling molecules. Because the ER is overexpressed in 40–70% of breast tumors, the cytoplasmic population of ER may play a more significant role in tumors than it does in nontumor tissues. In fact, nongenomic activities are proposed to not only contribute to, but to be critical for, ER activities including proliferation (16, 17). Nongenomic actions also occur through the PR and AR, either alone or in conjunction with the ER, and both of these receptors are present at significant levels in the cytoplasm (18, 19). Available data from ER knockout mice and human cell lines suggest that the bona fide genomic ER is responsible for cytoplasmic signaling (20). However, the cell surface G protein-coupled receptor 30 also binds E2 and through a c-Src-Shc-mediated pathway may contribute to E2 actions in various cell types (20, 21). Importantly, both antiestrogens, tamoxifen (TAM) and ICI 182,780, activate G protein-coupled receptor 30, and its knockdown in breast cancer cells does not interfere with E2-stimulated proliferation (22). Therefore, a major unresolved question is to determine not only the role of nongenomic ER and other steroid receptor actions in breast cancer cells, but also which of the downstream pathways are most important (23).
The 30–60% of breast tumors that do not overexpress the ER are termed ER-negative (ER–) and frequently overexpress members of the human EGF receptor (HER) family. In normal mammary gland development, the EGF receptor (EGFR) is essential for ductal morphogenesis, and HER2 is required for alveolar morphogenesis (24, 25). Ductal epithelial cells of the normal breast express the EGFR and HER2, as they are involved in normal processes of growth, differentiation, cell motility and survival (26, 27). Overexpression of these receptors either at the gene or protein level is well described in breast cancer (26). There is a significant correlation between expression of growth factor receptors of the EGFR (or HER family), and breast cancer growth, state, and aggressiveness (28). HER family members (EGFR/HER1, HER2/neu, HER3, and HER4) are overexpressed in 60% of different types of breast cancers (29, 30, 31). Approximately 10–20% of ER+ tumors overexpress HER2, and these are primarily ductal carcinomas (32). Increased HER2 expression is correlated with poor prognosis, but therapies to target both ER and HER2 may be beneficial. Whereas a basal level of the EGFR is expressed in all breast cancer cells and tumors, overexpression of the EGFR is rarely seen in ER+ tumors, but is highly expressed in more aggressive, ER– cancers (29). The EGFR is overexpressed in 16–48% of breast cancers, and patients whose tumors are EGFR positive have shorter relapse-free and overall survival times (30, 33).
Overexpression of HER family members alone does not result in efficient transformation and tumorigenesis in breast cancer cell models. However, co-overexpression with the intracellular/membrane-associated tyrosine kinase c-Src, which potentiates signaling from growth factor receptors, dramatically increases tumorigenesis (28, 34, 35). Because c-Src is overexpressed in a majority of breast cancers (>70%), many breast cancers that overexpress c-Src also overexpress members of the HER family (36, 37). Co-overexpression of EGFR and c-Src in human breast cancer cell lines results in their association, and the c-Src-mediated phosphorylation of the EGFR at a novel tyrosine (Y845) within its catalytic domain. These novel interactions result in increased proliferation, transformation, and, most dramatically, an increase in tumor formation in vivo (35, 38, 39). In a similar model, c-Src is also capable of interacting with, and augmenting the phosphorylation signal from, HER2 homodimers as well as HER2/HER3 heterodimers, resulting in increases in downstream signaling pathways that are pro-proliferative (40).
In addition to augmenting HER signaling, c-Src also mediates signaling through the ER in the cytoplasm/membrane and has been proposed to be important for E2-stimulated cellular proliferation (19). Protein complexes between ER and c-Src have been isolated but may occur physiologically via a variety of adaptor proteins including Shc, p130 Cas, and MNAR (moderator of nongenomic activity of ER). Binding of c-Src to these adapter proteins serves to either increase the catalytic activity of c-Src (by mediating conformational changes) and/or simply by bringing c-Src into a complex with potential substrates (41, 42). PR and AR similarly associate with c-Src but can do so directly via the SH3 domain of c-Src and the proline-rich region in the receptor N terminus (43, 44, 45). AR may also associate with MNAR to stimulate cytoplasmic signaling (46, 47) This ligand-stimulated association with c-Src or adaptor molecules similarly augments pathways such as MAPK. Shc binds to ER and c-Src in breast cancer cells, leading to E2-induced activation of the MAPK pathway and changes in the morphology of the cells (48). The adapter protein p130Cas binds to c-Src and increases its catalytic activity, whereas MNAR is an adapter protein that binds both ER and c-Src, thereby facilitating ER-mediated increases in both c-Src and MAPK activity (49, 50).
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INTEGRATION OF SIGNALING |
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TOPABSTRACTINTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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. This model demonstrates that cellular stoichiometry between the various receptors, and between groups of signaling molecules, is critically important and therefore, overall cell function and proliferation are determined by cell context. At present, it is unclear whether steroids and growth factors act together to help cells reach a critical threshold level of common critical signaling molecules or whether there are both common and complimentary pathways involved. However, a number of these signaling molecules, including MAPK, phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin, IKB kinase inhibitor of 
B/nuclear factor-B, protein kinase C-
(PKC), and phospholipase C-
(PLC), are known to play a role in both ER and GFR signaling (15, 16). In this review, we focus on the signal transducer and activator of transcription (STAT) pathway, not only as an example of a potential integrator of the numerous signaling pathways that originate from both growth factors and steroids, but also as a potential target to be exploited in tumor-specific directed therapies.
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STATs |
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TOPABSTRACTINTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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In addition to a well-described role in hematopoietic cancers, the overexpression and/or activation of STAT3, STAT5a, and STAT5b has been described in head and neck cancer (SCCHN), lung, prostate, and breast cancer (63, 64). A number of in vivo mouse models have demonstrated the integral role of these STAT proteins not only in the normal development of the mammary gland, but also in the process of breast tumorigenesis (2, 65, 66). The STAT5a knockout mouse shows loss of prolactin-dependent mammary gland development, whereas the STAT5b knockout shows no profound effect on the mammary gland but has severe growth retardation and the dysregulation of GH-regulated genes (67, 68). The knockout of STAT3 is embryonic lethal (69). However, the targeted knockout of STAT3 in the mammary gland results in a delayed involution of the mammary gland at the end of lactation (70). Consistent with these in vivo models, gene array analysis has shown that STAT5 functions to regulate genes involved in mammary gland proliferation, survival, and differentiation whereas STAT3 provides a proapoptotic signal in the normal gland (71).
A number of knockout and transgenic mouse models have demonstrated a role for the STAT3 and STAT5 proteins in breast cancer progression and development. In the T-antigen transgenic mouse model of mammary cancer progression, 86% of the tumors have activated STAT5a, 61% also have activated STAT5b, and 75% have activated STAT3. However, if these mice are crossed with 5a+/– heterozygous mice, there is a significant delay, not only in the age of cancer onset, but also a decrease in tumor volume (72). Similarly, crossing a mouse that overexpresses TGF in the mammary gland, and is predisposed to mammary tumorigenesis, with the STAT5a knockout mouse, results in an increase in tumor latency (73). Finally, targeting a constitutively active STAT5 to mammary epithelial cells predisposes the animal to mammary tumors (74). Together, these studies support an integral role for STAT3/5 signaling in breast tumorigenesis.
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STATs: COMMON DOWNSTREAM MEDIATORS OF GROWTH FACTOR AND ESTROGEN ACTIONS |
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TOPABSTRACTINTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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). Both mouse fibroblasts and human breast cancer cell line models demonstrate that the overexpression of HER family members, particularly HER1/EGFR and HER2/neu, results in constitutive and/or ligand-induced activation of STAT3, STAT5a, and STAT5b (75, 76). In addition, there is a large body of literature supporting the role of c-Src in activation of the STAT3/5 pathway (77). In some cases, the c-Src-mediated activation of the STAT signaling pathway can be direct, and increasing c-Src activity by overexpression, or by mutation (e.g. v-Src) leads to the tyrosine phosphorylation and activation of STAT3 and STAT5. However, in many cases, c-Src activation of STAT3/5 is mediated through GFR pathways. For example, in c-Src and EGFR co-overexpressing breast cancer cells, the c-Src-mediated, EGF-induced phosphorylation of tyrosine 845 (Y845) in the catalytic domain of the EGFR is crucial for EGF-induced DNA synthesis because a Y845F mutant abrogates this EGF-induced response (35). Furthermore, phosphorylation of this site mediates STAT5a/5b activation (75). This activation is distinct from other common signaling pathways because this same Y845F mutant does not abrogate the EGF-induced activation of STAT3, MAPK, or PLC (35). The integral role of STAT5 signaling in this paradigm is reflected by experiments that show that a dominant negative form of STAT5b inhibits EGF-induced proliferation of HER1/HER2/c-Src co-overexpressing breast cancer cells (60, 75). STAT3 has been defined as an oncogene because its critical role in v-Src-induced transformation was identified (78). STAT3 has been shown to be constitutively active in c-Src and EGFR co-overexpressing cells (76). Constitutively active STAT3 regulates genes involved in breast cancer cell proliferation and survival (e.g. bcl-xL, cyclin D1, c-myc) (64, 79). A dominant-negative STAT3 inhibits the growth of breast cancer cell lines, and knockdown of STAT3 inhibits induction of breast tumors in mice (80, 81, 82). Together, these data support an integral role for the STAT3/5 signaling pathway in growth factor-dependent tumors. It is becoming increasingly well documented that the STAT3/5 pathway also plays an important role in E2-dependent signaling. Functional and physical interactions between STAT3/5 and steroid receptors, including the ER, PR, and AR, have been described (83, 84, 85, 86, 87). Both cooperation through nuclear transcriptional pathways, and ER regulation of cytoplasmic stimulation of STAT phosphorylation may occur (88, 89). In vitro binding studies demonstrate direct protein interactions between ER and STAT3/5, occurring primarily through the DNA-binding domain (DBD) of ER. Interactions between the PR and STAT3/5, as well as the AR and STAT5, have been described through similar regions (83, 86, 87). In a mammary epithelial cell line (HC11), ER enhances prolactin-induced transcription from a STAT5-RE promoter (85). Studies in aortic epithelial cells showed that ER activates STAT5b, STAT5a, and STAT3 (90). Yamashita et al. (89) have detected the constitutive activation of STAT5b in a panel of breast cancer cell lines including the ER+ T47D and MCF-7 lines. A dominant-negative STAT5 inhibits E2-stimulated growth of T47D cells and increases apoptosis. Further studies demonstrated that introduction of a dominant-negative STAT5 into T47D ER+ human breast cancer cells suppresses E2-stimulated xenograft tumor growth in nude mice (91).
Although there is increasing evidence for the role of STAT signaling in proliferation of ER+ tumors, the mechanism by which STAT3/5 is activated by E2-ER has not been fully elucidated. Direct associations between ER and STAT3 and STAT5 have been demonstrated in immunoprecipitations and glutathione-S-transferase pull-down assays (92). However, because ER itself has no kinase activity, phosphorylation and thus activation of STATs must occur through another molecule that associates with ER or is activated by the receptor. In endothelial cells, 17ß-estradiol-bound ER stimulates Stat5 and Stat3 phosphorylation and nuclear translocation. Furthermore, transactivation of a Stat-regulated promoter has been suggested to require at least three different signal transduction pathways, including MAPK, Src-kinase, and phosphatidylinositol-3-kinase activities (90). Given the role of the c-Src tyrosine kinase in mediating ER signaling in the cytoplasm, one could speculate that activation of c-Src by E2-ER leads to c-Src-mediated activation of the STATs through phosphorylation of the activating tyrosine.
Just as the adapter proteins, Shc, p130Cas, and MNAR, play a role in E2-induced activation of c-Src, MAPK, and PI3-kinase, so there is also evidence that they are involved in E2-induced activation of the STAT3/5 pathway through c-Src. The Shc isoforms (p66 and p52) can induce c-Src-dependent phosphorylation of STATs1/3/5b (93). Furthermore, signaling through the PR results in increased HER1/2 signaling and increased association of STAT5 with tyrosine-phosphorylated JAK2 and Shc (83). The scaffolding protein MNAR [or proline-, glutamic acid-, and leucine-rich protein-1 (PELP1)] associates with the ER (through Y537), the AR, CREB-binding protein/p300 as well as c-Src, and STAT3 (94). Recent studies have shown that the MNAR-cSrc interaction increases the serine phosphorylation of STAT3 (95). This serine phosphorylation of STAT3 leads to increases in its transcriptional activity and a subsequent increase in the expression of target genes, (cyclin D1, c-myc, and c-fos). Of note, it was recently shown that this same serine phosphorylation of STAT3 is increased in ER-negative and GFR-overexpressing breast cancer through increases in the MAPK pathway (96). The adapter protein p130Cas interacts with c-Src and also plays a role in activation of the STAT pathway, because its overexpression results in an increase in STAT5b tyrosine phosphorylation and transcriptional activity (49). Activation of the ER-STAT pathway may be dependent on the relative expression levels of c-Src, the ER, and these adapter proteins, as well as the STAT proteins themselves. A threshold level of one or more of these signaling molecules could be required before E2 treatment is able to induce activation downstream to the STAT3/5 pathway.
Given the known role of growth factor receptors (particularly HER family but also IGF-I receptor) in mediating signaling through c-Src, cross talk between growth factors and E2 might occur through c-Src, alone or in various signaling complexes (15, 23, 48). GFRs could serve as docking proteins for adapter molecules such as Shc, c-Src itself, and/or the STAT3/5 proteins. As mentioned above, an example of this cross talk between ER and GFR has recently been documented. In the MCF7 ER+ breast cancer cell line, E2 induces the phosphorylation of Y845 in the catalytic domain of the EGFR. Because the phosphorylation of Y845 is mediated by the c-Src kinase, it is proposed that this activation occurs through ER-induced activation of c-Src (49). Although the E2-induced phosphorylation of Y845 is much less than that seen with EGF treatment, this event supports cross talk between these two receptors and c-Src. The role of GFRs in ER signaling in the cytoplasm, or at the membrane, has been described in many breast cancer models. For example, HER1, HER2, and IGF-I receptors have all been shown to provide docking sites for ER or c-Src and Shc (52). Finally, a G protein-linked activation of the EGFR through the ER has been proposed (20). E2 binding to the ER results in the activation of G proteins that activate the c-Src tyrosine kinase, leading to activation of matrix metalloproteinases-2 and -9. Matrix metalloproteinases then cleave heparin-binding EGF from the membrane, allowing the EGF to interact extracellularly with the EGFR, leading to activation of its signaling pathways. These pathways could include MAPK, PI3K/Akt/mTOR, inhibitor of B kinase/nuclear factor-B, and STAT3/5 as well as others; although the relative strength of the signal compared with that of direct binding of circulating, extracellular ligands has not been elucidated.
The other E2-induced signaling pathways discussed above also positively and independently influence STAT3/5 activity. In addition to the tyrosine phosphorylation required for activation of the STAT proteins, in most cases, there is a least one serine phosphorylation site in the TAD of the STATs. This serine phosphorylation leads to increases in transcriptional activation, presumably by allowing binding of coactivator proteins (59). Whereas this serine phosphorylation event is well characterized in STAT1 and STAT3, it is less well so for STAT5a/5b and, in most cases, the mechanisms of serine phosphorylation have been identified mainly in hematopoietic cell lines. Nevertheless, those pathways that have been shown to mediate the serine phosphorylation of STATs include MAPK, PI3K, and PKC, all of which are activated in breast cancer cells by both growth factor and steroid receptors such as the ER. For example, the MAPK pathway mediates the phosphorylation of a single serine (S727) in the TAD of STAT3 (59). The crucial role of this serine site in STAT3 activity is demonstrated by the fact that a S727A STAT3 knock-in is unable to compensate for the effects of STAT3 knockout on growth and survival (97). Furthermore, there is recent evidence that the phosphorylation of S727 is important in STAT3 function in breast cancer models. The adapter protein MNAR binds both ER and c-Src and has been shown to facilitate the MAPK-induced phosphorylation of STAT3 at S727 (95). A recent immunoblotting analysis of breast tumor tissue showed increased phosphorylation of S727 of STAT3 in breast cancer tissue compared with adjacent nontumor tissue (96). In this same study, detectable levels of S727 phosphorylation were seen in a panel of breast cancer cell lines, and this phosphorylation was increased by treatment with either EGF or E2. Although the serine kinase(s) that phosphorylate STAT5a and/or STAT5b has not been identified, there is evidence that MAPK family kinases, as well as PI3K and PKC kinases, are involved in this phosphorylation (98). Both STAT5a and STAT5b have been shown to be constitutively serine phosphorylated in the mouse mammary epithelial cell line, HC11, and in the MCF-7 human breast cancer cell line (99). Recently, it has been shown that hypoxia induces the serine phosphorylation of STAT3 and STAT5 in these same two cell lines (100). Together, these studies suggest a potentially important role of STAT3/5 serine phosphorylation in increasing STAT activity in breast cancer cells. Increases in serine kinase pathways induced by overexpression and activation of GFR and/or ER would facilitate this phosphorylation.
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IMPLICATIONS FOR ANTIESTROGEN THERAPY |
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TOPABSTRACTINTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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When a patient is diagnosed with breast cancer, the tumor is tested for presence of ER and the PR, an E2-stimulated gene, as the first part of the decision tree for adjuvant ER-targeted therapies such as TAM (104). However, despite ER expression, between 20–60% these tumors never respond to treatment, a phenomenon known as intrinsic resistance. A new class of drugs, the aromatase inhibitors, act to prevent E2 synthesis from many sites such as adrenal glands, adipose, and breast tissue, and thus effectively prevent ER activation. Clinical responses to aromatase inhibitors appear to be better than to TAM, but many ER+ tumors cannot respond to any of these ER-based therapies (105). Resistant tumors may sometimes respond to a second endocrine therapy, and responses to fluvestrant in tumors resistant to TAM or aromatase inhibitors have been described (106). Insight into mechanisms of resistance, or failure to respond, to ER-based therapies such as SERMS and aromatase inhibitors is critical to identify alternative therapeutic targets and treatments.
Although therapeutic resistance to TAM (and other antiestrogens) may involve several cellular pathways and might differ between intrinsic and acquired responses, neither mutations in ER nor altered metabolism of TAM or other anti-E2 appear to account for more than a small fraction of either acquired or intrinsic resistance (104). Results from numerous studies suggest that there are at least two major types of mechanisms for resistance: 1) hyperactivation of cytoplasmic signaling pathways and 2) dysregulation of coactivator or corepressor molecules that regulate transcription of proliferative and cell survival genes. These signaling pathways are coordinated by interactions between the ER and other signaling molecules depicted in Fig. 1, including the STAT3/5 transcription factors and involving both genomic and nongenomic activities of ER. The relative expression level of GFRs and ER, as well as the downstream signaling molecules (both cytoplasmic/membrane as well as nuclear), will not only dictate the strength of the proliferative signal, but will ultimately influence the response of a tumor to ER-based therapies.
Nongenomic Signaling Pathways
A noncancerous breast epithelial cell expresses basal levels of the signaling molecules in the pathways required for normal growth and development, including the ER, the c-Src tyrosine kinase, and members of the HER family. However, if these molecules/pathways become dysregulated by overexpression, the process of tumorigenesis results. This process is depicted in paradigms I, II, and III in Fig. 1, although in tumors, there no doubt exists a continuum of possibilities that spans these paradigms. In paradigm I, physiological levels of HER and c-Src tyrosine kinases are expressed whereas the ER is overexpressed. In this case, the ER provides the predominant proliferative signal, and antiestrogens or aromatase inhibitors are effective (at least initially) in preventing proliferation of these tumors. Breast tumor cells that overexpress HER family members and the c-Src tyrosine kinase, in addition to the ER, are depicted in paradigm II.
The relative level of HER family and c-Src overexpression will vary greatly depending on the particular tumor. However, as depicted in our model, the greater the overexpression of the GFR pathways, the stronger the downstream proliferative signal will be. As the GFR side of the signal is turned up, the ER pathway becomes less important. Such cells or tumors might respond best to joint therapies targeting both ER and GF pathways. We propose that paradigm II represents one subset of those 40–80% of ER+ tumors that are either intrinsically resistant to antiestrogen therapy due to overexpression and predominance of GFR signaling, or the remainder of ER+ tumors that acquire resistance over time because the GFR pathway has become increasingly predominant during antiestrogen therapy.
Support for this model comes from a number of in vitro models as well as human breast tumors. Overexpression of the HER and c-Src tyrosine kinases is often associated with resistance of the ER+ cancer to treatment with antiestrogens, providing strong evidence that the cell signaling context influences therapeutic responses (53). In an MCF-7 model that was developed to be resistant to the growth-inhibitory effects of TAM (simulating a tumor with acquired resistance), both the HER1/EGFR and HER2/neu pathways are up-regulated. There is an increase not only in the HER1/2 protein expression, but also in the tyrosine phosphorylation of these proteins and of signaling through the MAPK pathway (107, 108). A link between increases in c-Src activity and resistance to TAM has also been made since a more recent study demonstrated that expression of the phosphorylated (active) form of c-Src is increased not only in TAM-resistant cell lines, but also in metastatic tumors and those resistant to TAM therapy (109). In addition to the role of tyrosine kinase overexpression in TAM resistance, the role of the signaling molecules activated downstream of these kinases has also been shown to play a crucial role in the development of TAM resistance. The MAPK, PI3K/Akt, and PKC pathways have all been shown to be activated in models of antiestrogen resistance (107, 110, 111). The Cas/c-Src/ER complex has been shown to increase activity of the PI3K pathway, and the MNAR/c-Src/ER complex has been shown to increase the MAPK pathway (42, 112). Finally, by virtue of the fact that TAM has been used longer as a treatment for ER-positive breast cancers, mechanisms of its resistance have been most well studied. However, recently, it has been shown that similar pathways (HER1/2, MAPK, Akt) are activated in breast cancer cells resistant to ICI 182,780 (113) and raloxifene (114).
Finally, in paradigm III, the ER is no longer expressed (or at least is undetectable at the protein level), and the GFR and c-Src signaling molecules are overexpressed. This paradigm represents the most aggressive and metastatic tumors. These tumors would not be treated with antiestrogen-based therapies, but would require therapies targeted to the GFR/c-Src tyrosine kinase pathway and/or to those downstream signaling molecules that ultimately relay the proliferative signal (115). Support for this model also comes from studies in which expression of the ER is reintroduced into otherwise ER– cell lines. Early studies demonstrated that increased expression of ER led to inhibition of E2-induced proliferation and cell death (116). Studies in otherwise ER– breast cancer cell lines that are engineered to stably (MDA-MB231) or transiently (SKBr3) express the ER have shown that E2 inhibits not only EGF-induced STAT5 signaling (and not MAPK) but also DNA synthesis (88). Finally, a more recent study in which the ER mRNA expression was reactivated by treating ER– cells (MDA-MB231) with demethylating agents rendered these cells responsive to the growth-inhibitory effects of TAM (117).
Recent studies have suggested that the STAT3/5 signaling pathway is also integrally involved in antiestrogen resistance. As described above, the STAT3/5 pathway is activated downstream of both GFR and c-Src, and these signaling molecules are overexpressed in TAM-resistant tumors. Therefore, it is not surprising that recent studies support the integral role of STAT3/5 signaling in TAM resistance. Yeh et al. (96) have shown that TAM treatment of ER+ breast cancer cells results in a decrease in the serine (S727) phosphorylation of STAT3, suggesting a link between TAM sensitivity and decreased STAT3 transcriptional activity. From the result of this study, one could predict that development of TAM resistance may, in part, involve increased STAT3 signaling. A recent study provides such a link between TAM resistance and STAT5 signaling. The p130Cas adapter protein binds c-Src and increases the catalytic activity of this tyrosine kinase (118). The gene that encodes for p130Cas in humans (bcar1) was identified by insertional mutagenesis as a genetic factor that could lead to TAM resistance in vitro and in transfected T47D breast cancer cells (119). Furthermore, analysis of a large series of primary breast tumors showed that high Cas protein levels in ER+ tumors were associated with more rapid disease reoccurrence and greater risk for intrinsic antiestrogen resistance (120). Recent in vitro studies have shown that increasing Cas expression results in resistance of ER+ cells to TAM and that this effect is dependent on the ability of Cas to interact with c-Src (49). In this same cell model, increased Cas expression results in an increase in STAT5b tyrosine phosphorylation and transcriptional activation. Most importantly, it was also shown that a dominant-negative STAT5b inhibited the ability of Cas to induce TAM resistance. These data provide a link between TAM resistance, Cas overexpression, increased c-Src kinase activity, and increased STAT5b activity. Together, these studies suggest that STAT3/5 signaling may play an integral role in the development of TAM resistance and may provide an additional target for therapy.
Genomic Signaling Pathways
In addition to interactions between cytoplasmic signaling pathways described above, there is evidence to support interaction between these pathways in the nucleus. Because the ER and STAT proteins are both transcription factors, there is ample opportunity for them to interact once they translocate to the nucleus. Such interactions, either direct or indirect, could take place on DNA and involve protein-protein interactions between the transcription factors themselves, or via protein complexes, including common coregulatory proteins. STAT3, STAT5a, and STAT5b bind as homo- or heterodimers to STAT3/5-specific consensus elements, via the central DNA-binding domains, with STAT-specific gene regulation mediated through the C-terminal transcriptional activation domains (121). A number of coregulator proteins that modulate transcription have been documented to interact with the STAT3/5 proteins and include CREB-binding protein, p100, steroid receptor coactivator 1 (SRC-1), octamer transcription factor 1, protein inhibitor of activated STAT1/3, silencing mediator of retinoid and thyroid hormone receptor, and Nmi (122). Most of these also interact with the steroid receptors, including ER, and alter chromatin structure and control transcription (123). Furthermore, the STAT proteins (STAT3/5a/5b) and ER regulate many of the same genes, thus providing a site for their interaction at the DNA promoter. These genes are proproliferative and prosurvival, and include c-myc, cyclin D1, c-myb, p21waf/cip1, bcl-xL, and IGF-I (14). Many of these same genes are also regulated by the PR and AR and could involve STAT-receptor interactions in the nucleus (83, 87, 124).
Several studies have demonstrated both physical and functional interactions between STAT3/5 and steroid receptors, including the glucocorticoid receptor (84), the progesterone receptor (83), as well as the ER (125). Such interactions either enhance or inhibit STAT5 transcription in a cell-specific manner. Bjornstrom et al. (85) demonstrated that ER or ERß could potentiate prolactin-induced transcription through a STAT5RE. This response required specific residues within the DBD, but not the entire DBD of ER, suggesting a dependence on conformation of the ER rather than binding to DNA (90). In similar studies, another group reported that both the ER and ERß could repress STAT5a/5b transcriptional activation in ER-negative human embryonic kidney (HEK)293 cells, an effect that required both the DBD as well as the C terminus of the ER (126). Wang et al. (127) demonstrated a direct interaction between STAT5a and ER by glutathione-S-transferase-fusion protein pull-down experiments in HEK293 cells, as well as by coimmunoprecipitation in two ER+ human breast cancer cell lines (MCF-7 and T47D). However, E2 treatment attenuated prolactin-mediated signaling to STATs in ER-negative HEK293 cells but potentiated prolactin-mediated signaling in ER-positive MCF-7 cells. Interactions between STAT3 and the ER in a number of target types have also been reported (128).
Importantly, E2 and SERMs, such as TAM and raloxifene, differentially alter ER structure and therefore its ability to recruit coregulatory proteins. Altered ratios of coactivators to corepressors in a tumor or specific cell type may alter or prevent responsiveness to SERMs, and lower levels of coactivators or higher levels of corepressors have been associated with differential patient responses to TAM therapy (103, 106, 129). Because little is known about how STAT factors communicate with other transcription factors and the transcriptional machinery, any molecular mechanism for cell-specific ER-mediated repression of STAT5 activity must be speculative. One possibility may be that ER binds to STAT5 and inhibits necessary contact between STAT5 and other transcription factors or coregulatory proteins. In addition to activation of coactivators by cyptoplasmic signaling pathways, altered coactivator to corepressor ratios can contribute to inappropriate responses to SERMS. Transient overexpression of the coactivator SRC-1 reduces TAM suppression in MCF7 or T47D cells (130). The therapeutic consequences of the AIB1 gene, originally found amplified in approximately 10% of human breast tumors, were unknown but may be due to increased expression of the coactivator (131). Higher expression of SRC-1 was also associated with resistance to endocrine treatment of ER+ tumors. In contrast, a recent report suggests that lower expression of the corepressor, nuclear receptor corepressor, was the best predictor for TAM resistance compared with other coregulators tested in ER+ breast cancers (132). Redundancy in the actions and number of coactivators and corepressor proteins, and the lack of cell specificity in their expression, may make it difficult to measure a single protein to assess resistance. Finally, because ER and/or ERß may form functional complexes with AR or PR in breast or prostate cells, cross talk between sex steroid receptors with different preferences or specificities for coactivators may also influence response to therapy (43, 133).
One intriguing exception among transcriptional regulatory proteins may be observed changes in BRCA1 (breast cancer 1, early onset), the protein encoded by the breast cancer susceptibility gene 1. Mutations in Brca1 predispose women to familial breast and ovarian cancer; however, these account for only 5–10% of breast tumors (134). However, the vast majority of breast cancers are classified as sporadic, and up to 40% of these (including ER+ tumors) have up to an 80% decrease in BRCA1 mRNA and protein (135). BRCA1 plays a critical role in DNA-repair mechanisms and modulates gene transcription. It does not bind directly to DNA but, instead, exerts its influence by interacting directly with a subset of transcription factors, and these include ER, PR, AR, STAT3, and STAT5. Overexpression of BRCA1 in breast cancer cells suppresses ER-mediated transcription, as well as E2-stimulated nongenomic pathways (136, 137). Similarly, BRCA1 binds to STATs and inhibits transcription through STAT5a in mammary cells (138). It is not known how BRCA1 mediates this inhibition; however, it can associate with many protein partners, including histone-modifying proteins (p300 and histone deacetylases) and ATP-dependent chromatin remodeling enzymes (hSNF/SWI), which are important transcriptional regulatory proteins (134). Potential cross talk of BRCA1 with ER and cytoplasmic signaling molecules and the role of BRCA1 in therapeutic resistance in breast cancer have been largely unexplored.
BRCA1 actions on other steroid receptors, such as PR, in conjunction with ER may also have significant impact on mammary tumorigenesis and/or therapy. As with ER, BRCA1 binds to and suppresses PR transcription (139). Recent studies with Brca1/p53-deficient mice demonstrate more lateral branching and extensive alveologenesis in mammary glands of nulliparous females, as well as augmented progesterone-stimulated gene expression (140). PR, but not ER, levels are increased, and PR degradation through the proteasomal pathway is diminished; the E3 ligase activity of BRCA1 is required for this response. Both E2 and progesterone stimulate mammary tumorigenesis in these animals, and treatment with an antiprogestin, mifepristone, prevents these tumors. PR itself is an E2-stimulated gene, and BRCA1 may thus act at many levels in this model to control hormone-induced cancer. Given the potential for ER, PR, and AR to influence many common cellular pathways and to functionally interact in model systems, the entire molecular signature of breast cancers, including steroid receptors and coregulators, growth factor receptors, and cytoplasmic signaling molecules, will likely influence both tumor development and clinical responses.
As depicted in Fig. 1, STAT3/5a/5b proteins might also modulate ER-coregulator interactions, thus altering the response to E2 and antiestrogens. For example, Kim et al. (141) recently demonstrated that p300 mediates the acetylation of ER at two specific lysines (K266 and K268), resulting in an increase in DNA-binding of the ER in vitro and increased transcription of a reporter construct. Because STAT3 and STAT5 also bind p300 and are potentially present at the same promoter regions, increases in STAT3/5 binding at the promoter may recruit p300 away from the ER, thereby decreasing the ER-mediated response. Mechanisms similar to this one may contribute to E2-independent growth and eventual dependence of the tumor on growth factor signaling exclusively (paradigm III). Additional differences in cell-specific responses could be due to differential posttranslational modification of the transcription factors, cell-type specific complements of coregulatory proteins, or differential cross talk between cytoplasmic and nuclear signaling components.
Thus, there are a number of examples that illustrate the interactions between the ER and STAT3/5 that are regulated by both estrogen and growth factor pathways. The complexity of interactions that are modulated by the expression level of each of these proteins is just beginning to be elucidated. However, because many of these regulated genes are proproliferation and survival, they are also ultimately targets for the process of tumor formation. Critical differences in STAT5 responses to E2 and TAM depend on cell context, including the array and levels of coregulatory proteins. Therefore, further understanding of these processes, as well as those of other steroid and growth factor receptors, will provide insight into the potential novel targets for therapies.
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SUMMARY |
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TOPABSTRACTINTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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FOOTNOTES |
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Disclosure Statement: M.A.S receives a stipend as an Endocrine Society officer and antibody royalties from Upstate Biotechnologies. C.M.S receives antibody royalties from BD BioSciences.
First Published Online April 24, 2007
Abbreviations: AR, Androgen receptor; BRCA1, breast cancer 1, early onset; DBD, DNA-binding domain; E2, 17ß-estradiol; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; ERE, estrogen response element; GF, growth factor; GFR, growth factor receptor; HEK, human embryonic kidney; HER, human epidermal growth factor receptor; MNAR, moderator of nongenomic activity of estrogen receptor; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PR, progesterone receptor; SERMs, selective estrogen receptor modulators; SRC, steroid receptor coactivator; STAT, signal transducer and activator of transcription; STATRE, STAT response element; TAD, transcriptional activation domain; TAM, tamoxifen.
Received for publication February 26, 2007. Accepted for publication April 11, 2007.
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TOPABSTRACTINTRODUCTIONSTEROID AND GROWTH FACTOR...INTEGRATION OF SIGNALINGSTATsSTATs: COMMON DOWNSTREAM...IMPLICATIONS FOR ANTIESTROGEN...SUMMARYREFERENCES |
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. Cancer Res 59:1869–1876 and ß: regulation by selective estrogen receptor modulators and importance in breast cancer. Breast Cancer Res 2:335–344[CrossRef][Medline]
