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[Cancer Research 66, 8373-8381, September 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

AKT Regulation of Estrogen Receptor ß Transcriptional Activity in Breast Cancer

Bich N. Duong1, Steven Elliott1,3, Daniel E. Frigo1, Lilia I. Melnik1, Lyndsay Vanhoy3, Suzanne Tomchuck3, Helena P. Lebeau4, Odile David7, Barbara S. Beckman1,2, Jawed Alam5,6, Melyssa R. Bratton1, John A. McLachlan1,2,5 and Matthew E. Burow1,2,3

1 Center for Bioenvironmental Research, 2 Department of Pharmacology, 3 Department of Medicine, Section of Hematology and Medical Oncology, and 4 Department of Pathology, Tulane University; 5 Department of Environmental Health Sciences, Tulane University School of Public Health and Tropical Medicine; 6 Department of Molecular Genetics, Alton Ochsner Medical Foundation, New Orleans, Louisiana and 7 Department of Pathology, University of Illinois College of Medicine, Chicago, Illinois

Requests for reprints: Matthew Burow, Department of Medicine-Section of Hematology and Medical Oncology, Tulane University School of Medicine, 1430 Tulane Ave. SL-78, New Orleans, LA 70112. Phone: 504-988-6688; Fax: 504-988-5483; E-mail: mburow{at}tulane.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Growth factor activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway has been shown to activate the estrogen receptor (ER) {alpha} and to mediate tamoxifen resistance in breast cancer. Here, we investigated the regulation of the transcriptional activity of the newer ERß by PI3K-AKT signaling. Tissue arrays of breast cancer specimens showed a positive association between the expressions of AKT and ERß in the clinical setting. Reporter gene assays using pharmacologic and molecular inhibitors of AKT and constitutively active AKT revealed for the first time the ability of AKT to (a) potentiate ERß activity and (b) target predominantly the activation function-2 (AF2) domain of the receptor, with a requirement for residue K269. Given the importance of coactivators in ER transcriptional activity, we further investigated the possible involvement of steroid receptor coactivator 1 (SRC1) and glucocorticoid receptor-interacting protein 1 (GRIP1) in AKT regulation of ERß. Mammalian two-hybrid assays revealed that AKT enhanced both SRC1 and GRIP1 recruitment to the ERß-AF2 domain, and reporter gene analyses revealed that AKT and GRIP1 cooperatively potentiated ERß-mediated transcription to a level much greater than either factor alone. Investigations into AKT regulation of GRIP with mammalian one-hybrid assays showed that AKT potentiated the activation domains of GRIP1 itself, and in vitro kinase assays revealed that AKT directly phosphorylated GRIP1. The cross-talk between the PI3K-AKT and ERß pathways, as revealed by the ability of AKT to regulate several components of ERß-mediated transcription, may represent an important aspect that may influence breast cancer response to endocrine therapy. (Cancer Res 2006; 66(17): 8373-81)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The two isoforms of the estrogen receptor (ER; {alpha} and ß) are members of the nuclear receptor superfamily that function as ligand-inducible transcription factors, which mediate the biological effects of the steroid hormone estrogen [17ß-estradiol (E2)] on a variety of tissues (1). On ligand binding, the ER dissociates from heat shock proteins and dimerizes with another ER, and the two complexed receptors bind via their DNA-binding domains (DBD) to the estrogen response elements (ERE) in the promoter regions of target genes (1). Transcriptional activities of the receptors are mediated by two distinct activation domains: activation function-1 (AF1) at the NH2 terminus that exhibits constitutive ligand-independent activity and activation function-2 (AF2) at the COOH terminus that requires ligand binding for activity (1).

Various agents have been shown to regulate ER activity in addition to E2, including peptide growth factors (PGF), such as epidermal growth factor and insulin like growth factor-I (2). ER activity is often potentiated/elevated to a higher level with the combination of PGF and E2 than with either factor alone (3). Mechanistic studies have revealed that a cross-talk between the PGF and E2 signaling cascades occurs at the level of the ER{alpha}, such that PGF activation of the phosphatidylinositol 3-kinase (PI3K)-AKT and mitogen-activated protein kinase (MAPK) signaling cascades (4) ultimately leads to activation of ER{alpha} (5, 6).

Research into the role of AKT and ERß in breast cancer etiology has intensified in recent years. The PI3K-AKT pathway confers a potent survival signal (7), and researchers have shown that AKT, once activated by members of the HER family, may contribute to chemotherapeutic drug resistance (6, 8). Like the ER{alpha}, the more recently discovered ERß has emerged as an important determinant in breast cancer (9). ERß expression is a useful biomarker for breast cancer (9–11) in a manner that is independent of ER{alpha} expression (10). Studies have indicated that ERß expression may be an indicator of a favorable prognosis for breast cancer patients (12), because the loss of ERß expression has been associated with tamoxifen resistance (13, 14). However, studies evaluating ERß expression with certain tumor variables, such as tumor grade and disease-free survival, had produced conflicting results (15). Although some of the discrepancies may be resolved with detailed studies of the different ERß variants (9, 13), the complexity of ERß signaling, like that of the ER{alpha}, will require understanding of ERß cross-talk with kinase signaling molecules.

Although AKT potentiation of ER{alpha} has been shown to involve phosphorylation of the receptor (5, 6), recent works document that PGF signaling may target a nonreceptor protein, such as a coactivator (16–18). Members of the p160 family of coactivators, including steroid receptor coactivator 1 (SRC1) and glucocorticoid receptor-interacting protein 1 (GRIP1; ref. 19), have intrinsic histone acetyltransferase capabilities and are necessary for ER transcriptional activity. PGF, through activation of the MAPK signaling cascade, has been shown to phosphorylate and thereby to regulate the activities of p160 coactivators (16–18). Additionally, MAPK potentiation of ER{alpha} activity may be achieved through its ability to enhance coactivator recruitment to the receptor (18). Given the importance of coactivators in ER transcriptional activity, the PI3K-AKT signaling cascade may also regulate coactivator function to potentiate ER activity.

Many studies have focused on the regulation of ER{alpha} transcriptional activity by PI3K-AKT signaling, but much less is known about the regulation of the newer ERß. Given the similarities between the ER{alpha} and ERß (1) and the importance of p160 coactivators to their transcriptional activities (19), we hypothesized that PI3K-AKT signaling may also modulate ERß activity and that this modulation may be achieved through recruitment/regulation of p160 coactivators. To test this hypothesis, we evaluated the regulation of both ERß and coactivator activities by AKT. Here, we show that for the first time the ability of AKT to potentiate ERß transcriptional, to enhance coactivator recruitment to the receptor, and to potentiate coactivator activity. Our results extend the sphere of PI3K-AKT regulation to the ERß and to the coactivators that interact with the receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. DMEM, phenol red–free DMEM, fetal bovine serum (FBS), BME amino acids, MEM amino acids, L-glutamine, penicillin, streptomycin, and sodium pyruvate were obtained from Life Technologies (Gaithersburg, MD). Porcine insulin was purchased from Sigma (St. Louis, MO), and charcoal-stripped FBS was obtained from HyClone (Logan, UT). LipofectAMINE, Effectene, and Fugene were purchased from Life Technologies (Grand Island, NY), Qiagen (Valencia, CA), and Roche (Indianapolis, IN), respectively. E2 was obtained from Sigma and ICI 182,780 was obtained from Torcis (Ellisville, MO). AKT inhibitor II was purchased from Calbiochem (Darmstadt, Germany). Luciferase assay substrate and lysis buffer were purchased from Promega (Madison, WI). The Berthold AutoLumat Plus luminometer was obtained from Analytical Luminescence Laboratory (Ann Arbor, MI).

Plasmids. pGl2-ERE2X-Luc plasmid (pERE-Luc) contains two copies of the vitellogenin ERE linked to the luciferase gene (3). pGal4-Luc (also termed pFR-Luc and containing 5x Gal4-binding element upstream of the luciferase gene) was obtained from Stratagene (La Jolla, CA). Empty vector pcDNA3.1 and pcDNA3.1-ERß have been described previously (20). pRST7-ERß constructs (ERß-AF1 with alanine substituting for amino acids at positions 436, 440, and 443 and ERß-AF2 containing amino acids 90-477) were generously provided by Dr. Donald McDonnell (Duke University; ref. 21). ERß-K269A was a generous gift of Dr. Paul Webb (University of California San Francisco, San Francisco, CA). Empty vector pVP16 was provided by Dr. Jawed Alam (Ochsner Medical Foundation). The expression vectors pVP16-ERß-AF2 and pGal4-SRC1-nuclear receptor-interacting domain (NRID) were kind gifts of Dr. Jeff Northrop (Affymax Research Institute; ref. 22). Constitutively active AKT (AKT-CA) was provided by Dr. Anke Klippel (Chiron Corp., Emeryville, CA; ref. 23). pSG5-GRIP1-HA and pGal4-GRIP1 constructs (NRID, AD1, and AD2) were generously given by Dr. Michael Stallcup (University of California at Los Angeles; refs. 24, 25). Constitutively active MKK1 (MKK1-CA) was obtained from Stratagene. Dominant-negative MKK1 (MKK1-DN) was provided by Drs. Melanie Cobb (UT Southwestern Medical Center at Dallas, Dallas, TX) and Roger Davis (University of Massachusetts Medical School, Worcester, MA) and dominant-negative AKT (AKT-DN) was purchased from Upstate Biotechnology (Lake Placid, NY).

Cell culture. ER-negative HEK-293 cells were maintained in DMEM supplemented with 10% FBS, BME amino acids, MEM amino acids, L-glutamine, 100 units/mL penicillin, 100 units/mL streptomycin, sodium pyruvate, and 1 x 10–10 mol/L porcine insulin under Mycoplasma-free conditions at 37°C in humidified 5% CO2 and 95% air. For described studies, cells were grown in phenol red–free DMEM supplemented with 5% charcoal-stripped FBS and supplements as above but without insulin (5% charcoal-stripped DMEM) as described previously (3, 26).

Transient transfection and luciferase assay. Before all transient transfection experiments, cells were grown in 5% charcoal-stripped DMEM for 48 hours before seeding onto 24-well plates at a concentration of 1 x 104 per well. Cells were allowed to attach overnight and transfected with plasmids for 5 hours in the serum/supplement-free DMEM using either LipofectAMINE or Effectene and according to the manufacturers' protocols. Cells were then treated and harvested 24 hours later for luciferase assays as described previously (26).

Mammalian one- and two-hybrid assays. For both assays, 1 x 105 HEK-293 cells per well in 5% charcoal-stripped DMEM were seeded into 12-well plates and allowed to attach overnight. For mammalian one-hybrid assays, cells were transfected for 5 hours in serum/supplement-free DMEM with 100 ng pGal4-Luc reporter along with 200 ng pGal4-GRIP1-AD1 or pGal4-GRIP1-AD2, 400 ng pcDNA3.1 empty vector, or AKT using Fugene and following the manufacturer's protocol. For mammalian two-hybrid assays, cells were transfected for 5 hours in serum/supplement-free DMEM with 100 ng pGal4-Luc reporter along with 200 ng pGal4-SRC1-NRID or pGal4-GRIP1-NRID used as bait, 200 ng pVP16-ERß-AF2 or pVP16-empty vector used as prey, and 400 ng pcDNA3.1 empty vector, AKT, or MKK1 using Fugene and following the manufacturer's protocol. Cells were treated and harvested 24 hours later for luciferase assays as described previously (26).

Tissue microarray and immunohistochemistry. Breast slides (T-BO-1) were obtained from the tissue array research program (National Cancer Institute and National Human Genome Research Institute). Twenty-nine infiltrating breast carcinomas were represented in the array. Immunohistochemistry was done using ERß antibody from Novus Biologicals (Littleton, CO) and phosphorylated AKT (pAKT) antibody from Cell Signaling (Beverly, MA). For pAKT, the primary antibody was produced by immunizing rabbits with a synthetic phosphorylated Ser473 peptide corresponding to residues around Ser473 of mouse AKT. Nuclear and cytoplasmic staining is expected. A section from a block of infiltrating breast carcinoma from the files of the Tulane Histology Laboratory was used as a positive control. Negative controls consisted of both internal negative controls, including spots of normal liver, pancreas, and brain tissue incorporated into the array, and array slides to which only buffered saline but no primary antibody was applied. Immediately before each run, antigen retrieval was done by cooking the deparaffinized slides in a commercial rice cooker in preheated 95°C DAKO (Carpinteria, CA) target retrieval solution. After cooling and washing, slides were loaded onto a DAKO Autostainer instrument. The slides were treated with 3% H2O2 endogenous block for 5 minutes and then serum-free DAKO protein block for 5 minutes. Primary antibodies were applied at dilutions of 1:50 for the pAKT antibody and 1:40 for the ERß. Incubation time was 120 minutes for pAKT and 60 minutes for ERß. Secondary antibody (DAKO LSAB+ Link system) was applied for 30 minutes. Tertiary antibody (DAKO LSAB+ streptavidin-horseradish peroxidase) was applied for 30 minutes. DAKO 3,3'-diaminobenzidine chromogen substrate was applied until light brown staining was seen (~30 seconds).

With the ERß antibody, there was diffuse nuclear staining in positive tissues. With the pAKT antibody, staining was predominantly cytoplasmic with occasional nuclear staining in positive tissues. Staining intensity was scored as follows. For ERß, the percentage of tumor nuclei staining diffusely brown was assessed by counting tumor cell nuclei and calculating the percentage of positive tumor cell nuclei per total tumor cell nuclei in each spot on the array. A score of 0 was given if the percentage were <5%, a score of 1 was given if the percentage were 5% to 30%, and a score of 2 was given if the percentage were >30%. This system is based on current clinical practice of 5% as a cutoff and on previously reported scoring systems (27). pAKT immunostaining was scored as levels on an intensity scale ranging from 0 to 3. A tumor was scored as 0 if there were no appreciable staining in tumor cells compared with stromal elements, as 1 if there were barely detectable staining in cytoplasm and/or nucleus compared with stromal elements, as 2 if there were readily appreciable brown staining distinctly marking tumor cell cytoplasm and/or nucleus, and as 3 if there was dark brown staining in tumor cells completely obscuring cytoplasm and/or nucleus. This scoring system has been reported previously (27).

In vitro kinase assay. The glutathione S-transferase (GST) and GST-GRIP1 fusion proteins were generated using pGEX empty GST expression vector (Amersham Biosciences, Piscataway, NJ) by the researchers at Dr. McDonnell's laboratory and were transformed in BL21star cells. Bacteria were grown and GST fusion proteins were purified as described previously (28).

Eluted purified GST or GST-GRIP1 (~3-5 µg) and purified active AKT (500 ng) were incubated in the presence of magnesium/ATP cocktail containing {gamma}-32P for 30 minutes at 30°C with shaking according to the manufacturer's protocol. Reactions were stopped by the addition of 2x SDS sample buffer (20 µL) containing 0.1 mol/L phenylmethylsulfonyl fluoride, 10 µL/mL protease inhibitor cocktail, 10 µL/mL phosphatase inhibitor cocktail, 5% ß-mercaptoethanol, and 0.01% bromophenol blue. Samples were boiled for 5 minutes, resolved on 4% to 12% Tris-Bis NuPAGE, transferred to nitrocellulose, and subjected to both staining with Coomassie blue to monitor expression and autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AKT and ERß protein expressions are positively associated. Research into the roles of AKT and ERß in breast cancer etiology has intensified in recent years. To establish a connection, if any, between AKT and ERß in the clinical setting, tissue arrays of 29 unidentified patient samples were done using antibodies specific for the ERß and the phosphorylated/active form of AKT (Fig. 1A and B ). The array slides were then scored by a pathologist as per scoring systems above. Briefly, scoring was done by assessing the intensity of tumor cell staining relative to stromal elements in the same spot. For ERß, staining was nuclear and diffuse, as expected. For pAKT, staining was predominantly cytoplasmic with occasional nuclear staining. Our results reveal a positive association between phosphorylated/active AKT and ERß. The most frequent pattern for an identical set of spots from any given breast carcinoma was one in which ERß positivity occurred in >30% of the cells (2+) and AKT positivity was relatively weak (1+; Fig. 1A).


Figure 1
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  		Figure 1. A and B, pAKT and ERß protein expressions are positively associated. Breast cancer tissue specimens from 29 patients were stained with anti-ERß or anti-pAKT antibodies.

 
AKT and MKK1 potentiate ERß transcriptional activity. Previous reports have shown that PGF activation of the MAPK and PI3K-AKT signaling cascades potentiate ER{alpha} transcriptional activity (5, 6, 29). Our results above suggest a connection between AKT and ERß. To investigate kinase mediation of ERß activity, luciferase assays were preformed with ER-negative HEK-293 cells that transiently expressed ERß and AKT-CA or MKK1-CA (Fig. 2A and B ). Here, the MKK1-p44/p42 MAPK signaling pathway potentiated ERß by 1.6-fold over vector control in the presence of 1 nmol/L E2, similar to the levels observed by other researchers (30). More interestingly, we show here for the first time the ability of AKT to potentiate ERß activity by 1.5-fold over vector control.


Figure 2
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  		Figure 2. AKT and MKK1 potentiate ERß activity. HEK-293 and MDA-MB-231 cells were transfected with ERß, pERE-Luc reporter, and empty vector control (VEC), AKT-CA (A), or MKK1-CA (B). Cells were treated with 0 to 10 nmol/L E2 and harvested 24 hours later for reporter-based luciferase assays (n = 4). C, MCF-7, MDA-MB-231, and HEK-293 cells were transfected with pERE-Luc reporter, with HEK-293 cells additionally transfected with ERß. Cells were treated with vehicle control (Con), E2 (10 pmol/L) with or without ICI 182,780 (100 pmol/L), or AKT inhibitor II (5 µmol/L) and harvested 24 hours later for reporter-based luciferase assays (n = 3-4). D, HEK-293 cells were transfected with pERE-Luc reporter, ERß, and empty vector control or AKT-DN. Cells were treated with vehicle control or E2 (10 pmol/L) and harvested 24 hours later for reporter-based luciferase assays (n = 2). Columns, percent estrogen activity relative to untreated or E2-treated empty vector cells (set at 100%); bars, SE.

 
Although the results above showed the ability of AKT to potentiate ERß activity in the presence of E2, a requirement for this kinase was investigated with pharmacologic and molecular inhibitors of AKT. Here, luciferase assays were done with MCF-7 breast adenocarcinoma cells (ER{alpha} and ERß positive), MDA-MB-231 breast adenocarcinoma cells (transfected with ERß), and HEK-293 cells (ER negative but transiently expressing ERß; Fig. 2C). In the MDA-MB-231 and HEK-293 cell lines, AKT inhibitor caused a noticeable decrease in ERß potentiation (down to 73 ± 12% in MDA-MB-231 and 48 ± 21% in HEK-293). Additionally, in HEK-293 cells transfected with ERß and AKT-DN (Fig. 2D), the molecular inhibitor of AKT led to a dramatic decrease in ERß potentiation.

AKT and MKK1 target different activation domains of ERß. Previous works have shown that AKT and MKK1 potentiation of ER{alpha} occurs through targeted phosphorylation of the AF1 domain (5, 6, 29). To explore the targets of MKK1 and AKT on ERß, luciferase assays were done with HEK-293 cells that transiently expressed ERß mutants and AKT-CA or MKK1-CA (Fig. 3A and B ). In agreement with previous results (29, 31), MKK1 also targeted the AF1 domain in the absence of E2. However, in the presence of E2, MKK1 exclusively targeted the AF2 domain. On the other hand, AKT targeted both activation function domains of ERß in the presence of E2. Given that AF1 and AF2 synergize for maximal transcriptional activity (1), the ability of AKT to target both activation function domains may contribute to its potentiation of ERß activity. In the absence of E2, AKT also targeted the AF2 domain, but the magnitude of activation was minor compared with activation in the presence of E2. More importantly, however, the preferential targeting of AF2 domain by AKT and MKK1 in the presence of ligand suggests that coactivators may be involved in kinase potentiation of ERß.


Figure 3
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  		Figure 3. AKT and MKK1 target different activation function domain of ERß. HEK-293 cells were transfected with pERE-Luc reporter along with ERß-AF1 (A) or ERß-AF2 (B) and empty vector control, AKT-CA, or MKK1-CA. Cells were treated with 1 nmol/L E2 and harvested 24 hours later for reporter-based luciferase assays. Columns, percent estrogen activity relative to untreated empty vector cells (n = 4); bars, SE. C, HEK-293 cells were transfected with pERE-Luc reporter along with wild-type ERß or ERß-K269A and empty vector control or AKT-CA. Cells were treated with 1 nmol/L E2 and harvested 24 hours later for reporter-based luciferase assays. Columns, percent estrogen activity relative to untreated empty vector cells (n = 4); bars, SE.

 
To further confirm the involvement of coactivators in kinase potentiation of ERß activity, luciferase assays were done with HEK-293 cells that transiently expressed ERß-K269A, in which lysine residue 269 was mutated to alanine, and AKT-CA or MKK1-CA (Fig. 3C). In the presence of 1 nmol/L E2, AKT and MKK1 were able to potentiate wild-type ERß activity but not of the mutant ERß-K269A. Residue K269 on the ERß is equivalent to residue K362 on the ER{alpha}, which is located in the highly conserved loop between helices 3 and 4 (the so-called signature sequence) and is required for coactivator binding to the ER{alpha} (32). These results suggest that coactivator binding to the ERß may be essential for AKT potentiation of the receptor.

AKT and MKK1 enhance SRC1 and GRIP1 recruitment to ERß-AF2. To achieve full transcription potential, the ERs must recruit the histone-modifying activities of coactivators to overcome the repressive packaging imposed by chromatin. The AF1 and AF2 domains of both ERs have been shown to interact with the NRID of p160 members (19). Although the ER{alpha} must associate with p160 coactivators to achieve full transcriptional potential, the ability of AKT to regulate p160 recruitment to the ER has not been established (33). To further investigate kinase-mediated regulation of ERß-AF2 function, we sought to determine the role of coactivator recruitment on receptor activity. We used the mammalian two-hybrid assay that has as prey the ERß-AF2 domain fused to a potent transactivation domain of VP16 (VP16-ERß) and as bait the NRID of SRC1 or GRIP1 fused to the Gal4-DBD. Interaction between Gal4-CoA and VP16-ER was regulated by the presence or absence of 1 nmol/L E2 and quantified by measuring transcription from a Gal4-Luc construct (Fig. 4A and B ). AKT enhanced recruitment of both SRC1 and GRIP1 to the receptor, whereas MKK1 enhanced only SRC1 recruitment. Gal4-DBD alone was used as a control and was not affected by VP16-ERß, E2, AKT, or MKK1 (data not shown). These results suggest that AKT and MKK1 potentiation of ERß activity may be attributed to their ability to enhance coactivator recruitment to the ERß-AF2.


Figure 4
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  		Figure 4. AKT enhances SRC1 and GRIP1 recruitment, whereas MKK1 enhances SRC1 recruitment, to ERß-AF2 domain. HEK-293 cells were transfected with VP16-ERß-AF2, pGal4-Luc reporter, Gal4-SRC1 (A) or Gal4-GRIP1 (B), and empty vector control along with AKT-CA or MKK1-CA. Cells were treated with vehicle control, 1 nmol/L E2, and 100 nmol/L ICI 182,780 with or without 1 nmol/L E2 for 24 hours and harvested for the mammalian two-hybrid assays. Columns, percent estrogen activity relative to untreated empty vector-VP16 transfected cells (n = 4); bars, SE.

 
AKT and GRIP1 cooperatively potentiate ERß activity. Because our above results implicated a role for GRIP1 in AKT regulation of ERß activity, we aimed to assess the contribution of GRIP1 to AKT potentiation of ERß activity. Luciferase assays were done with HEK-293 cells that transiently expressed ERß and AKT-CA with or without GRIP1 (Fig. 5 ). In the presence of 1 nmol/L E2, AKT potentiated ERß activity, and although GRIP1 has been shown to potentiate ER{alpha} activity (25, 34, 35), GRIP1 did not by itself potentiate ERß activity in our system. Moreover, AKT and GRIP1 cooperatively potentiated ER{alpha}-mediated transcription to a level much greater than either factor alone, thus establishing for the first time the cooperation of AKT and GRIP1 in the potentiation of ERß activity. However, this cooperation was obliterated in the presence of AKT-DN, in which ERß activity dropped below baseline. These results suggest that AKT may target GRIP1 in its potentiation of ERß activity.


Figure 5
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  		Figure 5. AKT and GRIP1 cooperatively potentiate ERß activity. HEK-293 cells were transfected with ERß, pERE-Luc reporter, and AKT-CA with or without GRIP1. Cells were treated with vehicle control or 1 nmol/L E2 for 24 hours before harvesting for reporter-based luciferase assays. Columns, percent estrogen activity relative to untreated empty vector cells (n = 4); bars, SE.

 
AKT potentiates the AD2 of GRIP1. Although we have shown that AKT can target the ERß itself, our above results also suggest that AKT may target a coactivator. GRIP1 contains two activation domains, AD1 and AD2, which distinctly target and regulate transcriptional complex activity (19). Whereas GRIP1-AD1 functions through a CBP-dependent mechanism (35), GRIP1-AD2 acts through CARM1 (36). Using Gal4 fusions of GRIP1-AD1 and GRIP1-AD2, we examined the ability of AKT to regulate transcription from these two domains (Fig. 6 ). The results show that AKT increased transcription activation of the AD2-CARM1 pathway of GRIP1. Because the GRIP1-AD2 is required for GRIP1 coactivator function with the ER{alpha} (25), the ability of AKT to activate this domain suggests that AKT may potentate ERß activity through targeting of the coactivator.


Figure 6
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  		Figure 6. AKT regulates GRIP1. A, HEK-293 cells were transfected with pGal4-Luc reporter, Gal4-GRIP-AD1 or Gal4-GRIP-AD2, and empty vector control or AKT. Cells were treated with vehicle control or 1 nmol/L E2 for 24 hours before harvesting for reporter-based luciferase assays. Data are relative light units (RLU) normalized to untreated vector control (n = 4). B, diagram of putative AKT and MAPK phosphorylation sites on GRIP1. C, phosphorylation of purified GST-GRIP1 by recombinant AKT in vitro. Purified GST or GST-GRIP1 were incubated with recombinant active AKT and [{gamma}-32P]ATP. Kinase reactions were run on a SDS-PAGE, transferred to nitrocellulose, and subjected to both autoradiography (right) and staining with Coomassie blue (left).

 
AKT directly phosphorylates GRIP1. Recently, coactivators that interact with the ER{alpha} have been shown to be targeted by the MAPK signaling cascade. Extracellular signal-regulated kinase 2 (ERK2) phosphorylates amplified in breast cancer 1 (AIB1) in vivo (16) and SRC1 (17) and GRIP1 (18) in vitro. Here, we aimed to determine the ability of AKT to phosphorylate GRIP1 with in vitro kinase assay of purified GST-GRIP1184-766 (Fig. 6C; ref. 18). The molecular weight of GST-GRIP1184-766 was determined to be ~100 kDa. As shown on the Coomassie stain, the larger GST-GRIP1184-766 moieties were of the correct approximate size, with the smaller fragments being incompletely translated proteins for degradation products (lane 3; ref. 34). AKT did not phosphorylate GST, and the ability of AKT to autophosphorylate (37) may explain the pAKT bands (lanes 5 and 7). More importantly, AKT was able to directly phosphorylate GRIP1 (lanes 6 and 8), probably at multiple sites given the phosphorylation of both large and small GST-GRIP1 species.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ER{alpha} and ERß have been shown to regulate distinct biological functions (1). Whereas studies of the ER{alpha} have contributed vastly to our understanding of breast cancer biology, the precise role of the ERß in this complex disease remains unclear. Studies evaluating ERß expression with certain tumor variables, such as tumor grade and disease-free survival, have produced conflicting results (15). Although some of the discrepancies may be resolved with detailed studies of the different ERß variants (9), the complexity of ERß signaling, like that of the ER{alpha}, will require understanding of ERß cross-talk with kinase signaling molecules. Previous researchers have established a cross-talk between AKT and the ER{alpha}. Here, we provide evidence for a cross-talk between AKT and the full-length/wild-type ERß.

Our tissue array results indicate a positive correlation between ERß and pAKT protein levels in a clinical setting, in which the largest population consisted of high ERß and pAKT expressions. Because the tissue arrays were done on unidentified patient samples, we were unable to determine whether the combination of high ERß and pAKT expressions may affect patient outcome. However, some conclusions may be inferred when our results are combined with previous studies. ERß expression has been shown to predict response to tamoxifen (9), whereas the expression and/or activity of AKT has been implicated in the development of tamoxifen resistance (6, 38). Because a population of high expressers for ERß and pAKT has been detected in our clinical samples, it may be important to determine both ERß and pAKT protein levels to more accurately gauge patient response to tamoxifen.

The positive correlation between ERß and pAKT suggests that, in some clinical breast cancers, pAKT may be able to regulate ERß transcriptional activity. We show here for the first time the ability of PI3K-AKT signaling to regulate ERß activity. AKT specifically potentiated ERß activity in the HEK-293 and MDA-MB-231 cell lines, indicating that this kinase can target the ERß in cells where the ERß isoform dominates. Moreover, our results indicate that the ERß may be important for overall cellular response to E2 in cells that contain both isoforms of the receptor, because AKT was shown to up-regulate ERE activity (data not shown) and AKT inhibitor II was shown to down-regulate ERE activity in MCF-7 cells. Recently, Helguero et al. showed that, in a mouse mammary cell line that contains both ER isoforms, the proliferative drive of ER{alpha} in response to E2 may be attenuated by the apoptotic drive of ERß (39). Because both receptor isoforms coexist in some tissues (1) and altered expression patterns appear during tumorigenesis (40), ERß activation by kinase signaling pathways may contribute to overall ER transcriptional activity and, ultimately, to cell fate.

The AF1 of both ER{alpha} and ERß have been shown to be targeted by MAPK signaling (41), and the AF1 of ER{alpha} is targeted by PI3K-AKT signaling (5, 6). However, our results suggest that the AF2 domain of ERß may also be an important target for kinase signaling, as both MKK1 and AKT targeted this AF2 domain. Although AKT also targeted the AF1 domain, it may not be surprising that AKT predominantly targeted the AF2 domain, because the ERß has been shown to have a weak AF1 domain (21). Because AF1 and AF2 synergize for maximal transcriptional activity (42), the ability of AKT to target both activation function domains may contribute to its potentiation of ERß activity. Lastly, potentiation of ERß-AF2 domain, along with the requirement of residue K269 on the receptor, suggest the involvement of coactivators in AKT regulation of ERß activity.

Transcriptional activity of the ER{alpha} is dependent on its ability to recruit coactivators (33), and our coactivator studies further illustrate the importance of the AF2 domain in AKT regulation of the ERß. Coactivators are differentially recruited to ERß-AF2 domain by AKT and MKK1, such that MKK1 only enhanced SRC1 recruitment, whereas AKT enhanced SRC1 recruitment slightly and GRIP1 recruitment potently. Recently, Zhao et al. employed pull-down assays to show physical interaction between the mouse wild-type ERß and TIF2 in the presence of E2 (43). Because coactivator binding to the ER has been shown to enhance receptor activity (35), we sought to determine the role of GRIP1 in AKT potentiation of ERß transcriptional activity. AKT and GRIP1 cooperatively potentiated ERß-mediated transcription to a level greater than either factor alone, thus establishing for the first time the cooperation of AKT and GRIP1 in the potentiation of ERß activity. Although these results explained AKT regulation of ERß activity, works form other laboratories led us to speculate that AKT may be able to target GRIP1 itself.

The activation domains of GRIP1 may serve integral roles in AKT potentiation of ER{alpha} transcriptional activity because of their abilities to recruit other histone-modifying proteins, such as CBP/p300 (35) and CARM1 (44). Our results, indicating slight potentiation of GRIP1-AD1 and strong potentiation of GRIP1-AD2 by AKT, suggests that AKT may potentiate ERß in part through its ability to activate GRIP1 to recruit CARM1. Because transcriptional complexes require the coordinated efforts of numerous factors that must be recruited to the area, the ability of AKT to potentiate ERß activity may require this kinase to not only recruit coactivators to the receptor but also enhance recruitment of other histone-modifying proteins, such as CARM1, to the vicinity.

Coactivators that interact with the ER{alpha} have been shown to be targeted by the MAPK signaling cascade. ERK2 phosphorylates AIB1 in vivo (16) and SRC1 (17) and GRIP1 (18) in vitro. Here, we document for the first time the ability of AKT to phosphorylate GRIP1 in vitro. Interestingly, AKT phosphorylated GRIP1 extensively as documented by the numerous phosphorylated bands. Analysis of the GRIP1 protein sequence revealed three putative AKT phosphorylation sites within the GST-GRIP1184-766 plasmid, at T197, S360, and S671. Phosphorylation of T197, located at the beginning of the GRIP1 fusion protein, may explain the small phosphorylated bands toward the bottom of the gel.

Phosphorylation of GRIP1-NRID by AKT and MAPK may affect the level of GRIP1 recruitment to the ER{alpha}. Lopez et al. showed that ERK2 phosphorylation of S736, located within the NRID, is required for GRIP1 binding to the ER{alpha}-AF2 (18). Interestingly, the GST-GRIP1184-766 construct used for our in vitro kinase assays also contains the NRID of GRIP1. Our phosphorylation studies, combined with previously published results, may explain the ability of kinases to affect GRIP1 recruitment to the ERß. Our Gal4-GRIP1-NRID, which contains putative AKT and MKK1 phosphorylation sites, was recruited to the ERß-AF2 domain by AKT but surprisingly, not by MKK1. Hence, MKK1 phosphorylation of GRIP1 may enhance the ability of the coactivator to bind to the ER{alpha} but not to the ERß. Although the exact mechanism for this difference is currently unknown, we do know that the binding of coactivator to the ER is a complex process that depends on residues that flank the NR boxes (45) as well as the nature of the ligands that bind to the receptor (46), among other factors. Therefore, this coactivator-ER interaction most likely involves numerous phosphorylations by the coordinated efforts of multiple kinases.

Recently, clinical studies revealed that the interplay between growth factor signaling and the ER may involve coactivators (47, 48). Osborne et al. showed that tumors that were relatively resistant to tamoxifen treatment express high levels of both HER-2 and the coactivator AIB1 (48). These authors suggested that HER-2, possibly through activation of MAPK signaling, might target AIB1 for the promotion of tamoxifen resistance. Myers et al. showed that SRC1 expression was negatively associated with disease-free survival in breast cancer patients (47). Interestingly, these investigators also found that tumors that up-regulated SRC1 expression in the presence of estrogen were all positive for HER-2. These clinical studies indicate that ER coactivators may be important diagnostic and therapeutic targets (47, 48).

The ER is a conduit for numerous biological functions due to the many signaling pathways that converge on the receptor (19, 49). However, the two isoforms of the ER have both common and disparate functions (50). Thus, kinase regulation of these two receptors may influence overall cellular response to estrogenic compounds and reveal the need to study not only kinase regulation of the ER{alpha} but also regulation of the ERß. Perhaps not surprisingly, our studies reveal that the MAPK and PI3K-AKT signaling cascades differentially regulated ERß activity not only at the level of the receptor but also at the level of the coactivators that were recruited to the receptor. Thus, although both the PI3K-AKT and MAPK pathways can regulate ERß activity, the fine-tuning of biological responses may require these pathways to target not only the ERß but also the coactivators that are integral for ERß function.


   Acknowledgments
 
Grant support: NIH grant DK059389 (M.E. Burow), U.S. Department of Defense Breast Cancer Research Program grant DAMD-17-97-1-7024 (M.E. Burow), Office of Naval Research grant N00014-99-1-0763 (J.A. McLachlan and M.E. Burow), and Center for Disease Control grant RO6/CCR419466-02 (J.A. McLachlan and M.E. Burow).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/24/05. Revised 5/ 1/06. Accepted 6/29/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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