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Cancer Research 67, 10600, November 1, 2007. doi: 10.1158/0008-5472.CAN-07-0055
© 2007 American Association for Cancer Research
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Endocrinology

Interaction of the Tumor Metastasis Suppressor Nonmetastatic Protein 23 Homologue H1 and Estrogen Receptor {alpha} Alters Estrogen-Responsive Gene Expression

Carol D. Curtis1, Varsha S. Likhite2, Ian X. McLeod3, John R. Yates3 and Ann M. Nardulli1

Departments of 1 Molecular and Integrative Physiology and 2 Biochemistry, University of Illinois, Urbana, Illinois; and 3 Department of Cell Biology, The Scripps Research Institute, La Jolla, California

Requests for reprints: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana–Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, IL 61801. Phone: 217-244-5679; Fax: 217-333-1133; E-mail: anardull{at}life.uiuc.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Metastasis of cancer cells from the primary tumor is associated with poor prognosis and decreased overall survival. One protein implicated in inhibiting metastasis is the tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1). NM23-H1 is a multifunctional protein, which, in addition to limiting metastasis, has DNase and histidine protein kinase activities. We have identified new functions for NM23-H1 in influencing estrogen receptor {alpha} (ER{alpha})–mediated gene expression. Using a battery of molecular and biochemical techniques, we show that NM23-H1 interacts with ER{alpha} and increases the ER{alpha}–estrogen response element (ERE) interaction. When NM23-H1 expression is increased in U2 osteosarcoma and MDA-MB-231 breast cancer cells, transcription of a transiently transfected, estrogen-responsive reporter plasmid is decreased. More importantly, when endogenous NM23-H1 expression is knocked down in MCF-7 human breast cancer cells using small interfering RNA, estrogen responsiveness of the progesterone receptor (PR), Bcl-2, cathepsin D, and cyclin D1 genes, but not the pS2 gene, is enhanced. Furthermore, NM23-H1 associates with the region of the PR gene containing the +90 activator protein 1 site, but not with the ERE-containing region of the pS2 gene, indicating that NM23-H1 mediates gene-specific effects by association with endogenous chromatin. Our studies suggest that the capacity of NM23-H1 to limit the expression of estrogen-responsive genes such as cathepsin D and Bcl-2, which are involved in cell migration, apoptosis, and angiogenesis, may help to explain the metastasis-suppressive effects of this protein. The complementary abilities of ER{alpha} and NM23-H1 together to influence gene expression, cell migration, and apoptosis could be key factors in helping to determine tumor cell fate. [Cancer Res 2007;67(21):10600–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer is the second leading cause of cancer-related deaths in the United States. In 2005 alone, 210,000 women were diagnosed with breast cancer, and 40,000 died of the disease (1). Progression of breast cancer can result in migration of cells from the primary tumor through the blood stream to a secondary site where the cells colonize. This metastatic process is the leading cause of cancer-related morbidity and mortality (2, 3). A majority of these primary and secondary metastatic tumors are estrogen receptor (ER) positive and respond to estrogenic and antiestrogenic hormones (4, 5). Thus, although 17ß-estradiol (E2) is critical for normal growth, development, and maintenance of the mammary gland, exposure of ER-positive mammary tumors to E2 increases proliferation of breast cancer cells and enhances tumor progression (6, 7).

Interaction of ER{alpha} with E2 facilitates binding of the receptor to estrogen response elements (ERE) in target genes, which in turn leads to the recruitment of coregulatory proteins such as the p160 proteins steroid receptor coactivator-1, transcription intermediary factor 2, and amplified in breast cancer 1 (8). These coregulatory proteins interact with ER{alpha} to enhance receptor-mediated transcription in a ligand-dependent manner. Conversely, corepressor proteins, such as the silencing mediator of retinoid and thyroid receptors and nuclear receptor corepressor, inhibit transcription of estrogen-responsive genes (9, 10). Both ligand and DNA induce changes in ER{alpha} conformation that alter recruitment of coregulatory proteins and influence gene expression (11–15).

In this study, we have identified a novel interaction between ER{alpha} and the tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1), which has been classified as a metastasis suppressor, a histidine kinase, and a DNA repair protein (16–21). We show that NM23-H1 interacts with ER{alpha} in vitro and in MCF-7 cells increases ER{alpha}-ERE complex formation, influences ER{alpha}-mediated transcription, and associates with the promoter region of the endogenous estrogen-responsive progesterone receptor (PR) gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
HeLa pull-down assay. Novel ER{alpha}-associated proteins were isolated in agarose gel mobility shift experiments in the presence of E2 and identified by mass spectrometry analysis as previously described (14, 22) using baculovirus-expressed, purified ER{alpha} and HeLa nuclear extracts. Two peptides, TFIAIKPDGVQR and GDFCIQVGR, with an amino acid sequence identical to that found in NM23-H1 and NM23-H2, were identified. However, because initial experiments revealed that only NM23-H1 interacted with ER{alpha}, our studies were confined to NM23-H1.

Western blot analysis. Nuclear extracts from untransfected MCF-7 breast cancer cells, U2 osteosarcoma (U2OS) cells, HeLa cervical cancer cells, and MDA-MB-231 breast cancer cells were prepared as described (13). Twenty micrograms of nuclear extracts were fractionated on an 8% to 16% Tris-glycine gel and transferred to a nitrocellulose membrane. Proteins were detected by Western blot analysis using an antibody that recognized NM23-H1 and NM23-H2 (RB-116-P1, Neomarkers), ER{alpha} (sc-543, Santa Cruz Biotechnology) or Sp1 (sc-59, Santa Cruz Biotechnology). Blots were probed with horseradish peroxidase–conjugated secondary antibody, and the Supersignal West Femto Maximum Sensitivity Substrate chemiluminescent detection kit (Pierce Chemical Co.) was used to visualize the proteins as per the manufacturer's instructions.

Cell culture and transfections. For U2OS and MDA-MB-231 cell transfections, cells were maintained and transfected using Lipofectin as described (15) with 1 ng of the ptk-Renilla expression vector (Promega), 1 µg of the firefly luciferase reporter vector 2EREtkLUC, and 5 ng CMV5-hER{alpha} (both kindly provided by Benita Katzenellenbogen, University of Illinois, Urbana, IL). Increasing concentrations of the pcDNA3-NM23-H1 expression vector (kindly provided by Patricia Steeg, National Cancer Institute, Bethesda, MD) were added as indicated. After a 6-h incubation at 37°C, cells were treated with ethanol vehicle or 10 nmol/L E2 for 24 h. Luciferase assays were carried out as previously described (14). Data from three independent experiments, which had been done in duplicate, were combined, and significant differences were determined with SAS 9.1 (SAS Institute, Inc.) using a one-way ANOVA with a post hoc Student's t test.

NM23-H1-Flag purification. The NM23-H1-Flag expression vector, pcDNA3-NM23-H1-Flag (kindly provided by Patricia Steeg, National Cancer Institute, Bethesda, MD), was used for NM23-H1 expression in BL21(DE3)pLysS Escherichia coli. Transformed bacteria were induced with isopropyl-L-thio-ß-D-galactopyranoside, pelleted, resusupended, and homogenized in homogenization buffer [20 mmol/L Tris (pH 7.4), 0.5 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 10% glycerol, and protease inhibitors]. Flag-tagged NM23-H1 was immobilized on EZview Red Anti-Flag M2 Affinity resin (Sigma-Aldrich). After three washes with TBS [50 mmol/L Tris (pH 7.4), 50 mmol/L NaCl, 0.2 mmol/L EDTA], Flag-tagged NM23-H1 was eluted with elution buffer [20 mmol/L Tris (pH 7.4), 0.4 mol/L NaCl, 0.2 mmol/L EDTA, 10% glycerol, 0.1% NP40, 0.1 µg Flag-peptide, 2 mmol/L DTT, and protease inhibitors] for 20 min at 4°C.

Pull-down assay using in vitro translated proteins. To establish whether ER{alpha} interacted directly with NM23-H1, Flag-tagged NM23-H1 was immobilized on anti-Flag M2 resin and incubated with 200 fmol baculovirus-expressed, purified ER{alpha} in TBS [50 mmol/L Tris (pH 7.4), 50 mmol/L NaCl, 0.2 mmol/L EDTA] for 1 h at 4°C in the presence of ethanol or 1 µmol/L E2. DNA competition assays were processed in the same way, except that 100 pmol of either nonspecific or consensus ERE-containing oligos were included. After four washes with TBS, the NM23-H1–associated proteins were eluted with SDS sample buffer [250 mmol/L Tris (pH 6.8), 300 mmol/L SDS, 40% glycerol, 20% ß-mecaptoethanol], separated by SDS-PAGE, and transferred to a nitrocellulose membrane for Western blot analysis. An ER{alpha}-specific antibody (sc-543) was used to detect ER{alpha}.

Gel mobility shift assay. 32P-labeled, 50-bp ERE-containing oligos (20,000 cpm) were incubated with or without purified ER{alpha} in binding reaction buffer [15 mmol/L Tris (pH 7.9), 60 mmol/L KCl, 200 µmol/L EDTA, 4 mmol/L DTT, 10% glycerol, 50 ng deoxyinosinic-deoxycytidylic acid, 50 nmol/L E2] for 10 min at 25°C. Increasing amounts of purified Flag-tagged NM23-H1 were added as indicated. Protein concentrations were held constant at 2 µg by the addition of bovine serum albumin (BSA). ER{alpha}- or NM23-H1–specific antibody (sc-8005 or sc-465, respectively, Santa Cruz Biotechnology) was added and incubated at 25°C for an additional 10 min. The ER{alpha}-ERE complexes were fractioned on a 6% low-ionic-strength gel at 4°C with buffer recirculation. The levels of bound and free 32P-labeled DNA were quantitated using a Molecular Dynamics Phosphorimager and ImageQuant 5.0 software (Molecular Dynamics, Inc.).

Coimmunoprecipitation assay. MCF-7 cells were maintained on phenol-red containing minimal essential medium (MEM) with 5% calf serum. Two days before harvest, cells were transferred to phenol-red–free MEM containing 5% charcoal-dextran–treated calf serum. Approximately 1.25 x 106 cells were treated with ethanol vehicle or 10 nmol/L E2 for 20 min and then harvested in TNE [40 mmol/L Tris (pH 7.5), 140 mmol/L NaCl, 1.5 mmol/L EDTA], resuspended in lysis buffer [20 mmol/L Tris (pH 8.0), 200 mmol/L NaCl, 1 mmol/L EDTA, 0.2% NP40], and subjected to two freeze-thaw cycles. The lysed cells were diluted in TE [10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA] with protease inhibitors, and NaCl was added to a final salt concentration of 240 mmol/L. Protein-A slurry (GE Healthcare) was added to preclear the lysates for 1.5 h at 4°C. The ER{alpha}-specific antibody sc-8002 (Santa Cruz Biotechnology) or the NM23-H1–specific antibody sc-465 or sc-343 (Santa Cruz Biotechnology) was bound to Protein-A sepharose slurry for 1.5 h at 4°C. Unbound antibody was discarded, and the MCF-7 precleared lysates were added to the antibody-bound resin. The samples were incubated at 4°C for 4 h. The resin was washed, and bound proteins were eluted with SDS sample buffer. Proteins were fractioned on an 8% to 16% Tris-glycine gradient gel (Bio-Rad) and transferred to a nitrocellulose membrane for Western blot analysis with antibodies to ER{alpha} (sc-543) and NM23-H1 and H2 (RB-116-P1). Additional coimmunoprecipitations were done using a class-matched antibody as a control (Supplementary Data).

RNA interference. MCF-7 cells were maintained on phenol-red containing MEM with 5% calf serum. One day before plating, cells were transferred to phenol-red–free MEM containing 5% charcoal dextran-treated calf serum. Cells were resuspended in antibiotic-free, phenol-red–free MEM containing 5% charcoal dextran-treated calf serum and seeded into 12-well plates. The next day, cells were transfected with 50 pmol NM23-H1 specific (Ambion) or control small interfering RNA (siRNA) directed against Renilla luciferase (Ambion) using SiLentfect (Bio-Rad). After a 1 to 4 day incubation, plated cells were treated with ethanol vehicle or 10 nmol/L E2. After 24 h, cells were harvested in TNE, resuspended in lysis buffer, and subjected to two freeze-thaw cycles. Whole cell extracts (20 µg) were fractionated on an 8% to 16% Tris-glycine gel, transferred to a nitrocellulose membrane, and subjected to Western blot analysis with antibodies to NM23-H1 and NM23-H2 (RB-116-P1), PR-A and PR-B (RM-9102-S1, Neomarkers), ER{alpha} (sc-543), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc-20357, Santa Cruz Biotechnology).

To quantitate mRNA levels, total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions, and RQ1 DNase (Promega) was used to digest any contaminating DNA. cDNA was prepared using the Reverse Transcription System (Promega), and real-time quantitative PCR was done using iQ SYBR Green Supermix and the iCycler PCR thermocyler (Bio-Rad) with the following gene-specific primer sets that were designed using Beacon Designer 2.1 (Biosoft International): NM23-H1 (5'-GGCTGAATGTGGTGAAGAC-3' and 5'-TTCCTGCCAACTTGTATGC-3'), cathepsin D (5'-TGCCACCCTACCTGTTCAG-3' and 5'-TCTCACTCCTTCCAGCTCATC-3'), cyclin D1 (5'-GCTCACGCTTACCTCAAC-3' and 5'-CCCATCACGACAGACAAAG-3'), PR (5'-GTGCCTATCCTGCCTCTCAATC-3' and 5'-CCCGCCGTCGTAACTTTCG-3'), Bcl-2 (5'-ATCGCCCTGTGGATGACTG-3' and 5'-GCCTCAGCCCAGACTCAC-3'), Sp1 (5'-ACCCACAAGCCCAAACAATC-3' and 5'-GAGGAGAGTTGAGCAGCATTC-3'), pS2 (5'-GCTGTTTCGACGACACCGTT-3' and 5'-TTCTGGAGGGACGTCGATG-3'), and ER{alpha} (5'-TGCCCTACTACCTGGAGAAC-3' and 5'-CCATAGCCATACTTCCCTTGTC-3'). cDNA synthesized from 20, 2, 0.2, and 0.02 ng RNA was run in triplicate during each experiment to derive a standard curve for each primer set. Extrapolation from the standard curve indicated the nanogram equivalent value for each sample. Data from four independent experiments, which were carried out in triplicate, were combined and are presented as the mean ± SE. The level of mRNA present in the presence of NM23-H1–specific siRNA was compared with the level of mRNA present in the presence of control siRNA for each gene. Statistical differences were determined with SAS 9.1 using a one-way ANOVA with a post hoc Student's t test.

Chromatin immunoprecipitation assay. MCF-7 cells were maintained on phenol-red–containing MEM with 5% calf serum. Three days before treatment, cells were transferred to phenol-red–free MEM containing 5% charcoal-dextran–treated calf serum. Cells were treated with ethanol vehicle or 10 nmol/L E2 for 2 h, and chromatin immunoprecipitation (ChIP) assays were carried out essentially as recommended by Upstate, except that cell lysates were diluted in micrococcal nuclease buffer [10 mmol/L Tris (pH 7.5), 10 mmol/L NaCl, 3 mmol/L MgCl2, 0.5% NP40, 10 mmol/L CaCl2, and 4% NP40] and treated with 50 units micrococcal nuclease (U.S. Biochemical) at 37°C for 10 min before sonication. Protein-A sepharose (GE Healthcare) and a nonspecific fluorescein antibody (Immunological Resource Center, University of Illinois) were added to preclear the chromatin overnight at 4°C. An ER{alpha}- (sc-8002) or NM23-H1–specific antibody (sc-465) was used for immunoprecipitation of protein-DNA complexes.

PCR primers flanking the pS2 ERE, the +90 activator protein 1 (AP-1) site of the PR gene, or a nonspecific sequence in the 36B4 gene were used for semiquantitative PCR using iQ SyBr Green Supermix and the iCycler PCR thermocycler according to manufacturer's directions (Bio-Rad). The gene regions amplified and the primer sets used were 36B4 control (5'-GCTGTTTCGACGACACCGTT-3' and 5'-TTCTGGAGGGACGTCGATG-3'), pS2 ERE (5'-CCCGTGAGCCACTGTTGTC-3' and 5'-CCTCCCGCCAGGGTAAATAC-3'), and PR +90 AP-1 (5'-GCGTGTGGGTGGCATTCTC-3' and 5'-GGCGACAGTCATCTCCGAAG-3'). Standard curves using 100,000, 50,000, 10,000, 5,000, and 1,000 copies of each gene were run for each primer set during each experiment. Data from three independent experiments, which were carried out in triplicate, were combined and are presented as the mean ± SE. The number of copies associated at each gene region was compared with the number of copies associated with 36B4, and statistical differences were determined with SAS 9.1 using a one-way ANOVA with a post hoc Student's t test.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To better understand how estrogen-responsive genes are regulated, our laboratory identified a number of proteins that associate with the DNA-bound ER{alpha} using agarose gel mobility shift assays. Mass spectrometry analysis indicated that NM23-H1, NM23-H2, or both proteins might be associated with the ER{alpha}–ERE complex,4 but we were unable to determine from the peptide sequences whether one or both of these proteins was present.

Endogenously expressed ER{alpha} and NM23-H1 interact. We first assessed the levels of NM23-H1 and NM23-H2 expressed in a number of cultured cell lines that have been used to study estrogen responsiveness (13, 14, 23, 24). MCF-7 breast cancer, U2OS, and HeLa cervical cancer cells expressed higher levels of NM23-H1 and NM23-H2 than MDA-MB-231 breast cancer cells, but only MCF-7 cells expressed ER{alpha} (Fig. 1 ). Thus, the ER{alpha}-positive MCF-7 breast cancer cells expressed higher levels of NM23-H1 than the ER{alpha}-negative MDA-MB-231 breast cancer cell line. Similarly, others have reported that ER{alpha}-positive BT-474 breast cancer cells express higher levels of NM23-H1 than ER{alpha}-negative BCM-1 breast cancer cells (25). The level of Sp1, which was used as a loading control, was similar in each of the cell lines tested.


Figure 1
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  		Figure 1. Endogenous expression of NM23-H1. Nuclear extracts (20 µg) from MCF-7, U2OS, HeLa, or MDA-MB-231 cells were separated on a denaturing acrylamide gel and transferred to nitrocellulose. NM23-H1 and NM23-H2 were detected with an antibody that recognizes both proteins. ER{alpha} and Sp1, which was used as a loading control, were detected with ER{alpha}- and Sp1-specific antibodies, respectively.

 
To determine whether ER{alpha} interacted with NM23-H1 or NM23-H2 in a cell where these proteins were endogenously expressed, immunoprecipitation assays were done. MCF-7 breast cancer cells were exposed to ethanol or E2 for 20 min, and then the receptor and its associated proteins were immunoprecipitated with an ER{alpha}-specific antibody and subjected to Western blot analysis with an antibody that recognizes both NM23-H1 and NM23-H2. Although NM23-H2 is highly expressed in MCF-7 cells (Fig. 2A, lanes 1 and 2 ), only NM23-H1 was immunoprecipitated with the ER{alpha}-specific antibody regardless of whether cells had not (lane 5) or had (lane 6) been exposed to E2. Likewise, when MCF-7 proteins were immunoprecipitated with an NM23-H1–specific antibody (Fig. 2B), ER{alpha} was detected in the absence (lane 5) and in the presence (lane 6) of hormone. Thus, because ER{alpha} interacted specifically with NM23-H1, but not with NM23-H2, all subsequent experiments were limited to the characterization of NM23-H1.


Figure 2
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  		Figure 2. Interaction of ER{alpha} with NM23-H1. MCF-7 cells were treated with ethanol or 10 nmol/L E2 20 min before harvest as indicated. Lysates were prepared, and proteins were immunoprecipitated with an antibody directed against ER{alpha} (A) or NM23-H1 (B) and subjected to Western blot analysis with an antibody that detects NM23-H1 and NM23-H2 (A) or ER{alpha} (B). Lanes 1 and 2 contain 10% input. C, purified, baculovirus-expressed ER{alpha} (200 fmol) was incubated with M2 resin alone (lane 2) or with immobilized Flag-tagged NM23-H1 in the absence (lanes 3, 5, and 7) or presence (lanes 4, 6, and 8) of E2. Oligos containing nonspecific (NS) DNA sequence (lanes 5 and 6) or a consensus ERE (lanes 7 and 8) were included as indicated. Bound ER{alpha} was eluted and subjected to Western blot analysis with an ER{alpha}-specific antibody. Lane 1 contains 10% ER{alpha} input.

 
ER{alpha} interacts directly with NM23-H1 in vitro. Although endogenously expressed NM23-H1 and ER{alpha} were able to interact in MCF-7 cells, many proteins were present in the extracts used. To determine whether purified NM23-H1 and ER{alpha} could interact directly, Flag-tagged NM23-H1 was immobilized and incubated with baculovirus-expressed, purified ER{alpha} (Fig. 2C). ER{alpha} bound to NM23-H1 in the absence (lane 3) and in the presence (lane 4) of E2, but did not bind to the resin alone (lane 2). When nonspecific (lanes 5 and 6) or ERE-containing DNA (lanes 7 and 8) was included in the binding reaction, ER{alpha} was able to bind to NM23-H1 in the absence and in the presence of hormone. Thus, ER{alpha} interacts directly with NM23-H1, and this interaction is unaffected by hormone or DNA.

NM23-H1 fosters the ER{alpha}-ERE interaction. Thus far, we had shown that NM23-H1 was associated with the receptor-DNA complex, and that it interacted with ER{alpha} in vitro and in MCF-7 cells. To determine whether this ER{alpha}-NM23-H1 interaction might affect the ability of the receptor to bind oligos each containing a single consensus ERE, gel mobility shift assays were done. In the absence of NM23-H1, ER{alpha} bound to the ERE (Fig. 3A, lanes 2 and 3 ). As increasing amounts of NM23-H1 protein were added, the ability of ER{alpha} to bind to the ERE-containing oligo increased in a dose-dependent manner (lanes 4–8). Addition of NM23-H1 resulted in an 8- to 10-fold increase in ER{alpha}-ERE complex formation (Fig. 3B, lanes 6–8). This increase in binding was due to the presence of NM23-H1 and not to increased protein concentrations because the total protein concentration was held constant by the addition of BSA. The ability of an ER{alpha}-specific antibody (lane 9) to supershift the protein-DNA complex indicated that the receptor was present. In contrast, an NM23-H1–specific antibody (lane 10) failed to supershift the protein-DNA complex. Two additional antibodies directed against different NM23-H1 epitopes produced similar results (data not shown). Thus, although NM23-H1 was not present in the protein-DNA complex, it did increase the ER{alpha}-ERE interaction. The inability of coregulatory proteins to form a stable trimeric complex with the receptor and DNA in gel mobility shift assays has been reported previously by us and others (14, 15, 22, 26–29) and may result from the transient interaction of NM23-H1 with the receptor-DNA complex, the necessity of other proteins to stabilize the receptor-DNA interaction, and/or the extended period of electrophoresis required for gel mobility shift assays.


Figure 3
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  		Figure 3. Effect of NM23-H1 on the ER{alpha}-ERE interaction and ER{alpha}-mediated transcription. A, 32P-labeled oligos containing a consensus ERE were run alone (lane 1) or combined with 30 (lane 2) or 10 (lanes 3–10) fmol baculovirus-expressed, purified ER{alpha} and increasing (lanes 4–8) or constant (lanes 8–10) amounts of purified NM23-H1. Total protein concentrations were held constant by the addition of BSA. ER{alpha}- (lane 9) and NM23-H1 (lane 10)-specific antibodies were added as indicated. Lane 11 contains purified NM23-H1 and 32P-labeled ERE oligo in the absence of ER{alpha}. B, the percent of probe bound was determined from five individual experiments and is shown graphically as the mean ± SE. MDA-MB-231 (C) or U2OS (D) cells were transfected with an ER{alpha} expression vector, the luciferase reporter plasmid 2EREtkLUC, and increasing amounts of an NM23-H1 expression vector. Cells were exposed to ethanol (light-gray bars) or 10 nmol/L E2 (dark-gray bars). Data from three independent experiments, which had each been done in duplicate, were combined and are presented as the mean ± SE. *, P < 0.05, statistical differences in transcription in the presence and in the absence of the NM23-H1 expression vector were measured using a one-way ANOVA with a post hoc Student's t test.

 
NM23-H1 decreases transcription of an estrogen-responsive reporter plasmid. Because we had shown that NM23-H1 was able to increase ER{alpha}-ERE complex formation, we investigated whether this interaction had a functional consequence and could alter ER{alpha}-mediated transcription. When MDA-MB-231 cells were transfected with an ER{alpha} expression vector and a luciferase reporter plasmid containing two EREs, inclusion of 500 and 1,000 ng of an NM23-H1 expression vector elicited 1.8- and 2.7-fold decreases, respectively, in estrogen-induced transactivation (Fig. 3C). Likewise, transcription was decreased 3- and 4-fold in U2OS cells when 500 and 1,000 ng of the NM23-H1 expression vector were included (Fig. 3D). Thus, although NM23-H1 increased the ER{alpha}-ERE interaction, it decreased the transcription of an estrogen-responsive reporter plasmid in two different cell lines. Similar decreases in estrogen-responsive gene expression have been observed with other ER{alpha}-associated DNA repair proteins (14, 22, 29). It has been suggested that these proteins may help to stabilize the transcription complex so that DNA repair and transcription can occur simultaneously.

NM23-H1 alters transcription of endogenous estrogen-responsive genes. Given that increased NM23-H1 expression decreased ER{alpha}-mediated transcription of a simple, estrogen-responsive reporter plasmid in transient transfections, we were interested in determining whether expression of endogenous, estrogen-responsive genes might also be affected by decreasing NM23-H1 expression. RNA interference experiments were carried out in MCF-7 cells, which express endogenous NM23-H1 and ER{alpha} (Fig. 1). A time course was initially done to determine the length of time needed to decrease NM23-H1 expression. Adequate knockdown of NM23-H1 was achieved after a 96-h exposure of cells to 50 pmol siRNA (Fig. 4A, lanes 5–8 ). In contrast, siRNA directed against Renilla luciferase did not affect NM23-H1 expression (lanes 1–4).


Figure 4
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  		Figure 4. Effect of knocking down NM23-H1 on protein levels. MCF-7 cells were transfected with NM23-H1 or control siRNA for 24 to 96 h (A) or 96 h (B). Cells were then treated with ethanol vehicle or E2 for 24 h, lysed, and subjected to Western blot analysis with a NM23-H1–specific antibody (A) or antibodies that recognize NM23-H1 and NM23-H2, PR-A and PR-B, ER{alpha}, or GAPDH (B).

 
Western blot analysis revealed that the NM23-H1 siRNA was specific because NM23-H1 expression was dramatically decreased, but NM23-H2 was unaffected (Fig. 4B). E2 treatment of MCF-7 cells increased PR expression in the presence of control siRNA as has been observed in untransfected E2-treated MCF-7 cells (30). However, when NM23-H1 expression was decreased, PR protein levels increased to even higher levels in the presence of E2 compared with control siRNA. In contrast, neither ER{alpha} nor GAPDH, which was used as an internal control, was affected by control or NM23-H1–specific siRNA.

To determine the effect of decreased NM23-H1 expression on endogenous, estrogen-responsive gene expression at the mRNA level, total RNA was isolated from MCF-7 cells that had been transfected with control or NM23-H1–specific siRNA, and real-time quantitative reverse transcription-PCR analysis was done. NM23-H1 mRNA was significantly decreased with the NM23-H1–specific siRNA in the absence and in the presence of E2 (Fig. 5 ). As expected, E2 treatment of MCF-7 cells increased PR mRNA levels when control siRNA was used. However, when NM23-H1 was knocked down, the level of PR mRNA was enhanced compared with the control siRNA. Thus, the increased PR mRNA level we observed when NM23-H1 levels were knocked down corresponded well to the increased level of PR protein (Fig. 4B). We also examined the expression of the estrogen-responsive Bcl-2, cathepsin D, cyclin D1, and pS2 genes (31–34). The mRNA levels of Bcl-2, cathepsin D, and cyclin D1 were significantly increased in the presence of E2 when NM23-H1 was knocked down. In addition, cyclin D1 mRNA levels were decreased in the absence of E2 when NM23-H1 was knocked down. Although estrogen responsiveness of these four genes was increased when NM23-H1 mRNA levels were decreased, neither the widely characterized estrogen-responsive pS2 gene nor the adhesion protein E-cadherin (data not shown) was affected by decreased NM23-H1 expression. Likewise, no significant changes were observed in ER{alpha}, Sp1 or GAPDH (data not shown) expression when NM23-H1 levels were reduced.


Figure 5
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  		Figure 5. Effect of knocking down NM23-H1 on estrogen-responsive gene expression. MCF-7 cells were transfected with NM23-H1 or control siRNA, treated with ethanol vehicle or E2 for 24 h, and lysed. RNA was isolated, cDNA was synthesized, and real-time PCR was carried out with gene-specific primers. Standard curves were derived for each primer set in each experiment, and the relative amount of RNA obtained for each sample was calculated from the standard curve. Data from four independent experiments, which had been done in triplicate, were combined and are presented as the mean ± SE. *, P < 0.05, statistical differences in mRNA levels in the presence of NM23-H1–specific siRNA compared with the corresponding mRNA levels in the presence of control siRNA were determined with SAS 9.1 using a one-way ANOVA with a post hoc Student's t test. Light- and dark-gray bars, ethanol and E2 treatment, respectively.

 
Interestingly, we noted what seemed to be a consistent increase in NM23-H1 mRNA levels when MCF-7 cells were treated with E2. Additional experiments revealed a consistent 1.6-fold increase in NM23-H1 mRNA expression after 24 h of hormone treatment (data not shown). These findings are in agreement with two earlier studies, which reported that NM23-H1 mRNA levels were increased upon E2 treatment of MCF-7 cells (25, 35). However, although NM23-H1 mRNA levels increased with E2 treatment, neither we nor others observed a concomitant increase in NM23-H1 protein levels in these cells in the presence of E2 (Figs. 2A and 4B; ref. 36).

NM23-H1 associates with the endogenous estrogen-responsive PR gene. The ability of NM23-H1 to interact with ER{alpha}, increase ER{alpha}-ERE binding, and influence ER{alpha}-mediated transcription suggested that NM23-H1 might associate with the regulatory regions of estrogen-responsive genes to influence transcription. ChIP assays were done with MCF-7 cells that had been treated with ethanol vehicle or 10 nmol/L E2. Hormone treatment elicited a dramatic increase in the association of ER{alpha} with the ERE-containing region of the pS2 gene (Fig. 6A ), but no change was observed in the association of NM23-H1 with this region of the pS2 gene in the absence or in the presence of hormone (Fig. 6B). This failure of NM23-H1 to associate with the pS2 gene is consistent with the inability of NM23-H1 to alter pS2 gene expression in our siRNA experiments (Fig. 5). We also determined whether NM23-H1 might associate with a region of the PR gene that we previously showed associates with ER{alpha}, c-Fos, and c-Jun and helps confer estrogen responsiveness to the PR gene, the +90 AP-1 site (37). More ER{alpha} and NM23-H1 were associated with this region of the PR gene in the presence than in the absence of E2 (Fig. 6B). Furthermore, more ER{alpha} and NM23-H1 were associated with the +90 AP-1 site than with the control (36B4) gene in the absence or in the presence of hormone. Combined with our siRNA experiments, which showed that PR gene expression was altered by decreased NM23-H1 expression, our findings suggest that NM23-H1 influences PR gene expression by associating with gene regions involved in conferring estrogen responsiveness.


Figure 6
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  		Figure 6. NM23-H1 association with endogenous estrogen-responsive genes. MCF-7 cells were treated with ethanol vehicle or 10 nmol/L E2 for 2 h. Chromatin was prepared and immunoprecipitated with an antibody directed against ER{alpha} (A) or NM23-H1 (B). DNA was isolated and amplified by quantitative PCR to determine the association of ER{alpha} and NM23-H1 with the ERE-containing region of the pS2 gene or the region of the PR gene containing the +90 AP-1 site. Standard curves were derived for each primer set in each experiment, and the relative number of copies was calculated from the standard curve. Data from three independent experiments, which had been done in triplicate, were combined and are presented as the mean ± SE. *, P < 0.05, statistical differences in the association of ER{alpha} and NM23-H1 with estrogen-responsive genes compared with the control (36B4) gene were determined with SAS 9.1 using a one-way ANOVA with a post hoc Student's t test. Light- and dark-gray bars, ethanol and E2 treatment, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NM23-H1 is a multifunctional protein that has been identified as a metastasis suppressor, a histidine protein kinase, and a granzyme A–activated DNase (17–20). We have now identified additional new functions for NM23-H1. We showed that NM23-H1 interacts with ER{alpha} in vitro and in MCF-7 cells increases ER{alpha}-ERE complex formation, influences ER{alpha}-mediated transcription, and associates with the promoter region of an endogenous estrogen-responsive gene.

The original intent of these studies was to identify ER{alpha}-associated proteins involved in regulating estrogen-responsive gene expression. We discovered that NM23-H1 altered expression of transiently transfected and endogenous, estrogen-responsive genes. When endogenous NM23-H1 expression was decreased in MCF-7 cells, the E2-induced levels of Bcl-2, cathepsin D, PR, and cyclin D1 mRNA were enhanced. In contrast, pS2 mRNA levels were unaffected by reduced NM23-H1 expression, suggesting that although NM23-H1 alters expression of a number of estrogen-responsive genes, its effects are not universal. Furthermore, we have shown that NM23-H1 associates with the region of the PR gene containing the +90 AP-1 site, yet does not interact with the ERE-containing region of the pS2 gene. These studies underscore the complex nature of differentially regulating an entire genome of estrogen-responsive genes in a single cell and reflect the gene-specific contributions of multiple cis-elements and their associated coregulatory proteins in determining the responsiveness of individual genes to hormonal cues.

NM23-H1 has been identified by a number of groups as a member of the multiprotein SET or INHAT complex (20, 21, 38). We also identified four other members of this complex, template activating factor-Iß (TAF1ß), pp32, apurinic endonuclease (APE1), and high mobility group 2 (HMG2), and characterized the abilities of TAF1ß and pp32 to interact with ER{alpha} and influence ER{alpha}-mediated transcription (14, 15). In addition, we and others have shown that HMG1/2 alters ER{alpha} activity and transactivation (28, 39, 40). Thus, the individual and collective actions of these SET complex members may help to differentially regulate expression of the estrogen-responsive genes in target cells. However, the ability of SET complex proteins to modulate gene expression is not limited to ER{alpha} and has been reported for a number of other nuclear receptors as well (14, 15, 41).

It has been suggested that NM23-H1 plays a role in DNA repair (21). Interestingly, we have identified other DNA repair proteins, 3-methyladenine DNA glycosylase (MPG) and flap endonuclease (FEN-1), which associate with ER{alpha} and influence estrogen-responsive gene expression (22, 29). Like NM23-H1, MPG and FEN-1 increase ER{alpha}-ERE binding in gel mobility shift assays and decrease transcription of a reporter plasmid in transient transfection assays. The DNA repair protein O(6)-methylguanine-DNA methyltransferase has also been identified as a regulator of ER{alpha}-mediated transcription (42). These combined studies suggest that ER{alpha}-mediated gene expression and DNA repair may be functionally linked. A common feature of transcription and DNA repair is that both processes depend on chromatin remodeling to make the DNA accessible to transcription factors and DNA repair proteins (43). Coupling of these two cellular processes is apparent in the decreased transcription and compromised genomic integrity that results when damaged DNA is not repaired (44, 45).

NM23-H1 was originally identified as a protein involved in decreasing metastasis in a mouse tumor model (16, 17). A loss of NM23-H1 expression has been linked to increased metastasis of breast, gastric, ovarian, cervical, and liver cancers (36), and a number of studies have shown that reduced expression of NM23-H1 in mammary tumors is linked to decreased survival (36, 46). However, the mechanism by which NM23-H1 limits metastasis is unclear.

Elegant studies by J. Lieberman et al. (20, 21) have suggested two ways in which NM23-H1 might be involved in regulating metastasis. First, NM23-H1 has the intrinsic capacity to nick DNA and, in so doing, to initiate DNA repair and limit aberrant cell migration. Second, metastasis could be avoided altogether by detecting and eliminating mammary tumor cells before they begin to migrate. The DNase activity of NM23-H1 could help to ensure that a tumor cell proceeds toward cell death, thereby limiting metastasis (20, 21).

Another way that NM23-H1 might decrease metastasis is through NM23-H1–induced phosphorylation of the kinase suppressor of Ras, which in turn inhibits Ras-mediated phosphorylation and signaling (18). Although we tested the ability of NM23-H1 to alter phosphorylation of ER{alpha}, we were unable to detect any NM23-H1–induced changes in receptor phosphorylation (data not shown). It is possible, however, that NM23-H1 may alter the phosphorylation state of other ER{alpha}-associated proteins and thereby influence ER{alpha}-mediated transcription and metastasis.

We have uncovered a novel process through which NM23-H1 might influence metastasis. Our studies showed that decreased expression of NM23-H1 led to increased expression of the acidic lysosomal protease cathepsin D, which plays a role in the degradation of the extracellular matrix and tumor cell invasion, and Bcl-2, which inhibits apoptosis, enhances angiogenesis, and helps to provide the vascular support needed to sustain newly colonized metastatic tumor cells (47–50). Thus, our studies would predict that decreased expression of NM23-H1 in ER{alpha}-positive mammary tumor cells would enhance the E2-induced expression of Bcl-2 and cathepsin D and thereby limit apoptosis, favor cell migration, increase angiogenesis, and ultimately enhance metastasis. This is a prediction that is consistent with current clinical evidence.


   Acknowledgments
 
Grant support: NIH grants R01 DK 53884 (to A.M. Nardulli) and P41 RR11823-10 (to J.R. Yates).

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.

We thank P. Steeg and B. Katzenellenbogen for expression vectors; L. Kraus and J. Kadonaga for viral stock used in ER{alpha} production; and J. Lieberman, A. Rao, and S. Marzouk for advice and helpful discussions.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

4 V.S. Likhite and A.M. Nardulli, unpublished data. Back

Received 1/ 4/07. Revised 8/ 8/07. Accepted 8/31/07.


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 Introduction
 Materials and Methods
 Results
 Discussion
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