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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2011 December 16; 286(50): 43091–43102.
Published online 2011 October 21. doi:  10.1074/jbc.M111.295865
PMCID: PMC3234857

Progesterone Receptor Inhibits Proliferation of Human Breast Cancer Cells via Induction of MAPK Phosphatase 1 (MKP-1/DUSP1)*An external file that holds a picture, illustration, etc.
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Abstract

The roles of progesterone (P4) and of progesterone receptor (PR) in development and pathogenesis of breast cancer remain unclear. In this study, we observed that treatment of T47D breast cancer cells with progestin antagonized effects of fetal bovine serum (FBS) to stimulate cell proliferation, whereas siRNA-mediated knockdown of endogenous PR abrogated progestin-mediated anti-proliferative effects. To begin to define mechanisms for the anti-proliferative action of P4/PR, we considered the role of MAPK phosphatase 1 (MKP-1/DUSP1), which catalyzes dephosphorylation and inactivation of MAPKs. Progestin treatment of T47D cells rapidly induced MKP-1 expression in a PR-dependent manner. Importantly, P4 induction of MKP-1 was associated with reduced levels of phosphorylated ERK1/2, whereas siRNA knockdown of MKP-1 blocked progestin-mediated ERK1/2 dephosphorylation and repression of FBS-induced cell proliferation. The importance of PR in MKP-1 expression was supported by findings that MKP-1 and PR mRNA levels were significantly correlated in 30 human breast cancer cell lines. By contrast, no correlation was observed with the glucocorticoid receptor, a known regulator of MKP-1 in other cell types. ChIP and luciferase reporter assay findings suggest that PR acts in a ligand-dependent manner through binding to two progesterone response elements downstream of the MKP-1 transcription start site to up-regulate MKP-1 promoter activity. PR also interacts with two Sp1 sites just downstream of the transcription start site to increase MKP-1 expression. Collectively, these findings suggest that MKP-1 is a critical mediator of anti-proliferative and anti-inflammatory actions of PR in the breast.

Keywords: Breast cancer, MAP kinases (MAPKs), Nuclear receptors, Progesterone, Proliferation, MAPK phosphatase, Progesterone receptor

Introduction

Progesterone (P4)3 exerts its primary actions in reproductive tissues, including the uterus, ovary, and breast. In the mammary gland, P4 plays an important role in ductal proliferation and lobuloalveolar differentiation during pregnancy (1). The actions of P4 are mediated by progesterone receptors (PR), PR-A and PR-B, which are products of a single gene and ligand-activated members of the nuclear receptor family. Classically, binding of P4 to PR induces a conformational change in the receptor, followed by dimerization and translocation from cytoplasm into nucleus where it binds to progesterone response elements (PRE) in PR-target gene promoters. In addition to directly binding to PREs in DNA, PR also can act via non-classical mechanisms by tethering to other transcription factors bound to their respective response elements in the promoter regions of target genes to modulate transcriptional activity. Transcription factors with which PR interacts include specificity protein 1 (Sp1) (2), nuclear factor κB (NF-κB) (3), signal transducer and activator of transcription 5 (4, 5), and activator protein-1 (AP-1) (6). After binding to PREs or tethering to other DNA-bound transcription factors, PRs recruit co-activators/co-repressor complexes, which contain activities for histone modification, chromatin remodeling, and RNA splicing and facilitate interactions with the basic transcriptional machinery to induce/repress target gene expression (7).

The roles of P4/PR in breast cancer are controversial. Levels of estrogen and progesterone receptors in a breast tumor are strong prognostic indicators for responsiveness to endocrine therapy and for survival (8, 9). Thus, many advanced breast cancers that are negative for PR and estrogen receptor (ER) are more aggressive and fail to respond to endocrine therapy. On the other hand, clinical findings of the Women's Health Initiative indicate that hormone replacement therapy using estrogen with progestin significantly increased breast cancer risk, as compared with use of estrogen alone (10, 11). Depending on the physiological state and incubation conditions, P4 can act either in a proliferative or differentiative capacity in cultured breast cancer cells (1). Moreover, both growth-stimulatory and -inhibitory actions of P4 have been reported in cultured breast epithelial cells and on cancer development in animal tumor models (13, 14). Studies using PR-positive mammary carcinoma cell lines as a model have demonstrated a biphasic cellular response to either P4 or synthetic progestins (R5020 or ORG 2058), with an immediate proliferative burst followed by a sustained growth arrest (1517). Moreover, treatment with P4 has been reported to suppress cell proliferation in response to different mitogens, such as estrogens, serum, and insulin-like growth factor, alone or in combination (18). P4 has been found to inhibit cell cycle progression of breast cancer cells by transient induction of the cyclin-dependent kinase inhibitors (CDKIs) p21Cip1/WAF1 (p21) and p18INK4c (p18), followed by a sustained induction of p27Kip1 (p27), leading to association of these CDKIs with the different G1 CDK complexes. CDKI·CDK complex formation results in down-regulation of CDK activity leading to decreased pRb phosphorylation and cell cycle arrest in the late G1 phase (1922).

Mitogen-activated protein kinase (MAPK) activation has been implicated in breast cancer progression and metastasis. MAPK signal transduction pathways are evolutionarily conserved and play a central role in conveying information from the extracellular environment to the cytoplasm and finally into the nuclear compartment (23). There are at least three known MAPK signaling pathways, including the extracellular signal-regulated protein kinase (ERK) pathway, the p38 MAPK pathway, and the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase pathway (24). The ERK pathway plays a critical role in cell proliferation, and its activity is enhanced by growth factors, integrins, activation of G protein-coupled receptor systems, products of several proto-oncogenes (25), as well as by estrogen (2628) and P4 (16, 29). ERK1 and ERK2 protein kinases (ERK1/2) are activated by phosphorylation on threonine and tyrosine residues within a conserved Thr-X-Tyr motif via a kinase cascade involving Ras/Raf/Mek proteins. Activation of ERK1/2 commonly causes its translocation from the cytoplasm to the nucleus. Once inside the nucleus, the active ERK1/2 are capable of directly phosphorylating a number of transcription factors and cell cycle regulators (24).

Recently, a family of protein phosphatases, the dual-specificity phosphatases (DUSPs), were discovered to have the ability to interact and catalyze dephosphorylation of active MAPK (30). To date, ten members of the DUSP family have been identified in mammals (31, 32). DUSP1 (also known as MAPK phosphatase 1 (MKP-1), CL100, 3CH134, Erp, and hVH-1) was observed to inactivate ERK1/2 by dephosphorylation of both threonine and tyrosine residues within the activation motif (33). MKP-1 has been shown to inhibit a number of cellular responses mediated by ERK1/2, JNK, and p38 MAPK (3436). Moreover, increasing MKP-1 protein expression has been shown to suppress growth of MCF-7 breast cancer cells (37). Recent studies have identified MKP-1 as a glucocorticoid receptor (GR) target gene, which mediates GR anti-inflammatory activity (3841). Progestins, acting through PR, also have been found to serve an anti-inflammatory role in both the uterus (42, 43) and in the breast (44, 45). We observed that the anti-inflammatory actions of PR are mediated by ligand-dependent and -independent mechanisms, resulting in an inhibition of NF-κB activation with consequent down-regulation of hCOX-2, aromatase/hCYP19, and HER-2/neu expression (44). Notably, MKP-1 mRNA expression was observed to be induced by P4/PR in human breast cancer cells (22).

In consideration of the potential role of MKP-1 as an important PR target gene in the breast that mediates some of its anti-inflammatory/anti-proliferative actions, in the present study, we investigated the mechanisms whereby P4/PR modulates MKP-1. We observed that the PR acts in a ligand-dependent manner to suppress serum-induced T47D cell proliferation and that these anti-proliferative actions were associated with PR induction of MKP-1 expression. In addition, P4/PR induction of MKP-1 promoter activity was mediated via PR binding to PREs in DNA and by PR-Sp1 interactions. Finally, using an siRNA approach, we verified that MKP-1 serves as a PR target gene that mediates P4 repression of ERK1/2 activation by serum growth factors and the subsequent increase in cell proliferation.

MATERIALS AND METHODS

Reagents and Cell Culture

T47D breast cancer cells and HEK293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). T47D cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) with phenol red and supplemented with 7.5% fetal bovine serum (FBS) plus antibiotic-antimycotic solution (Sigma). HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) with phenol red and supplemented with 5% FBS plus antibiotic-antimycotic solution. Cells were cultured and grown in an air-carbon dioxide (95:5) atmosphere at 37 °C. For transient transfection studies, cells were seeded in medium without phenol red and supplemented with 2.5% FBS stripped with dextran-coated charcoal (Invitrogen). For RNA and protein expression experiments, cells were seeded in maintenance medium; the next day cells were changed to serum-free medium without phenol red for another 24 h before treatment. For treatment with various reagents, cells were incubated in serum-free medium without phenol red for times indicated. Progesterone (Sigma), Mifepristone (RU486, Sigma), and all other chemicals were the highest quality available from commercial sources.

Cloning and Plasmids

The cDNA for human MKP-1 was purchased from Origene (Rockville, MD) and subcloned into pcDNA3 expression vector (Invitrogen). The pMKP1-A-Luc plasmid, which contains −403 bp of sequence upstream and +490 bp downstream of the transcription start site (TSS) of the human MKP-1 gene was amplified from human genomic DNA and cloned into pGL4 vector (Promega, Madison, WI). pMKP1-B (−403/+216), pMKP1-C (−403/+113), and pMKP1-D (−403/+18) were made by PCR amplification using pMKP1-A as template and subcloned into pGL4 vector. Site-directed mutagenesis was performed using a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's protocol.

Transient Transfection, RNA Interference, and Reporter Assay

For MKP-1 overexpression experiments, T47D cells were transfected with pcDNA3 or MKP-1 expression vector using Neon® Transfection System (Invitrogen) according to the manufacturer's recommendations. After transfection, cells were seeded in 6-well plates with growth medium for 24 h and then placed in fresh RPMI 1640 medium without phenol red or FBS. For RNA interference (RNAi) experiments, small inhibitory RNA (siRNA) oligonucleotides against PR-A and PR-B (43, 46), human MKP-1 (Invitrogen), and silencer-negative control oligonucleotides (Ambion, Austin, TX) were transfected using the Neon® Transfection System (Invitrogen). For luciferase reporter assays, T47D and HEK 293 cells were seeded in 24-well plates and transfected using FuGENE® HD transfection reagent (Roche Applied Science) with MKP-1 reporter constructs (100 ng), PR-B expression vectors (100 ng), and Renilla luciferase plasmid (20 ng, Promega). One day after transfection, cells were treated with DMSO or P4 (100 nm) for 24 h in medium without phenol red or FBS. Cells from each experiment were then harvested in 100 μl of 1× Passive Lysis Buffer (Promega). Firefly luciferase and Renilla luciferase activities were assayed using a Dual-Luciferase assay system (Promega). Relative luciferase activities were calculated by normalizing Firefly luciferase activity to Renilla luciferase activity in the same samples to correct for transfection efficiencies.

Immunoblot Analysis

T47D cells were harvested at designated time points and lysed in ice-cold lysis buffer (50 mm HEPES (pH 7.5), 500 mm NaCl, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA) supplemented with protease inhibitor mixture (Sigma). Equal amounts of protein from each treatment group were separated by SDS-10% PAGE and transferred to a PVDF membrane. Membranes were blocked with Blotto (5% (w/v) nonfat dry milk, Tris-buffered saline (10 mm Tris-HCl, pH 8.0, 150 mm NaCl), and 0.05% Tween-20) and probed with primary antibodies that recognize PR-A and PR-B (R&D Systems, Minneapolis, MN); phospho-ERK1/2 and ERK1/2 (Cell Signaling Technology, Beverly, MA); Sp1, MKP-1, α-tubulin, and β-actin (Upstate, Lake Placid, NY). Following incubation with peroxidase-conjugated secondary antibody, immune complexes were visualized using the ECL detection system (PerkinElmer Life Sciences).

RT-qPCR

Total RNA from T47D and 30 other breast cancer cell lines was extracted using TRIzol reagent (Invitrogen). The 30 breast cancer cell lines, derived from primary breast cancer biopsies at the Harold C. Simmons Comprehensive Cancer Center (University of Texas Southwestern) were kindly provided by Dr. John Minna. RNA was treated with deoxyribonuclease to remove any contaminating DNA and reverse transcribed using random primers and Superscript III reverse transcriptase (Invitrogen). The relative abundance of each RNA transcript was determined by quantitative PCR (qPCR) using the ABI Prism 7900 Detection System (Applied Biosystems, Foster City, CA) and the DNA-binding dye SYBR Green (PE Applied Biosystems). Relative arbitrary units were determined by comparative cycle times (Ct) of each transcript to Ct of human acidic ribosomal phosphoprotein P0 (h36B4) (endogenous control) and calculated using (2−ΔΔCt) method. Validated primer sets directed against hMKP-1 mRNA (forward: 5′-GGG CAG TGG AAT GAC AGG AA-3′; reverse: 5′-CTG AAG GCC AGA CAG GAT CC-3′), h36B4 (forward: 5′-TGC ATC AGT ACC CCA TTC TAT CA-3′; reverse: 5′-AAG GTG TAA TCC GTC TCC ACA GA-3′), hPR (forward: 5′-TCA GTG GGC AGA TGC TGT ATT T-3′; reverse: 5′-GCC ACA TGG TAA GGC ATA ATG A-3′), hGR (forward: 5′TCC CTG GTC GAA CAG TTT TTT-3′; reverse: 5′-AGC TGG ATG GAG GAG AGC TT-3′), hp21 (forward: 5′-CAT GGG TTC TGA CGG ACA T-3′; reverse: 5′-AGT CAG TTC CTT GTG GAG CC-3′), hSGK (forward: 5′-TTC CTA TCG CAG TGT TTC AGT TCT T-3′; reverse: 5′-CAC ACT CAC ACG ACG GTT CAC-3′), and hIκBα (forward: 5′-TTG GGT GCT GAT GTC AAT GC-3′; reverse: 5′-AGG TCC ACT GCG AGG TGA AG-3′) were used for qPCR amplification.

Cell Proliferation

For cell proliferation assays, ethynyl-2′-deoxyuridine (EdU) incorporation assays were performed. T47D cells were seeded in 4-well chamber slides, and cells were placed in medium without serum and phenol red on the next day. After 24-h incubation, cells were treated with DMSO or P4 (100 nm) in the absence or presence of 5% FBS in phenol red-free medium for another 24 h. EdU (10 μm, Invitrogen) was added to the medium for the last 4 h of the treatment. EdU incorporation was assayed using a Click-iT EdU Alexa Fluor® 488 cell proliferation assay kit (Invitrogen). Cell fixation, permeabilization, and EdU detection were performed according to the manufacturer's instructions. 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to identify nuclei for determination of cell number. EdU and DAPI signals were captured with a Zeiss Axiovert 100M fluorescence microscope, and captured images were processed and analyzed with ImageJ software (National Institutes of Health). The EdU and DAPI signals for each sample were analyzed in six different fields. The relative intensity of the EdU signal was calculated by normalizing the fluorescence signal of EdU with the DAPI staining.

ChIP

Chromatin immunoprecipitation (ChIP) was carried out, as described in detail previously (43). ChIP assays were performed using a ChIP kit (CHIP, Upstate), according to the manufacturer's recommendations. Briefly, confluent 100-mm dishes of T47D cells were treated with DMSO or P4 (100 nm) for 1 h and then cross-linked with 1% formaldehyde. Cells were washed with ice-cold 1× PBS, lysed, and sonicated on ice to produce sheared soluble chromatin. The soluble chromatin was precleared with Protein A/G Plus agarose beads (60 μl) at 4 °C for 1 h and then incubated with antibodies for PR (R&D Systems), Sp1 (Upstate), or with non-immune IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight. Immune complexes were collected on protein A/G-agarose and eluted. Cross-linking of immunoprecipitated chromatin complexes and of input controls was reversed by heating at 65 °C for 4 h, followed by proteinase K (Invitrogen) treatment. The purified DNA was subjected to PCR amplification and analyzed by electrophoresis on an ethidium bromide-stained agarose gel. The purified DNA also was analyzed by quantitative PCR.

Statistical Analysis

Statistical significance was determined by analysis of variance and Student's t test, and the levels of probability were noted. The data were expressed as means ± S.E. for at least three separate (replicate) experiments for each treatment.

RESULTS

Progesterone Acting via PR Induces MKP-1 mRNA Expression in T47D Cells

As noted above, MKP-1 is a known target of GR and has been shown to be induced by P4 in breast cancer cells (47). The mechanism whereby P4 modulates MKP-1 expression and the role of MKP-1 in mediating effects of P4 to alter cell proliferation remain undefined. To analyze the temporal induction of MKP-1 expression by progestin/PR, real-time PCR was used to measure mRNA in T47D cells cultured in the absence or presence of the synthetic progestin, R5020 (Fig. 1A). Treatment of T47D cells with R5020 induced MKP-1 mRNA expression within 4 h and reached maximal levels after 24 h of treatment. In association with mRNA induction, MKP-1 protein levels were rapidly induced following P4 treatment (Fig. 1B). Furthermore, upon knockdown of PR levels using a siRNA targeting PR (Fig. 1C) or by decreasing PR function by co-treatment with the PR antagonist RU486 (Fig. 1D), P4 induction of MKP-1 mRNA expression was significantly impaired. Taken together, these data suggest that P4 induction of MKP-1 expression in T47D cells is mediated by PR.

FIGURE 1.
Progesterone induces MKP-1 expression in breast cancer cells. T47D cells cultured in phenol red-free medium in the absence of FBS were treated with DMSO (V), R5020, or progesterone (P4, 100 nm) for 2–36 h. A, RNA was isolated and reverse-transcribed, ...

To examine the relationship between PR and MKP-1 further in vivo, we analyzed expression levels of MKP-1, PR, and GR in 30 human breast cancer cell lines. As shown in Fig. 1E, there was a positive correlation between MKP-1 and PR mRNA levels in these cell lines (p < 0.0001; r = 0.515; Pearson correlation). Interestingly, no correlation between MKP-1 and GR mRNA was observed. Previously, we reported that levels of PR mRNA in these human breast cancer cell lines were correlated with mRNA levels of the NF-κB inhibitor IκBα (44), which also is a PR target gene in breast cancer cells (44, 48). Interestingly, the correlation between MKP-1 and IκBα mRNA in the breast cancer cell lines also was found to be highly significant (Fig. 1E).

We also searched the Oncomine 4.4 database for breast cancer datasets to interrogate the relationship between MKP-1 mRNA expression with PR status. The Richardson breast cancer dataset shows that MKP-1 mRNA levels were higher in PR(+) breast carcinoma samples, as compared with samples with negative PR status (49). Other datasets also indicated that MKP-1 mRNA levels were higher in PR(+) breast cancer cells and tissue samples than in those negative for PR (5053).

Overexpression of MKP-1 in T47D Cells Suppresses Phosphorylation of ERK1/2 and Cell Proliferation

To examine the potential anti-proliferative function of MKP-1 in breast cancer cells, T47D cells were transiently transfected with MKP-1 expression vector, and EdU incorporation was employed to assess cell proliferation. After 24 h of serum (5% FBS) stimulation, EdU incorporation was significantly increased as compared with cells maintained in serum-free medium (Fig. 2A). However, overexpression of MKP-1 in T47D cells significantly inhibited the increase in cell proliferation caused by FBS, as compared with vector control (Fig. 2A). The anti-proliferative activity of MKP-1 also was observed using BrdU and thymidine incorporation assays (data not shown). It should be noted that the relative intensity of fluorescence signal observed in the EdU assay was determined by normalizing the EdU signal to the cell number, as determined by DAPI staining. This excludes the possibility that the reduced incorporation of EdU observed upon MKP-1 overexpression was caused by the cytotoxicity and cell death.

FIGURE 2.
Overexpression of MKP-1 represses proliferation of T47D breast cancer cells and inhibits ERK1/2 phosphorylation. T47D cells were transiently transfected with pcDNA3 (empty vector) or MKP-1 expression vector, and then cultured in RPMI 1640 medium with ...

The effect of FBS to increase T47D cell proliferation was associated with increased phosphorylation of ERK1 and -2 (Fig. 2B), whereas the action of overexpressed MKP-1 to inhibit cell proliferation was associated with an inhibition of FBS-induced ERK1/2 phosphorylation. Taken together, these results suggest that increased MKP-1 expression suppressed T47D cell proliferation in part, due to its action to decrease levels of phospho-ERK1/2.

Progesterone and Progesterone Receptors Suppress T47D Cell Proliferation and Repress ERK1/2 Phosphorylation by Induction of MKP-1 Expression

In light of these collective findings, we postulated that P4 exerts an inhibitory effect on cell proliferation in breast cancer cells via its effect to up-regulate MKP-1 expression. To explore the effects of P4/PR on T47D cell proliferation and the role of MKP-1, T47D cells were transfected with non-targeting siRNA, or with siRNAs for PR-A and PR-B or for MKP-1 to knock down the endogenous levels of these proteins. The cells were then synchronized in RPMI 1640 medium without FBS for 24 h and then treated with DMSO (vehicle (V)) or P4 (100 nm) with or without 5% FBS for another 24 h. Cell proliferation was analyzed using the EdU staining assay. As can be seen in Fig. 3A, when T47D cells were incubated with non-targeting control siRNA, FBS significantly increased the rate of cell proliferation, whereas treatment with P4 in FBS-containing medium significantly inhibited the cell proliferation rate, as compared with cells cultured with FBS alone. However, when cells were transfected with siRNAs to knock down PR-A and PR-B and cultured in medium containing FBS plus P4, the inhibitory effect of P4 on cell proliferation was abrogated. This suggests that PR mediates the inhibitory effect of P4 on T47D cell proliferation. In cells transfected with siRNA for MKP-1, we also observed that co-treatment with P4 did not inhibit cell proliferation induced by FBS, indicating that MKP-1 expression is crucial for the anti-proliferative action of P4 in T47D cells. Moreover, when these cells were transfected with siRNA targeting PR-A and PR-B or MKP-1 and cultured in FBS-containing medium, cell proliferation was significantly increased over that of cells transfected with non-targeting siRNA (Fig. 3A). This suggests that both PR and MKP-1 play a role to maintain the cell proliferation rate.

FIGURE 3.
Anti-proliferative effect of P4/PR on T47D cells is mediated by induction of MKP-1. A, effect of P4/PR to inhibit T47D cell proliferation is prevented by knockdown of PR-A/-B or MKP-1. T47D cells were synchronized in phenol red-free RPMI 1640 medium without ...

To further elucidate the underlying mechanisms for the anti-proliferative activity of P4/PR and of MKP-1, T47D cells were transfected with a non-targeting siRNA or with siRNAs for PR-A/-B or MKP-1 and serum-starved for 24 h, as above. The cells were then cultured in medium containing FBS with or without P4 for 2, 6, and 12 h, and ERK1/2 expression and phosphorylation state, as well as PR-A/B and MKP-1, were analyzed by immunoblotting. As shown in Fig. 3B, in T47D cells transfected with non-targeting control siRNA, incubation in medium containing FBS markedly induced phosphorylation of ERK1/2 at 2, 6, and 12 h. Co-treatment with P4 reduced the levels of ERK1/2 phosphorylation in FBS-treated cells after 6 and 12 h of treatment. Importantly, this decrease in ERK1/2 phosphorylation was associated with P4 induction of MKP-1 protein expression at these time points. On the other hand, siRNA knockdown of PR-A/B expression caused an apparent increase in ERK1/2 phosphorylation as compared with FBS-treated cells transfected with non-targeting siRNA, impaired the capacity of P4 to reduce ERK1/2 phosphorylation, and blocked the action of P4 to induce MKP-1 expression (Fig. 3B). Interestingly, incubation of T47D cells transfected with non-targeting or PR-A/B siRNA in FBS-containing medium caused a marked and rapid decline in MKP-1 expression (Fig. 3B). The effect of FBS to markedly reduce MKP-1 protein levels is likely due to activated ERK-mediated phosphorylation and subsequent ubiquitination of MKP-1, resulting in its proteolytic degradation (54). Notably, this effect of FBS was antagonized by P4 treatment in cells transfected with non-targeting siRNA but not in cells transfected with PR-A/B siRNA.

In cells transfected with MKP-1-specific siRNA, the ability of P4 to repress ERK1/2 phosphorylation also was blocked; this was correlated with a loss of P4 induction of MKP-1 expression (Fig. 3B). Taken together, these findings suggest that the effect of P4/PR to suppress FBS-induced cell proliferation is mediated by induction of MKP-1 expression.

Progesterone Up-regulates MKP-1 Expression via Recruitment of PR to PRE and Sp1 Response Elements

To further elucidate the molecular mechanisms for P4 induction of MKP-1 expression, we performed in silico analysis to define putative response elements within the regulatory regions of the hMKP-1 gene. Using the MatInspector program from Genomatix, we identified four potential PREs scattered throughout a 4-kb region surrounding the hMKP-1 TSS (Fig. 4A). ChIP assays were then employed to assess PR occupancy of MKP-1 genomic regions containing the four PREs in cultured T47D cells. As can be seen in Fig. 4B, PRs were recruited to two of the PREs, PRE1 and PRE2, located at +192 and +434 bp downstream of TSS, respectively. PR also was bound to a region (P0), which lies proximal to the TSS and does not contain a predicted PRE. To quantify in vivo binding of PR to these three sites, we used ChIP-qPCR (right panel). Treatment of the T47D cells with P4 for 60 min significantly increased the recruitment of PR to the PRE1 and PRE2 sites but had no effect to increase PR binding to the P0 site (Fig. 4C).

FIGURE 4.
Endogenous PR binds to two putative PREs in the hMKP-1 promoter. A, schematic of hMKP-1 gene (−1000 to +3000 bp) and genomic regions amplified for ChIP analysis. “+1” is the TSS; the filled boxes and circles indicate coordinates ...

To determine the functional roles of the identified PR-binding regions in P4 induction of MKP-1, we generated a luciferase reporter construct containing a genomic region of ~0.9 kb from −403 bp upstream to +490 bp downstream of the MKP-1 gene TSS (pMKP1-A). In HEK293 cells co-transfected with pMKP1-A and an expression vector containing PR-B, P4 treatment significantly induced luciferase activity compared with vehicle, suggesting that PREs are contained within this region (Fig. 5A). In HEK293 cells co-transfected with PR-B and reporter constructs containing deletions of this MKP-1 reporter construct, including pMKP1-B (−403/+216) and pMKP1-C (−403/+113), P4 retained the ability to induce luciferase activity. However, after further deletion of the MKP-1 first exon from +113 to +18, P4 lost its ability to induce luciferase activity. This suggests that the region between +18 and +113 contains critical progesterone-responsive elements. As mentioned, using ChIP, this region was observed to bind endogenous PR in the T47D cells (Fig. 4, B and C), although no consensus PREs were present. On the other hand, within the +18 to +113 bp genomic region, we identified two putative Sp1 sites at +20 and +52 bp. We considered that these may be responsible for P4 induction of MKP-1 promoter activity, because PR-Sp1 interaction has been reported to mediate rapid effects of P4 on PR transactivation (2, 55). As indicated in Fig. 5B, point mutations introduced individually into either of the two Sp1 sites in the MKP-1C-luciferase reporter did not affect P4/PR-B responsiveness of MKP-1 promoter activity in co-transfected HEK293 cells. However, when both Sp1 sites were mutated, both basal and P4 induction of MKP-1 promoter activity were greatly reduced, suggesting that these Sp1 sites act together to mediate basal and PR induction of MKP-1 expression. Furthermore, in T47D cells co-transfected with PR-B and the MKP-1C-luciferase reporter, mithramycin A, an inhibitor of Sp1 transcription factor binding to GC-rich elements in DNA (56), blocked P4 induction of MKP1-C-luciferase reporter expression (Fig. 5C). Also, treatment of T47D cells with mithramycin A significantly reduced P4 induction of endogenous MKP-1 (Fig. 5D). Treatment with mithramycin A, by contrast, did not affect the induction of SGK mRNA expression by P4 in T47D cells (supplemental Fig. S1). These collective findings suggest that Sp1 may play a role in P4/PR-mediated MKP-1 expression.

FIGURE 5.
Progesterone induces MKP-1 expression through Sp1 response elements. A, HEK293 cells were transiently transfected with PR-B expression vector or control vector without (w/o) PR-B, hMKP-1-Luc reporter constructs containing various deletions and with Renilla ...

To determine whether the putative PREs at +197 and +434 bp are required for P4-induced MKP-1 expression, these were mutated singly and together in the context of the MKP-1A-luciferase reporter construct containing the MKP-1 genomic region spanning −403 to +490 bp. These mutated constructs were co-transfected with a PR-B expression vector into HEK293 cells cultured with or without P4. Constructs containing mutations in both Sp1 sites, with or without mutations in both PREs, also were tested. In contrast to our findings using HEK293 cells transfected with the MKP-1C-luciferase reporter construct, in which mutation of both Sp1 sites markedly reduced basal and P4 induction of promoter activity, mutations in both Sp1 sites in the MKP-1A construct had little effect on basal or P4 stimulation of MKP-1 promoter activity (Fig. 6A). Although mutagenesis of one or both PREs resulted in a reduction in basal and P4-induced MKP-1 promoter activity, P4 responsiveness was partially retained. On the other hand, when both PREs and both Sp1 sites were mutated, P4 induction of MKP-1 promoter activity was greatly reduced. This suggests that both Sp1 sites and the two PREs cooperatively act to mediate P4 induction of MKP-1 promoter activity. To further study the mechanism of P4/PR induction of MKP-1 expression, HEK 293 cells were transiently transfected with a PR-B wild type (WT), a PR-B S345A mutant (2), which abrogates PR-Sp1 interaction, or with a PR-B DNA binding-defective mutant (mDBD) containing three point mutations in the DNA-binding domain (G585E-S586G-V589A). In HEK293 cells transfected with equivalent amounts of either the PR-B S345A mutant or the PR-B mDBD mutant expression vectors (supplemental Fig. S2), P4 partially retained the ability to induce endogenous MKP-1 expression, as compared with PR-B WT (Fig. 6B). However, in parallel HEK293 cell transfection studies, the PR-B S345A mutant was unable to induce expression of p21, cyclin-dependent kinase inhibitor 1 (Fig. 6B), whereas, PRB-mDBD mediated P4 induction of p21 expression at a level comparable to that of PRB-WT. Notably, p21 has been shown to be up-regulated by P4/PR via PR interaction with Sp1 (2). Importantly, the PRB-mDBD expression vector failed to mediate P4 induction of a mouse mammary tumor virus-luciferase reporter (MMTV-Luc), which was markedly induced by P4 in the presence of co-transfected PRB-WT and PRB-S345A expression vectors (supplemental Fig. S2). Taken together, these results suggest that PR acts in a ligand-dependent manner through binding to two PREs (+197 and +434) downstream of the MKP-1 TSS to up-regulate MKP-1 promoter activity. PR also acts via non-classical mechanisms to increase levels of MKP-1 expression by interaction with Sp1 transcription factor bound to two putative Sp1 response elements just downstream of the TSS.

FIGURE 6.
Progesterone induces MKP-1 expression through both Sp1 and PR response elements. A, HEK 293 cells were co-transfected the PR-B expression plasmid, MKP-1A luciferase reporter constructs containing various mutations and with Renilla luciferase plasmid. ...

DISCUSSION

The role of P4 and PR in the development and pathogenesis of breast cancer remains controversial. On the one hand, as mentioned above, P4 treatment of human breast cancer cell lines has been reported to cause a rapid and transient increase in cell proliferation via up-regulation of MAPK signaling and increased cell cycle progression (1517). On the other hand, the presence of PR in a breast tumor serves as an independent predictor for benefit from adjuvant endocrine therapy and of disease-free survival (8, 9). Moreover, breast tumors that are PR(−) have a much higher proliferation rate and are more likely to manifest increased expression of the tumorigenic prognostic indicators, HER-2/neu and EGFR, than PR(+) tumors (5760). In postmenopausal women with advanced metastatic breast cancer that had progressed during tamoxifen therapy, medroxyprogesterone acetate (MPA) inhibited disease progression (6164). In addition, in postmenopausal women with early stage (I–IIB) breast cancer, MPA significantly decreased the recurrence rate after a median follow-up of 37 months (65). Further, using clinical data obtained from human breast tumor specimens, we observed that PR expression levels serve as an important predictor of a lower stage of breast cancer at diagnosis, a measure of breast tumor progression (66). Using human breast cancer cell lines as a model, it was demonstrated that, in ER(−)/PR(−) MDA-MB-231 cells, overexpression of PR inhibited cell growth and induced spreading and adherence (67). Moreover, in ER(+)/PR(+) T47D breast cancer cells, P4 treatment suppressed cell proliferation stimulated by different mitogens (18) and inhibited cytokine activation of cyclooxygenase 2 (43).

The objective of the present study was to elucidate the underlying mechanisms for the anti-proliferative actions of P4/PR in human breast cancer cells. We focused on the role of MKP-1/DUSP1, a dual-specificity MAPK phosphatase (33), which has been reported to be up-regulated by glucocorticoids (3841) and progestins (47) in a number of cell types. Herein, we observed that treatment with P4 repressed proliferation of ER+/PR+ T47D cells induced by serum. The anti-proliferative action of P4 was associated with a rapid induction of mRNA and protein expression of MKP-1. Importantly, P4 stimulation of MKP-1 was associated with reduced levels of phosphorylated ERK1 and -2. Overexpression of MKP-1 in these cells also antagonized the stimulatory effect of serum on T47D cell proliferation and inhibited ERK1/2 phosphorylation, whereas serum induction of T47D proliferation was enhanced by knockdown of endogenous MKP-1. The role of PR was supported by the finding that the P4-mediated induction of MKP-1 was abrogated in T47D cells by PR knockdown or upon treatment with the PR antagonist, RU486. Moreover, serum induction of T47D proliferation was enhanced by knockdown of endogenous PR.

Our findings of an anti-proliferative effect of P4 in T47D cells differ from those of Lange and colleagues who observed a biphasic effect of P4, with stimulation of cell proliferation at time points of 18 and 24 h, followed by a subsequent decline (16). We suggest that these disparities are primarily due to differences in the culture conditions utilized. In their studies (16), T47D cells that stably expressed PR-B were serum-starved, then cultured with or without progestin in medium with 5% charcoal-stripped FBS. In our study, T47D cells were cultured with or without P4 in medium containing 5% FBS (not charcoal-stripped). After 24 h, we observed such a pronounced stimulatory effect of FBS on EdU incorporation in the absence of P4 (Fig. 3A) that any stimulatory effect of P4/PR on EdU incorporation was likely masked. Under these conditions, the predominant effect of P4 was to antagonize the effect of FBS on cell proliferation. Moreover, the marked stimulatory effect of FBS on cell proliferation was due, in part, to its combined actions to increase ERK1/2 phosphorylation and to decrease MKP-1 expression. This inhibitory effect on MKP-1 was previously reported to be due to ERK-mediated phosphorylation of MKP-1 followed by MKP-1 degradation (54). However, when cells were incubated with P4 plus FBS, progesterone/PR inhibited ERK phosphorylation and blocked the decline in MKP-1 protein levels (Fig. 3B).

Our data suggest that PR acts to increase MKP-1 expression and to inhibit MAPK activation in T47D cells both by classical and non-classical mechanisms. Findings from ChIP and luciferase reporter assays suggest that PR acts in a ligand-dependent manner through binding to two PREs (+197 and +434) downstream of the MKP-1 TSS to up-regulate MKP-1 promoter activity. These putative PREs do not correspond in sequence or location to GR half-sites observed to mediate glucocorticoid induction of the mouse MKP-1 gene (68). PR also acts via non-classical mechanisms to increase levels of MKP-1 expression by interaction with two putative Sp1 response elements just downstream of the TSS. Binding of endogenous PR to the Sp1 sites appears to occur in a ligand-independent manner. These alternative mechanisms of PR-mediated activation of MKP-1 promoter activity are supported by the findings that P4 induction of MKP-1 expression is only partially reduced upon transient transfection of HEK293 cells with a PR-B S345A mutant (2), which abrogates PR-Sp1 interaction, or with a PR-B DNA binding-defective mutant (mDBD) containing three point mutations in the DNA-binding domain (G585E-S586G-V589A).

Studies by Lange and colleagues using T47D cells suggested that progestin/PR rapidly activates EGF receptor (EGFR), c-Src, and MAPK signaling, resulting in increased phosphorylation of PR on Ser-345 (2). PR phosphorylated on Ser-345, in turn, tethers to Sp1 sites within specific PR-regulated promoters (e.g. EGFR), resulting in a rapid burst of cell proliferation (2). Based on the present findings, we suggest that P~345-PR interaction with Sp1 response elements within the MPK-1 promoter may serve a critical and protective role in the breast by inactivating MAPKs and switching off this proliferative signal. The tethering of PR to promoter-bound Sp1 may further enhance and stabilize PR binding to the two downstream PREs to increase the magnitude and duration of MKP-1 induction and maintain a reduced rate of cell proliferation.

In the present study, we observed that the increase in T47D cell proliferation and in the levels of phosphorylated ERK1/2 caused by siRNA-mediated knockdown of endogenous PR-A and PR-B occurred in the absence of exogenous P4. Our previous findings also revealed that, in T47D cells, siRNA-mediated knockdown of endogenous PR-A and -B caused a marked induction of cyclooxygenase 2, aromatase/CYP19, and Her-2/neu expression in the absence of P4 (43, 44). This was associated with a pronounced increase in nuclear levels of NF-κB p65 (44), suggesting that decreased PR expression promotes activation of NF-κB. Thus, PR likely suppresses NF-κB activation, in part, via a ligand-independent mechanism that may involve direct interaction with p65 (3). Our previous findings suggest that PR also inhibits NF-κB activation in a ligand-dependent manner via P4/PR induction of IκBα expression. However, in other studies using T47D cells, it was observed that, although the progestin R5020 increased IκBα expression, it had no effect on p65 nuclear localization (45).

In studies using T47D cells, the progestin R5020 was observed to inhibit estradiol-induced cell proliferation in a PR-dependent manner (69). The action of estrogen to stimulate breast cancer growth is mediated, in part, by non-genomic mechanisms involving interaction of ERα with growth factor receptors and signaling cascades that originate at the plasma membrane. These include activation of MAPK and phosphatidylinositol 3-kinase (PI3K) pathways, with enhanced estrogen-independent phosphorylation and activation of nuclear and membrane-associated ERα (70). Interestingly, the ERα co-activator SRC-3 (AIB1), which is up-regulated in metastatic human breast cancer, is phosphorylated in response to E2, MAPK (71), and Her-2/neu activation (72). SRC-3 phosphorylation promotes its nuclear translocation, increased interaction with ER, and enhancement of ER transcriptional activity (71). The HER-2/neu proto-oncogene is amplified in ~30% of invasive ductal carcinomas of the breast (57). Her-2/neu overexpression triggers activation of MAPK, PI3K, and NF-κB signaling pathways (73, 74). Moreover, MAPK overexpression in breast cancer cells promotes activation/phosphorylation of NF-κB (75), ERα (76), and AP-1 (77), which enhance breast cancer initiation, growth, and survival. Previously, we observed that Her-2/neu expression was markedly inhibited by P4 treatment of T47D cells and by up-regulation of PR expression in MCF-7 cells (44). Thus, in addition to its action to inhibit MAPK activation via induction of MKP-1, P4/PR may also suppress expression of Her-2/neu, a major activator of MAPK.

Besides its potential action to inhibit breast cancer cell proliferation, MKP-1 also likely plays an anti-inflammatory role. Overexpression of MKP-1 in macrophages blocks lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines by inhibiting p38 MAPK (78). Conversely, MKP-1−/− mice manifest an exacerbated response to LPS, manifesting elevated cytokine levels in serum and prolonged activation of p38 MAPK and JNK in macrophages (79). The anti-inflammatory actions of glucocorticoids in a variety of cell types have been attributed, in part, to their actions to increase expression of MKP-1 and decrease MAPK activation, resulting in decreased expression of genes encoding inflammatory mediators, such as cyclooxygenase 2. Although MKP-1 inhibition of p38 MAPK activity can cause decreased NF-κB and AP-1 activation of pro-inflammatory gene transcription, decreased p38 activity also has been found to cause destabilization of pro-inflammatory mRNAs (80).

Despite the anti-inflammatory, immunosuppressive, and anti-proliferative actions of glucocorticoids, trials of glucocorticoid therapy in breast cancer have lacked efficacy (81). Interestingly, estradiol has been reported to inhibit GR transactivation activity by protein phosphatase 5-mediated dephosphorylation of GR at Ser-211 (82), a ligand-induced phosphorylation site associated with GR transcriptional activation (12). Consequently, treatment of human breast cancer cells with estradiol inhibited glucocorticoid induction MKP-1 and serum glucocorticoid kinase (SGK) expression (82). On the other hand, we observed that, whereas estradiol treatment of T47D cells blocked dexamethasone induction of MKP-1 and SGK, it failed to inhibit P4 induction of MKP-1 and SGK expression (supplemental Fig. S3). Notably, the Ser-211 phosphorylation site present in GR is not conserved in PR. These intriguing findings suggest that P4/PR signaling is not affected by estrogen-mediated inhibition. Notably, whereas, expression levels of MKP-1 and PR were positively correlated in 30 human breast cancer cell lines, no correlation between the levels of MKP-1 and GR were observed (Fig. 1E). Similarly, we previously found that expression levels IκBα and PR were positively correlated in these breast cancer cell lines, whereas no correlation was found between levels of IκBα and GR (44). These collective findings suggest that MKP-1 is an important target of P4/PR in human breast cancer cells and may play a significant role in its anti-proliferative and anti-inflammatory actions.

Supplementary Material

Supplemental Data:

*This work was supported, in whole or in part, by National Institutes of Health Grants 5-R01-DK031206 and 2-R56-DK031206 (to C. R. M.). This work was also supported by a postdoctoral fellowship from the Susan G. Komen Foundation (to C.-C. C. and D. B. H.).

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThe on-line version of this article (available at http://www.jbc.org) contains supplemental “Methods” and Figs. S1–S3.

3The abbreviations used are:

P4
progesterone
PR
progesterone receptor
MKP-1
MAPK phosphatase 1
DUSP1
dual-specificity phosphatase 1
PRE
progesterone response element
Sp1
specificity protein 1
ER
estrogen receptor
CDK
cyclin-dependent kinase
GR
glucocorticoid receptor
TSS
transcription start site
qPCR
quantitative PCR
h36B4
human ribosomal phosphoprotein P0
EdU
ethynyl-2′-deoxyuridine
DAPI
4′,6-diamidino-2-phenylindole
IκBα
NF-κB inhibitor, alpha
SGK
serum glucocorticoid kinase
mDBD
DNA binding-defective mutant.

REFERENCES

1. Conneely O. M., Jericevic B. M., Lydon J. P. (2003) J. Mammary Gland Biol. Neoplasia 8, 205–214 [PubMed]
2. Faivre E. J., Daniel A. R., Hillard C. J., Lange C. A. (2008) Mol. Endocrinol. 22, 823–837 [PubMed]
3. Kalkhoven E., Wissink S., van der Saag P. T., van der Burg B. (1996) J. Biol. Chem. 271, 6217–6224 [PubMed]
4. Richer J. K., Lange C. A., Manning N. G., Owen G., Powell R., Horwitz K. B. (1998) J. Biol. Chem. 273, 31317–31326 [PubMed]
5. Cerliani J. P., Guillardoy T., Giulianelli S., Vaque J. P., Gutkind J. S., Vanzulli S. I., Martins R., Zeitlin E., Lamb C. A., Lanari C. (2011) Cancer Res. 71, 3720–3731 [PubMed]
6. Tseng L., Tang M., Wang Z., Mazella J. (2003) DNA Cell Biol. 22, 633–640 [PubMed]
7. Lonard D. M., O'Malley B. W. (2006) Cell 125, 411–414 [PubMed]
8. Osborne C. K., Yochmowitz M. G., Knight W. A., 3rd, McGuire W. L. (1980) Cancer 46, 2884–2888 [PubMed]
9. Young P. C., Ehrlich C. E., Einhorn L. H. (1980) Cancer 46, 2961–2963 [PubMed]
10. Chlebowski R. T., Hendrix S. L., Langer R. D., Stefanick M. L., Gass M., Lane D., Rodabough R. J., Gilligan M. A., Cyr M. G., Thomson C. A., Khandekar J., Petrovitch H., McTiernan A. (2003) JAMA 289, 3243–3253 [PubMed]
11. Chlebowski R. T., Anderson G. L., Gass M., Lane D. S., Aragaki A. K., Kuller L. H., Manson J. E., Stefanick M. L., Ockene J., Sarto G. E., Johnson K. C., Wactawski-Wende J., Ravdin P. M., Schenken R., Hendrix S. L., Rajkovic A., Rohan T. E., Yasmeen S., Prentice R. L. (2010) JAMA 304, 1684–1692 [PMC free article] [PubMed]
12. Ismaili N., Garabedian M. J. (2004) Ann. N. Y. Acad. Sci. 1024, 86–101 [PubMed]
13. Lin V. C., Eng A. S., Hen N. E., Ng E. H., Chowdhury S. H. (2001) Clin. Cancer Res. 7, 2880–2886 [PubMed]
14. van der Burg B., Kalkhoven E., Isbrücker L., de Laat S. W. (1992) J. Steroid Biochem. Mol. Biol. 42, 457–465 [PubMed]
15. Groshong S. D., Owen G. I., Grimison B., Schauer I. E., Todd M. C., Langan T. A., Sclafani R. A., Lange C. A., Horwitz K. B. (1997) Mol. Endocrinol. 11, 1593–1607 [PubMed]
16. Skildum A., Faivre E., Lange C. A. (2005) Mol. Endocrinol 19, 327–339 [PubMed]
17. Musgrove E. A., Swarbrick A., Lee C. S., Cornish A. L., Sutherland R. L. (1998) Mol. Cell. Biol. 18, 1812–1825 [PMC free article] [PubMed]
18. Gill P. G., Tilley W. D., De Young N. J., Lensink I. L., Dixon P. D., Horsfall D. J. (1991) Breast Cancer Res. Treat. 20, 53–62 [PubMed]
19. Gizard F., Robillard R., Gervois P., Faucompré A., Revillion F., Peyrat J. P., Hum W. D., Staels B. (2005) FEBS Lett. 579, 5535–5541 [PubMed]
20. Swarbrick A., Lee C. S., Sutherland R. L., Musgrove E. A. (2000) Mol. Cell. Biol. 20, 2581–2591 [PMC free article] [PubMed]
21. Owen G. I., Richer J. K., Tung L., Takimoto G., Horwitz K. B. (1998) J. Biol. Chem. 273, 10696–10701 [PubMed]
22. Gizard F., Robillard R., Gross B., Barbier O., Révillion F., Peyrat J. P., Torpier G., Hum D. W., Staels B. (2006) Mol. Cell. Biol. 26, 7632–7644 [PMC free article] [PubMed]
23. Robinson M. J., Cobb M. H. (1997) Curr. Opin. Cell Biol. 9, 180–186 [PubMed]
24. Pearson G., Robinson F., Beers Gibson T., Xu B. E., Karandikar M., Berman K., Cobb M. H. (2001) Endocr. Rev. 22, 153–183 [PubMed]
25. Cobb M. H., Goldsmith E. J. (1995) J. Biol. Chem. 270, 14843–14846 [PubMed]
26. Yue W., Fan P., Wang J., Li Y., Santen R. J. (2007) J. Steroid Biochem. Mol. Biol. 106, 102–110 [PMC free article] [PubMed]
27. Santen R. J., Song R. X., McPherson R., Kumar R., Adam L., Jeng M. H., Yue W. (2002) J. Steroid Biochem. Mol. Biol. 80, 239–256 [PubMed]
28. Fox E. M., Andrade J., Shupnik M. A. (2009) Steroids 74, 622–627 [PMC free article] [PubMed]
29. Daniel A. R., Knutson T. P., Lange C. A. (2009) Mol. Cell. Endocrinol. 308, 47–52 [PMC free article] [PubMed]
30. Alonso A., Sasin J., Bottini N., Friedberg I., Friedberg I., Osterman A., Godzik A., Hunter T., Dixon J., Mustelin T. (2004) Cell 117, 699–711 [PubMed]
31. Jeffrey K. L., Camps M., Rommel C., Mackay C. R. (2007) Nat. Rev. Drug Discov. 6, 391–403 [PubMed]
32. Owens D. M., Keyse S. M. (2007) Oncogene 26, 3203–3213 [PubMed]
33. Sun H., Charles C. H., Lau L. F., Tonks N. K. (1993) Cell 75, 487–493 [PubMed]
34. Liu Y., Gorospe M., Yang C., Holbrook N. J. (1995) J. Biol. Chem. 270, 8377–8380 [PubMed]
35. Franklin C. C., Kraft A. S. (1997) J. Biol. Chem. 272, 16917–16923 [PubMed]
36. Slack D. N., Seternes O. M., Gabrielsen M., Keyse S. M. (2001) J. Biol. Chem. 276, 16491–16500 [PubMed]
37. Chen Y. W., Huang S. C., Lin-Shiau S. Y., Lin J. K. (2005) Carcinogenesis 26, 1296–1306 [PubMed]
38. King E. M., Holden N. S., Gong W., Rider C. F., Newton R. (2009) J. Biol. Chem. 284, 26803–26815 [PMC free article] [PubMed]
39. Bladh L. G., Johansson-Haque K., Rafter I., Nilsson S., Okret S. (2009) Biochim. Biophys. Acta 1793, 439–446 [PubMed]
40. Kassel O., Sancono A., Krätzschmar J., Kreft B., Stassen M., Cato A. C. (2001) EMBO J. 20, 7108–7116 [PubMed]
41. Shipp L. E., Lee J. V., Yu C. Y., Pufall M., Zhang P., Scott D. K., Wang J. C. (2010) PLoS One. 5, e13754. [PMC free article] [PubMed]
42. Tibbetts T. A., Conneely O. M., O'Malley B. W. (1999) Biol. Reprod. 60, 1158–1165 [PubMed]
43. Hardy D. B., Janowski B. A., Corey D. R., Mendelson C. R. (2006) Mol. Endocrinol. 20, 2724–2733 [PubMed]
44. Hardy D. B., Janowski B. A., Chen C. C., Mendelson C. R. (2008) Mol. Endocrinol. 22, 1812–1824 [PubMed]
45. Kobayashi S., Stice J. P., Kazmin D., Wittmann B. M., Kimbrel E. A., Edwards D. P., Chang C. Y., McDonnell D. P. (2010) Mol. Endocrinol. 24, 2292–2302 [PubMed]
46. Janowski B. A., Kaihatsu K., Huffman K. E., Schwartz J. C., Ram R., Hardy D., Mendelson C. R., Corey D. R. (2005) Nat. Chem. Biol. 1, 210–215 [PubMed]
47. Quiles I., Millán-Ariño L., Subtil-Rodriguez A., Miñana B., Spinedi N., Ballaré C., Beato M., Jordan A. (2009) Mol. Endocrinol. 23, 809–826 [PubMed]
48. Deroo B. J., Archer T. K. (2002) J. Steroid Biochem. Mol. Biol. 81, 309–317 [PubMed]
49. Richardson A. L., Wang Z. C., De Nicolo A., Lu X., Brown M., Miron A., Liao X., Iglehart J. D., Livingston D. M., Ganesan S. (2006) Cancer Cell 9, 121–132 [PubMed]
50. Schuetz C. S., Bonin M., Clare S. E., Nieselt K., Sotlar K., Walter M., Fehm T., Solomayer E., Riess O., Wallwiener D., Kurek R., Neubauer H. J. (2006) Cancer Res. 66, 5278–5286 [PubMed]
51. Huang E., Cheng S. H., Dressman H., Pittman J., Tsou M. H., Horng C. F., Bild A., Iversen E. S., Liao M., Chen C. M., West M., Nevins J. R., Huang A. T. (2003) Lancet 361, 1590–1596 [PubMed]
52. Ginestier C., Cervera N., Finetti P., Esteyries S., Esterni B., Adélaïde J., Xerri L., Viens P., Jacquemier J., Charafe-Jauffret E., Chaffanet M., Birnbaum D., Bertucci F. (2006) Clin. Cancer Res. 12, 4533–4544 [PubMed]
53. Chin K., DeVries S., Fridlyand J., Spellman P. T., Roydasgupta R., Kuo W. L., Lapuk A., Neve R. M., Qian Z., Ryder T., Chen F., Feiler H., Tokuyasu T., Kingsley C., Dairkee S., Meng Z., Chew K., Pinkel D., Jain A., Ljung B. M., Esserman L., Albertson D. G., Waldman F. M., Gray J. W. (2006) Cancer Cell 10, 529–541 [PubMed]
54. Lin Y. W., Yang J. L. (2006) J. Biol. Chem. 281, 915–926 [PubMed]
55. Goldhar A. S., Duan R., Ginsburg E., Vonderhaar B. K. (2011) Mol. Cell. Endocrinol. 335, 148–157 [PMC free article] [PubMed]
56. Blume S. W., Snyder R. C., Ray R., Thomas S., Koller C. A., Miller D. M. (1991) J. Clin. Invest. 88, 1613–1621 [PMC free article] [PubMed]
57. Slamon D. J., Clark G. M., Wong S. G., Levin W. J., Ullrich A., McGuire W. L. (1987) Science 235, 177–182 [PubMed]
58. Tandon A. K., Clark G. M., Chamness G. C., Ullrich A., McGuire W. L. (1989) J. Clin. Oncol. 7, 1120–1128 [PubMed]
59. Lebeau A., Unholzer A., Amann G., Kronawitter M., Bauerfeind I., Sendelhofert A., Iff A., Löhrs U. (2003) Breast Cancer Res. Treat. 79, 187–198 [PubMed]
60. Arpino G., Weiss H., Lee A. V., Schiff R., De Placido S., Osborne C. K., Elledge R. M. (2005) J. Natl. Cancer Inst. 97, 1254–1261 [PubMed]
61. Hultborn R., Johansson-Terje I., Bergh J., Glas U., Hallsten L., Hatschek T., Holmberg E., Idestrom K., Norberg B., Ranstam J., Soderberg M., Wallgren U. B. (1996) Acta Oncol. 35 (Suppl. 5), 75. [PubMed]
62. Parazzini F., Colli E., Scatigna M., Tozzi L. (1993) Oncology 50, 483–489 [PubMed]
63. Byrne M. J., Gebski V., Forbes J., Tattersall M. H., Simes R. J., Coates A. S., Dewar J., Lunn M., Flower C., Gill P. G., Stewart J. (1997) J. Clin. Oncol. 15, 3141–3148 [PubMed]
64. Buzdar A., Jonat W., Howell A., Jones S. E., Blomqvist C., Vogel C. L., Eiermann W., Wolter J. M., Azab M., Webster A., Plourde P. V. (1996) J. Clin. Oncol. 14, 2000–2011 [PubMed]
65. Pannuti F., Martoni A., Cilenti G., Camaggi C. M., Fruet F. (1988) Eur. J. Cancer Clin. Oncol. 24, 423–429 [PubMed]
66. Coyle Y. M., Xie X. J., Hardy D. B., Ashfaq R., Mendelson C. R. (2007) Cancer Lett. 258, 253–261 [PubMed]
67. Lin V. C., Ng E. H., Aw S. E., Tan M. G., Ng E. H., Bay B. H. (2000) Mol. Endocrinol. 14, 348–358 [PubMed]
68. Tchen C. R., Martins J. R., Paktiawal N., Perelli R., Saklatvala J., Clark A. R. (2010) J. Biol. Chem. 285, 2642–2652 [PMC free article] [PubMed]
69. Vignon F., Bardon S., Chalbos D., Rochefort H. (1983) J. Clin. Endocrinol. Metab. 56, 1124–1130 [PubMed]
70. Song R. X., Barnes C. J., Zhang Z., Bao Y., Kumar R., Santen R. J. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 2076–2081 [PubMed]
71. Wu R. C., Qin J., Yi P., Wong J., Tsai S. Y., Tsai M. J., O'Malley B. W. (2004) Mol. Cell 15, 937–949 [PubMed]
72. Osborne C. K., Bardou V., Hopp T. A., Chamness G. C., Hilsenbeck S. G., Fuqua S. A., Wong J., Allred D. C., Clark G. M., Schiff R. (2003) J. Natl. Cancer Inst. 95, 353–361 [PubMed]
73. Yarden Y., Sliwkowski M. X. (2001) Nat. Rev. Mol. Cell Biol. 2, 127–137 [PubMed]
74. Merkhofer E. C., Cogswell P., Baldwin A. S. (2010) Oncogene 29, 1238–1248 [PMC free article] [PubMed]
75. Viatour P., Merville M. P., Bours V., Chariot A. (2005) Trends Biochem. Sci. 30, 43–52 [PubMed]
76. Arnold S. F., Obourn J. D., Jaffe H., Notides A. C. (1995) J. Steroid Biochem. Mol. Biol. 55, 163–172 [PubMed]
77. Dérijard B., Hibi M., Wu I. H., Barrett T., Su B., Deng T., Karin M., Davis R. J. (1994) Cell 76, 1025–1037 [PubMed]
78. Chen P., Li J., Barnes J., Kokkonen G. C., Lee J. C., Liu Y. (2002) J. Immunol. 169, 6408–6416 [PubMed]
79. Zhao Q., Wang X., Nelin L. D., Yao Y., Matta R., Manson M. E., Baliga R. S., Meng X., Smith C. V., Bauer J. A., Chang C. H., Liu Y. (2006) J. Exp. Med. 203, 131–140 [PMC free article] [PubMed]
80. Lasa M., Abraham S. M., Boucheron C., Saklatvala J., Clark A. R. (2002) Mol. Cell. Biol. 22, 7802–7811 [PMC free article] [PubMed]
81. Keith B. D. (2008) BMC Cancer 8, 84. [PMC free article] [PubMed]
82. Zhang Y., Leung D. Y., Nordeen S. K., Goleva E. (2009) J. Biol. Chem. 284, 24542–24552 [PMC free article] [PubMed]

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