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[Cancer Research 65, 3586-3595, May 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Breast Cancer Metastasis Suppressor 1 Inhibits Gene Expression by Targeting Nuclear Factor-{kappa}B Activity

Muzaffer Cicek1, Ryuichi Fukuyama1, Danny R. Welch2, Nywana Sizemore1 and Graham Casey1

1 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Lerner School of Medicine, Cleveland, Ohio and 2 Department of Pathology, Comprehensive Cancer Center, and National Foundation for Cancer Research Center for Metastasis Research, University of Alabama at Birmingham, Birmingham, Alabama

Requests for reprints: Graham Casey, Department of Cancer Biology, ND50, Lerner Research Institute, Cleveland Clinic Lerner School of Medicine, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: 216-445-9754; Fax: 216-445-0610; E-mail: caseyg{at}ccf.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Breast cancer metastasis suppressor 1 (BRMS1) functions as a metastasis suppressor gene in breast cancer and melanoma cell lines, but the mechanism of BRMS1 suppression remains unclear. We determined that BRMS1 expression was inversely correlated with that of urokinase-type plasminogen activator (uPA), a prometastatic gene that is regulated at least in part by nuclear factor-{kappa}B (NF-{kappa}B). To further investigate the role of NF-{kappa}B in BRMS1-regulated gene expression, we examined NF-{kappa}B binding activity and found an inverse correlation between BRMS1 expression and NF-{kappa}B binding activity in MDA-MB-231 breast cancer and C8161.9 melanoma cells stably expressing BRMS1. In contrast, BRMS1 expression had no effect on activation of the activator protein-1 transcription factor. Further, we showed that suppression of both constitutive and tumor necrosis factor-{alpha}–induced NF-{kappa}B activation by BRMS1 may be due to inhibition of I{kappa}B{alpha} phosphorylation and degradation. To examine the relationship between BRMS1 and uPA expression in primary breast tumors, we screened a breast cancer dot blot array of normalized cDNA from 50 breast tumors and corresponding normal breast tissues. There was a significant reduction in BRMS1 mRNA expression in breast tumors compared with matched normal breast tissues (paired t test, P < 0.0001) and a general inverse correlation with uPA gene expression (P < 0.01). These results suggest that at least one of the underlying mechanisms of BRMS1-dependent suppression of tumor metastasis includes inhibition of NF-{kappa}B activity and subsequent suppression of uPA expression in breast cancer and melanoma cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer metastasis suppressor 1 (BRMS1) is a known metastasis suppressor gene, and high expression of BRMS1 in both breast cancer and melanoma cell lines significantly reduces their metastatic potential without affecting primary tumor growth (1). BRMS1 is a nuclear protein that contains an imperfect leucine zipper motif and coiled-coiled domains (1), suggesting that it may function as part of a transcriptional complex. Recent studies suggest that BRMS1 inhibits metastasis in part through gene regulation via interaction with histone deacetylases (HDAC; refs. 2, 3). However, the underlying mechanism of BRMS1 metastasis gene regulation remains unclear.

An important component of metastatic dissemination is the proteolysis and degradation of the extracellular matrix (ECM), and among the key mediators of ECM remodeling is the urokinase-type plasminogen activator (uPA), a serine protease that stimulates the conversion of inactive plasminogen to the broad-spectrum protease plasmin (4, 5). Plasmin mediates cellular invasion directly by degrading members of the matrix proteins (6, 7) and indirectly by activating matrix metalloproteinases (MMP; ref. 8). Several groups have shown that uPA plays a critical role in tumor metastasis (9) and that elevated levels of uPA as well as its inhibitor plasminogen activator inhibitor-1 (PAI-1) are strong indicators of poor prognosis in breast cancers (10–14).

Recent studies have shown that the nuclear factor-{kappa}B (NF-{kappa}B) transcription factor positively regulates uPA expression as well as several other genes implicated in angiogenesis and metastasis (15–17). NF-{kappa}B is a heterodimeric transcription factor composed predominantly of p50 (NF-B1) and p65 (RelA) subunits and activation is regulated through interaction with the I{kappa}B{alpha} kinase (IKK) complex (18–20). NF-{kappa}B is sequestered in the cytoplasm through its interaction with inhibitors of NF-{kappa}B (I{kappa}B). Following certain stimuli, a cascade of events leads to the phosphorylation of I{kappa}B by the IKK complex and subsequent degradation of I{kappa}B through ubiquitination. This in turn leads to the liberation of NF-{kappa}B and its translocation to the nucleus where it transactivates NF-{kappa}B-responsive genes (21–24). NF-{kappa}B is constitutively activated in many cancers, and several studies suggest that it plays a critical role in apoptosis, deregulation of cell growth, and propensity of tumors to metastasize (16, 17, 25–32).

The present study was done to determine mechanisms underlying the suppression of metastasis by BRMS1 in human breast cancer and melanoma cells. We show that highly invasive MDA-MB-231 breast cancer and C8161.9 melanoma cell lines show constitutive NF-{kappa}B activity and that BRMS1 expression inversely correlates with suppression of constitutive and tumor necrosis factor-{alpha} (TNF-{alpha})–induced activation of NF-{kappa}B-dependent uPA gene expression. Furthermore, BRMS1 seems to suppress NF-{kappa}B activity by blocking I{kappa}B{alpha} phosphorylation and degradation. Finally, we show a general inverse correlation between BRMS1 and uPA expression in primary breast tumors. These data suggest that BRMS1 regulates metastatic potential at least in part through the down-regulation of NF-{kappa}B-dependent metastasis-related gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The MDA-MB-231 estrogen receptor–negative human breast cancer cell line is highly metastatic to the lung when injected i.v. (33). The C8161 amelanotic human melanoma cell line is metastatic to the lung when injected s.c., i.d., or i.v. in nude mice (34). The C8161.9 is a highly metastatic clone obtained by limiting dilution cloning of parental C8161 cells (35). MDA-MB-231 and C8161.9 cells were grown in DMEM (Life Technologies, Grand Island, NY) supplemented with 5% FCS, 1% L-glutamine, and 1% penicillin and streptomycin in 5% CO2 and 95% air at 37°C. All cultures were tested and shown free of Mycoplasma contamination. MDA-MB-231 cells were passaged at 80% to 90% confluence using a solution of 0.125% trypsin and 2 mmol/L EDTA in Ca2+/Mg2+-free DMEM. C8161.9 cells were passaged at 80% to 90% confluence using Ca2+/Mg2+-free PBS containing 2 mmol/L EDTA. Full-length BRMS1 cDNA was cloned into the mammalian constitutive expression vector pcDNA3 (Invitrogen, San Diego, CA) and transfected stably into parental cells by electroporation (GenePulser, Bio-Rad, Hercules, CA; refs. 36, 37). Control cells were transfected with empty pcDNA vector. The vector control MDA-MB-231/pcDNA was a single transfectant that was shown to retain a high metastatic phenotype equivalent to parental cells (1). The vector control C8161.9/pcDNA is a pooled transfectant source shown to retain a highly metastatic phenotype similar to parental cells (37).

Electrophoretic mobility shift assay and nuclear factor-{kappa}B activation. Electrophoretic mobility shift assay (EMSA) was done to determine the effect of BRMS1 expression on NF-{kappa}B activation (38). Briefly, nuclear and cytoplasmic proteins were extracted from exponentially growing cells, and protein concentration was quantified using BCA protein assay (Pierce, Rockford, IL). Nuclear proteins (10 µg) were incubated at room temperature for 20 minutes with 32P-end-labeled nucleotide derived from a NF-{kappa}B binding sequence (5'-AGTTGAGGGGACTTTCCCAGG-3') from the immunoglobulin gene promoter (38) and the activator protein-1 (AP-1) transcription factor consensus oligonucleotide (5'-CGCTTGATGAGTCAGCCGGAA-3'; Promega, Madison, WI). For TNF-{alpha}–dependent NF-{kappa}B activation, exponentially growing cells were rinsed twice with 1x PBS and treated with TNF-{alpha} (20 ng/mL) in serum-free DMEM for various times (0-2 hours). Nuclear and cytoplasmic protein extracts were isolated and EMSA was done as described above. Samples were resolved on 5% native polyacrylamide gels, which were dried and exposed to Kodak BioMax film (Rochester, NY) at –80°C.

I{kappa}B{alpha} phosphorylation and degradation. The effect of BRMS1 expression on I{kappa}B{alpha} phosphorylation and degradation with and without TNF-{alpha} (20 ng/mL) treatment was examined in a time-dependent manner (0-2 hours) in cells stably expressing exogenous BRMS1. Cytoplasmic protein extracts were prepared from untreated and TNF-{alpha} (20 ng/mL)–treated cells and quantitated and 50 µg were resolved on 12% SDS-polyacrylamide gels. Following electrotransfer, polyvinylidene difluoride (PVDF) filters were probed with rabbit polyclonal antibodies specific to I{kappa}B{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-I{kappa}B{alpha} (Ser32; Cell Signaling Technology, Inc., Beverly, MA), and ß-actin (Sigma-Aldrich, St. Louis, MO), and immunoreactive proteins were detected by enhanced chemiluminescence (ECL; Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions.

Transient expression of breast cancer metastasis suppressor 1 and ssI{kappa}B{alpha}. Transient transfections were done on exponentially growing cells using LipofectAMINE 2000 (Invitrogen) in serum-free DMEM according to the manufacturer's instructions with pcDNA.901BRMS1 and ssI{kappa}B{alpha} (ssI{kappa}B{alpha} is a mutant I{kappa}B{alpha} containing serine-to-alanine substitutions at amino acid positions 32 and 36) containing plasmids at a range of concentrations (0-5 µg). Transfected cells were incubated in serum-free DMEM for 24 hours and medium was collected for uPA and PAI-1 analysis.

Urokinase-type plasminogen activator activity assay. uPA activity was assayed using the synthetic substrate Pyro-Glu-Gly-Arg-pNA.HCl (Chemicon, Temecula, CA) in conditioned medium from MDA-MB-231 and C8161.9 cells following 12-hour treatment with or without TNF-{alpha} (20 ng/mL). Cells were grown in serum-free DMEM over a time course (0-24 hours) and supernatant was collected and centrifuged at 4,000 x g for 10 minutes. The supernatant was concentrated 10-fold using Amicon ultra 10K filters (Millipore, Bedford, MA) at 4,000 x g for 30 minutes. The supernatant (80 µL) was incubated with 100 µL buffer containing 100 mmol/L Tris and 0.5% Triton X-100 (pH 8.8) and 20 µL substrate for 24 hours at 37°C in 96-well microplates. Reactions were monitored at 405 nm using a microplate reader.

Northern blot analysis. Total RNA was extracted from cell lines with TRIzol reagent (Life Technologies) according to the manufacturer's instructions. RNA (15 µg) was separated on 1.5% agarose gels containing 2.2 mol/L formaldehyde, transferred to nylon membranes (Nytran Super Charge, Schleicher & Schuell, Dassel, Germany), and UV cross-linked. Probes for BRMS1 and uPA were amplified by PCR from MCF-7- and MDA-MB-231-derived cDNA, respectively. PCR products were radiolabeled with [32P]dATP by random priming using a DNA labeling kit (Ambion Inc., Austin, TX). Northern blot hybridization was done using QuickHyb (Clontech Laboratories, Inc., Palo Alto, CA) following the manufacturer's instructions. After hybridization, membranes were washed twice in 0.2% SSC (1x SSC is 0.15 mol/L NaCl/0.015 mol/L sodium citrate) containing 0.1% (w/v) SDS at room temperature for 1 hour and finally in 0.1% SSC/0.1% SDS at 50°C for 45 minutes. Membranes were exposed overnight at –80°C to Kodak BioMax film. RNA levels were quantified and normalized against 28S RNA for loading.

Real-time PCR quantification. The following uPA primers were designed using Primer Express version 1.5 (Applied Biosystems, Foster City, CA): uPA (forward) 5'-GCCTTGCTGAAGATCCGTTC-3' and uPA (reverse) 5'-GGATCGTTATACATCGAGGGGCA-3'. A single PCR-amplified band was observed on ethidium-stained gels using standard PCR conditions. Real-time PCR experiments were done using the SYBR Green PCR Core kit (Applied Biosystems) according to the vendor's instructions and an ABI 7900HT (Applied Biosystems) real-time PCR instrument. uPA expression levels were calibrated using endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels as an internal control. The following GAPDH primers were designed using Primer Express version 1.5: GAPDH (forward) 5'-GAAGGTGAAGGTCGCAGT-3' and GAPDH (reverse) 5'-GAAGATGGTGATGGGATTTC-3'. Cycle conditions were 95°C for 10 minutes (AmpliTaq Gold activation) followed by 45 cycles of 95°C for 15 seconds (denaturation) and 60°C for 60 seconds (annealing and extension) and fluorescence was quantified as a Ct value. The differences between the mean Ct values of uPA, I{kappa}B{alpha}, and GAPDH were denoted ({Delta}Ct) for each time course and the difference between {Delta}Ct and the {Delta}Ct value of the GAPDH was calculated as {Delta}{Delta}Ct. The log2({Delta}{Delta}Ct) gave the relative quantification value of uPA expression.

Western blot analysis. Exponentially growing cells were rinsed twice with 1x PBS and lysed in buffer containing 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 1% Triton X-100, and 1x cocktail of proteinase inhibitors (Boehringer, Mannheim, Germany). Protein concentration was determined using the BCA protein assay kit. Proteins were separated on 12% SDS-PAGE gels. The resolved proteins were electroblotted on to PVDF membranes (Millipore) using transfer buffer containing 48 mmol/L Tris, 39 mmol/L glycine, 0.1% SDS, and 20% methanol at pH 9.2. Nonspecific binding was blocked by incubation in 1x PBS containing 5% fat-free dry milk for 1 hour at room temperature. Blots were incubated with the primary antibodies specific to p65 (Santa Cruz Biotechnology), p50 (Santa Cruz Biotechnology), phospho-p65 (Cell Signaling Technology), uPA (Santa Cruz Biotechnology), and PAI-1 (Santa Cruz Biotechnology) for 1 hour and subsequently horseradish peroxidase–conjugated secondary antibody (Amersham Biosciences) for 1 hour at room temperature. Signals were visualized using the ECL detection system according to the manufacturer's instructions. To monitor secreted uPA, cells were grown in serum-free medium with or without treatment for 24 hours. Supernatant was concentrated using Amicon 30 microconcentration columns and total protein was extracted. Western blots were done as above and equal loading of protein was determined by estimating the total number of cells at the end of the experiment.

Adenovirus infections. Parental cells and BRMS1 transfectants were infected with adenovirus expressing IKKß and green fluorescence protein (GFP; kindly provided by Frank Mercurio, Celgene, San Diego, CA) for 36 hours in Opti-MEM medium (Life Technologies) at 104 plaque-forming units/mL. After 36 hours, medium was collected and concentrated 10-fold using Amicon ultra 10K filters at 4,000 x g for 30 minutes for Western blot analysis. The remaining infected cells were washed twice with 1x PBS and harvested for total protein. Expression of IKK and GFP was examined by the Western blot analysis using polyclonal antibodies to IKK and GFP.

Cancer profiling arrays. The cancer profiling array membrane (Clontech Laboratories) was hybridized with 32P-labeled cDNA probes according to the manufacturer's instructions. Briefly, the membrane was prehybridized for 30 minutes with 10 mL ExpressHyb solution (Clontech Laboratories) at 65°C. The 32P-labeled cDNA probes were mixed with 30 µg of Cot-1 DNA (Roche Molecular Biochemicals, Indianapolis, IN), 150 µg of sheared salmon sperm DNA, and 50 µL of 20x SSC in a total volume of 200 µL. The mixture was heated to 95°C for 5 minutes, incubated at 65°C for 30 minutes, and mixed with 5 mL fresh ExpressHyb solution. The filter was hybridized overnight at 65°C and washed four times at 65°C for 20 minutes with 2x SSC-0.5% SDS, once with 0.2x SSC-0.5% SDS, and finally with 2x SSC for 5 minutes at room temperature. For consecutive hybridizations, the filter was stripped at 68°C using probe degradation and removal reagents (Ambion) and hybridized with a human uPA cDNA probe followed by a human ubiquitin cDNA probe as a quantitation control. Filters were washed and exposed to X-ray film. For quantitation of gene expression, the film was scanned using Storm imager (Molecular Dynamics), and densitometric values of the spots were calculated using ImageQuant 1.2 software (Amersham Biosciences, Sunnyvale, CA).

Statistical analysis. Gene expression levels determined by PhosphorImager analysis of BRMS1 and uPA in normal and tumor tissues were compared with paired t test using the Statview (J4.02; Abacus Concepts, Berkley, CA) computer program (39). P was set at <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Breast cancer metastasis suppressor 1 and urokinase-type plasminogen activator gene expression levels are inversely correlated. To provide insight into possible mechanisms underlying BRMS1-regulated metastasis suppression, we examined the expression of uPA, a key metastasis-related gene implicated strongly in breast cancer metastasis. Here, we show an inverse correlation between BRMS1 mRNA expression and uPA mRNA and protein expression (Fig. 1). BRMS1 and uPA mRNA expression was examined in highly metastatic breast cancer cell line MDA-MB-231/pcDNA and melanoma-derived C8161.9/pcDNA cells and compared with mRNA levels in clonally derived stably expressing BRMS1 transfectants MDA-MB-231/BRMS1-11 and MDA-MB-231/BRMS1-13 and C8161.9/BRMS1-6 and C8161.9/BRMS1-11, respectively, which exhibited low metastatic potential (1, 37). Northern blot analysis revealed that uPA mRNA expression was reduced in BRMS1 transfectants compared with parental cells and that the extent of reduction in expression generally correlated with levels of BRMS1 mRNA expression. Secreted uPA protein levels in supernatants were examined from the same cells grown for 24 hours. A similar inverse correlation between BRMS1 mRNA expression and levels of secreted uPA protein was observed. Parental MDA-MB-231 and C8161.9 cells exhibited similar levels of BRMS1 and uPA mRNA expression to MDA-MB-231/pcDNA and C8161.9/pcDNA cells, respectively (data not shown).



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  		Figure 1. BRMS1 expression and uPA expression are inversely correlated. Top, level of mRNA expression of uPA was examined by Northern blot analysis in MDA-MB-231/pcDNA and C8161.9/pcDNA cells and two MDA-MB-231/BRMS1 and two C8161.9/BRMS1 transfectants stably expressing BRMS1. Total mRNA (15 µg) was run on formaldehyde-agarose gels, transferred on nylon membrane, and UV cross-linked. The membrane was probed with 32P-labeled uPA cDNA, stripped, and reprobed with 32P-labeled BRMS1 cDNA. Bottom, secreted uPA protein was shown by Western blot analysis in the same cells described above. Cells were grown in serum-free medium for 24 hours. Supernatant was concentrated using Amicon 30 microconcentration columns and total protein was extracted. Equivalent amounts of protein were loaded in each lane based on the total cell numbers. Proteins were separated by SDS-PAGE and electroblotted on to PVDF membranes. Blots were incubated with uPA-specific antibody (Santa Cruz Biotechnology) and visualized using the ECL detection system.

 
NF-{kappa}B has been shown previously to regulate uPA expression in MDA-MB-231 cells (16), suggesting that BRMS1 may affect uPA expression at least in part directly or indirectly through interaction with NF-{kappa}B. NF-{kappa}B is activated in cells in response to several extracellular signals, including treatment with TNF-{alpha}. Therefore, to further examine the potential relationship between BRMS1 and NF-{kappa}B, we determined whether BRMS1 expression inhibited NF-{kappa}B–dependent uPA induction by TNF-{alpha}. In Fig. 2A, we show that induction of uPA mRNA expression was reduced and/or delayed in cells expressing high levels of BRMS1 compared with parental MDA-MB-231/pcDNA and C8161.9/pcDNA cells. In these experiments, we examined levels of mRNA expression using real-time PCR amplification following exposure of cells to TNF-{alpha} for different lengths of time. uPA gene expression was induced by TNF-{alpha} in MDA-MB-231/pcDNA and C8161.9/pcDNA cells as early as 1 hour following exposure, whereas induction was delayed and/or reduced in both MDA-MB-231/BRMS1-13 and C8161.9/BRMS1-11 transfectants. GAPDH was used as an internal control and calibrator in these experiments. Experiments were done in triplicate.



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  		Figure 2. A, TNF-{alpha}–induced, NF-{kappa}B–dependent uPA mRNA expression is suppressed by BRMS1. Total RNA was isolated from exponentially growing cells treated with TNF-{alpha} for 0 to 6 hours and total RNA (2 µg) was reverse transcribed. At the indicated time points, gene expression of uPA was analyzed by real-time quantitative PCR and normalized to GAPDH expression as described in Materials and Methods. Relative expression levels of uPA and GAPDH were determined in highly metastatic MDA-MB-231/pcDNA and C8161.9/pcDNA cells and metastasis-suppressed transfectants MDA-MB-231/BRMS1-13 and C8161.9/BRMS1-11 expressing high levels of BRMS1. B, TNF-{alpha} induction of NF-{kappa}B–dependent uPA protein expression is inhibited by BRMS1. Following exposure of the same cells to increasing concentrations of TNF-{alpha}, total secreted uPA protein was determined by Western blot analysis.

 
We next determined whether BRMS1 affected dose-dependent TNF-{alpha} induction of NF-{kappa}B–dependent uPA protein expression and secretion (Fig. 2B). MDA-MB-231/pcDNA and C8161.9/pcDNA and BRMS1 transfectants were exposed to increasing amounts of TNF-{alpha} for 24 hours. Western blot analyses revealed a TNF-{alpha} dose-dependent increase in the level of secreted uPA protein in MDA-MB-231/pcDNA and C8161.9/pcDNA cells, which was substantially reduced in both MDA-MB-231/BRMS1-13 and C8161/BRMS1-11 transfectants expressing high levels of BRMS1. TNF-{alpha} induced uPA protein expression as early as 6 hours in MDA-MB-231 cells and as early as 3 hours in C8161.9 cells (Fig. 3A). BRMS1-expressing MDA-MB-231 and C8161.9 cells showed substantially reduced uPA protein expression compared with parental cells. In addition, we also examined the effect of BRMS1 on TNF-{alpha}–induced uPA activity using the synthetic uPA substrate Pyro-Glu-Gly-Arg-pNA.HCl (Fig. 3B). Here, we also observed a BRMS1-dependent reduction in uPA enzyme activity over 12 hours in both MDA-MB-231/BRMS1-13 and C8161.9/BRMS1-11 cells compared with parental cells.



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  		Figure 3. Constitutive and TNF-{alpha}–dependent NF-{kappa}B activation of uPA activity is inhibited by BRMS1 expression. TNF-{alpha}–dependent NF-{kappa}B activation of uPA activity was examined in MDA-MB-231/pcDNA, C8161.9/pcDNA, and BRMS1-expressing cells treated with TNF-{alpha} (20 ng/mL) for 0 to 12 hours in serum-free medium. A, BRMS1-expressing cells show a marked reduction in TNF-{alpha}–dependent uPA protein secretion compared with parental cells in both MDA-MB-231 and C8161.9 cells. Relative levels of secreted uPA protein were detected by Western blot analysis. B, BRMS1-expressing cells show a marked reduction in TNF-{alpha}–dependent uPA enzyme activity compared with parental cells at all doses of TNF-{alpha}. uPA activity was determined using a synthetic substrate Pyro-Glu-Gly-Arg-pNA.HCl in MDA-MB-231, C8161.9, and BRMS1-expressing cells. Relative levels of uPA activity in parental and BRMS1 transfectants are shown graphically.

 
Breast cancer metastasis suppressor 1 suppresses constitutive and tumor necrosis factor-{alpha}–induced nuclear factor-{kappa}B activity in breast and melanoma cancer cell lines. In the present study, we showed that both MDA-MB-231 and C8161.9 cells showed constitutive NF-{kappa}B DNA binding activity (data not shown). To examine the effect of BRMS1 expression on NF-{kappa}B binding activity, we did EMSA. We determined that constitutive NF-{kappa}B binding activity was suppressed in BRMS1 transfectants compared with MDA-MB-231/pcDNA and C8161.9/pcDNA cells (Fig. 4) and nontransfected parental cells (data not shown). In general, the level of suppression of NF-{kappa}B binding activity inversely correlated with the level of BRMS1 mRNA expression in both MDA-MB-231 and C8161.9 cells. Nuclear protein extracts were prepared from exponentially growing cells following growth of cells for 24 hours in serum-free medium and treatment with or without TNF-{alpha} and subjected to EMSA. Activation of NF-{kappa}B DNA binding was observed within 20 minutes of treatment with TNF-{alpha} in both parental breast cancer and melanoma cell lines, whereas BRMS1 transfectants showed a marked delay and reduction in TNF-{alpha}–induced NF-{kappa}B activation (Fig. 4).



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  		Figure 4. NF-{kappa}B DNA binding activity but not AP-1 binding activity is inhibited by BRMS1 expression. Nuclear proteins were extracted from exponentially growing MDA-MB-231/pcDNA, C8161.9/pcDNA, and corresponding BRMS1 transfectants treated with or without TNF-{alpha} (20 ng/mL) for increasing lengths of time in serum-free medium. Nuclear proteins (10 µg) were incubated with 32P-end-labeled NF-{kappa}B oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGG-3') from the immunoglobulin gene promoter and an AP-1 consensus oligonucleotide (5'-CGCTTGATGAGTCAGCCGGAA-3') and EMSA was done. NF-{kappa}B and AP-1 DNA binding activities were examined in the same nuclear extracts. NS, nonspecific interaction. Left, MDA-MB-231/pcDNA and transfectant MDA-MB-231/BRMS1-13. NF-{kappa}B DNA binding activity but not AP-1 binding activity was reduced and delayed in BRMS1-expressing cells. Right, C8161.9/pcDNA and transfectant C8161.9/BRMS1-11. As with MDA-MB-231, in C8161.9 cells, NF-{kappa}B DNA binding activity but not AP-1 binding activity was substantially reduced and delayed in BRMS1-expressing cells.

 
The AP-1 transcription factor has also been reported to regulate uPA gene expression in MDA-MB-231 cells on treatment with insulin-like growth factor-I (40). To confirm the specificity of suppression of NF-{kappa}B binding activity by BRMS1, we also determined the effect of BRMS1 expression on the DNA binding activity of AP-1 (Fig. 4). BRMS1 expression had no effect on AP-1 DNA binding activity in the presence or absence of TNF-{alpha}.

Breast cancer metastasis suppressor 1 inhibits I{kappa}B{alpha} phosphorylation and degradation. Phosphorylation and subsequent proteolytic degradation of I{kappa}B{alpha} facilitates NF-{kappa}B translocation to the nucleus where NF-{kappa}B in turn binds to consensus sequences on promoters and activates NF-{kappa}B-regulated genes. We therefore examined whether constitutive I{kappa}B{alpha} phosphorylation and degradation was reduced by BRMS1 expression. We found that there was a reduction in I{kappa}B{alpha} phosphorylation in transfectants compared with MDA-MB-231/pcDNA and C8161.9/pcDNA cells (Fig. 5), consistent with the reduction in constitutive NF-{kappa}B activation seen in these cells.



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  		Figure 5. I{kappa}B{alpha} phosphorylation was inhibited by BRMS1 expression. MDA-MB-231/pcDNA, C8161.9/pcDNA, and corresponding BRMS1 transfectants were treated with or without TNF-{alpha} (20 ng/mL) for different lengths of time (0-2 hours) in serum-free medium. Cytosolic protein fractions were extracted from exponentially growing cells and analyzed by Western blot analysis using antibodies specific for I{kappa}B{alpha} and phospho-I{kappa}B{alpha} (Ser32). Equal protein loading was confirmed using ß-actin antibody. Left, on TNF-{alpha} activation of NF-{kappa}B, there is a time-dependent increase in phospho-I{kappa}B{alpha}, which was substantially reduced in BRMS1-expressing MDA-MB-231/BRMS-13 cells. Right, similar data for C8161.9 cells. Although there is an increase in phospho-I{kappa}B{alpha} following TNF-{alpha} incubation, this increase is reduced relative to parental cells in BRMS1-expressing cells.

 
To determine whether BRMS1 inhibition of constitutive and TNF-{alpha}–induced NF-{kappa}B binding was due to the inhibition of proteolytic degradation of I{kappa}B{alpha}, we examined cytoplasmic I{kappa}B{alpha} phosphorylation and degradation by Western blot analysis using I{kappa}B{alpha} and phospho-I{kappa}B{alpha} antibodies (Fig. 5). Whereas I{kappa}B{alpha} phosphorylation and degradation was seen over time in MDA-MB-231/pcDNA cells, there was a notable reduction in I{kappa}B{alpha} phosphorylation and degradation in MDA-MB-231/BRMS1-13 cells. We observed little difference in total I{kappa}B{alpha} levels between C8161.9 and C8161.9/BRMS1-11 cells following TNF-{alpha} stimulation (Fig. 5). However, a prolonged I{kappa}B{alpha} degradation was consistently observed in parental C8161.9 compared with C8161.9/BRMS1-11 cells. In addition, we observed reduced I{kappa}B{alpha} phosphorylation in C8161.9/BRMS1-11 compared with parental C8161.9 cells.

We also examined the effect of BRMS1 on TNF-{alpha}–induced p65 phosphorylation and translocation of p65/p50 proteins to the nucleus. Consistent with the effect of BRMS1 expression on I{kappa}B{alpha} phosphorylation and degradation, we observed a time-dependent, TNF-{alpha}–dependent induction of p65 phosphorylation and nuclear translocation of p65/p50 as early as 20 minutes in MDA-MB-231/pcDNA (Fig. 6) and MDA-MB-231 cells (data not shown). In contrast, p65 phosphorylation and p65/p50 translocation were almost completely abolished in MDA-MB-231/BRMS1-13 cells even following treatment with TNF-{alpha}. C8161.9/BRMS1-11 cells also showed a suppression of p65 phosphorylation and p65/p50 translocation to the nuclear fraction when treated with TNF-{alpha} compared with C8161.9/pcDNA cells, although the level of suppression was not as robust as that seen in MDA-MB-231/BRMS1-13 cells (Fig. 6).



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  		Figure 6. Nuclear translocation of NF-{kappa}B/p50 and NF-{kappa}B/p65 is reduced in BRMS1-expressing cells. MDA-MB-231/pcDNA, C8161.9/pcDNA, and corresponding BRMS1-expressing cells were treated with or without TNF-{alpha} (20 ng/mL) for different lengths of time (0-2 hours) in serum-free medium. Nuclear protein was extracted from exponentially growing cells. Western blot analyses were done and filters were probed with antibodies against p50 and phospho-p65 (Ser32). Left, MDA-MB-231 cells show a reduced nuclear p50 fraction in BRMS1-expressing cells with or without TNF-{alpha} treatment. In addition, although there is an increase in phospho-p65 in nuclear extracts following TNF-{alpha} treatment, this is not observed in BRMS1-expressing cells. Right, results for C8161.9/pcDNA. Although not as substantial as in MDA-MB-231 cells, there is a reduction or delay in both constitutive and TNF-{alpha}–induced p50 nuclear protein levels in BRMS1-expressing cells. A similar reduction or trend in both constitutive and TNF-{alpha}–induced phospho-p65 levels is also observed.

 
Transient expression of breast cancer metastasis suppressor 1 and ssI{kappa}B{alpha} and inhibitor studies. To more directly evaluate the effect of BRMS1 on the expression uPA through NF-{kappa}B activity, we did transient transfections of BRMS1 cDNA. The effect of increasing concentrations of BRMS1 cDNA on uPA expression was compared with the effect of inhibiting NF-{kappa}B through mutant I{kappa}B{alpha} (ssI{kappa}B{alpha}). To accomplish this, we transiently expressed increasing concentrations of BRMS1 cDNA or ssI{kappa}B{alpha} in MDA-MB-231 and C8161.9 cells. Western blot analyses were done on proteins isolated from serum-free medium 24 hours following transient transfection. Increasing concentrations of BRMS1 and ssI{kappa}B{alpha} both led to a similar concentration-dependent reduction in uPA protein expression (Fig. 7) and uPA activity (data not shown) compared with untreated MDA-MB-231 and C8161.9 cells. PAI-1 expression levels were also examined and were unaffected by BRMS1 and ssI{kappa}B{alpha} expression. Transfection efficiencies for MDA-MB-231 and C8161.9 were 37% and 43.4%, respectively.



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  		Figure 7. Increasing concentrations of BRMS1-expressing plasmid inhibit uPA protein secretion in a similar manner to inhibiting NF-{kappa}B activation of uPA using mutant I{kappa}B{alpha}-expressing plasmid (ssI{kappa}B{alpha}). Increasing concentrations of pcDNA.901BRMS1 cDNA and mutant I{kappa}B{alpha} plasmid (ssI{kappa}B{alpha}) were transiently transfected into MDA-MB-231, C8161.9, and corresponding BRMS1 transfectants using LipofectAMINE 2000. Transfected cells were grown in serum-free medium for 24 hours. Secreted proteins in supernatants were concentrated and analyzed by Western blot analysis using antibodies specific for uPA and PAI-1 as a loading control. In both MDA-MB-231 and C8161.9 cells, a corresponding dose-dependent reduction in uPA expression was observed in both BRMS1 and ssI{kappa}B{alpha} transfected cells. As expected, there was no effect on NF-{kappa}B-independent PAI-1 secretion.

 
Overexpression of I{kappa}B{alpha} kinase ß activates nuclear factor-{kappa}B and induces urokinase-type plasminogen activator expression in the absence or presence of breast cancer metastasis suppressor 1 expression. To more directly assess whether BRMS1 down-regulates uPA expression through inhibition of NF-{kappa}B signaling, we artificially activated NF-{kappa}B signaling by overexpressing IKKß. This was achieved by infecting cells with adenovirus expressing IKKß or GFP as a negative control. We monitored I{kappa}B{alpha} degradation and NF-{kappa}B-regulated uPA and cyclooxygenase-2 (COX-2) expression in infected parental and BRMS1 cells (Fig. 8). We found that overexpression of IKKß led to I{kappa}B{alpha} degradation and induction of uPA and COX-2 expression, suggesting that IKKß is one of the major factors controlling NF-{kappa}B signaling. These data support a role for BRMS1 in the regulation of uPA through upstream inhibition of NF-{kappa}B signaling.



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  		Figure 8. Activation of NF-{kappa}B signaling through overexpression of IKKß results in an increase in uPA expression in BRMS1-expressing cells. Both parental MDA-MB-231 and C8161.9 cells and BRMS1 transfectants MDA-MB-231/BRMS1-13 and C8161.9/BRMS1-11 cells were infected with adenovirus expressing either IKKß or GFP as negative control for 36 hours with 104 plaque-forming units/mL. After 36 hours, medium was collected and concentrated for analysis of secreted uPA protein. Cells were harvested and cellular protein expression levels of IKKß and GFP were examined by Western blot analysis. We found that all cell lines expressed IKKß and GFP proteins and that overexpression of IKKß leads to I{kappa}B{alpha} phosphorylation and degradation and activation of NF{kappa}B signaling shown by induction of COX-2 expression. Whereas secreted uPA levels in infected parental cells showed limited elevation, infected BRMS1-expressing clones were highly elevated, suggesting that the inhibition of uPA expression by BRMS1 is NF-{kappa}B–dependent.

 
Cancer profiling array: expression of breast cancer metastasis suppressor 1 and urokinase-type plasminogen activator in breast tumors. To determine the relationship between BRMS1 and uPA expression in human cancers, we analyzed a cancer profiling array containing normalized cDNA from tumors and corresponding normal tissues of 241 subjects with different cancers. Included in the array were cDNAs of 50 breast tumors and matched normal breast tissues. We hybridized the array in turn with human BRMS1, uPA, and ubiquitin cDNA probes, and relative expression levels were quantitated using a PhosphorImager (Fig. 9A). We observed a generally consistent reduced expression of BRMS1 only in breast tumors compared with normal tissues, suggesting that loss of BRMS1 expression may be a specific marker for breast cancer progression (melanoma was not represented on the arrays; Fig. 9B). Scatter plot analysis revealed a >2-fold reduction in BRMS1 mRNA expression levels in ~90% of the breast tumor samples compared with matched normal tissues (Fig. 9C). We observed an inverse correlation between BRMS1 and uPA expression in breast tumors with a reduction in BRMS1 mRNA expression levels in 90% (P < 0.0001) of the tumor versus matched normal tissues. In contrast, 88% (P < 0.01) of the breast tumors showed higher uPA mRNA expression levels than corresponding normal tissues (Fig. 9D).



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  		Figure 9. Expression analysis of BRMS1 and uPA are inversely correlated in primary human breast tumors and normal tissues. A, a cancer profiling array containing cDNA from 241 human tumor (T) and corresponding normal (N) tissues from individual patients was probed consecutively with 32P-labeled cDNA probes of BRMS1, uPA, and ubiquitin and relative expression levels were quantitated using PhosphorImager. A, we observed a generally consistent reduced expression of BRMS1 only in breast tumors compared with normal tissues, suggesting that the BRMS1 may only affect breast cancer progression (melanoma was not represented on the arrays). B, breast tumor and normal cDNAs in expanded view to show the inverse correlation between expression levels of BRMS1 and uPA. Ubiquitin is shown as a control for cDNA loading. C, a scatter plot analysis shows a >2-fold reduction in BRMS1 mRNA expression in ~90% of the breast tumor samples. X axis, all corresponding breast tumor ({bullet}) and normal tissue pairs ({circ}) labeled 1 to 50; Y axis, relative PhosphorImager values of BRMS1 and uPA hybridization. D, graphical summary. A general inverse correlation between BRMS1 and uPA mRNA expression is observed in breast tumors. BRMS1 showed reduced mRNA expression levels in 90% of the tumors versus matched normal tissues (P < 0.0001). In contrast, 88% of the breast tumors showed higher uPA mRNA expression levels than corresponding normal tissues (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NF-{kappa}B is known to modulate the expression of genes involved in cancer cell invasion, metastasis, angiogenesis, and apoptosis, and constitutive NF-{kappa}B activation has been reported in several different cancers (26, 30, 31, 41–45). This study is the first to implicate NF-{kappa}B signaling in the suppression of the metastatic phenotype by the BRMS1 metastasis suppressor gene. We show an inverse correlation between BRMS1 expression and that of the NF-{kappa}B-regulated gene uPA in both highly metastatic MDA-MB-231 breast cancer and C8161.9 melanoma cell lines, with elevated BRMS1 expression leading to a reduction in levels of both secreted uPA protein and uPA enzyme activity in these cells. We also show an inverse correlation between BRMS1 expression and both constitutive and TNF-{alpha}–induced NF-{kappa}B binding activity in these cells. Phosphorylation and degradation of I{kappa}B{alpha} facilitates NF-{kappa}B translocation to the nucleus where it binds to promoter consensus sequences to activate NF-{kappa}B-regulated genes, and we show an inverse correlation between BRMS1 expression and levels of constitutive and TNF-{alpha}–induced I{kappa}B{alpha} phosphorylation and degradation. This leads to decreased nuclear translocation of NF-{kappa}B p50 and phospho-p65 subunits that would in turn be expected to reduce expression of NF-{kappa}B-regulated genes, such as uPA. Related to these findings, the expression of the KiSS-1 metastasis suppressor gene has been reported to inhibit NF-{kappa}B-regulated MMP9 gene expression, suggesting that inhibition of NF-{kappa}B signaling may be a common target of metastasis suppressor genes (46).

The underlying mechanism of regulation of NF-{kappa}B signaling by BRMS1 remains to be elucidated. However, our finding that BRMS1 expression leads to the inhibition of I{kappa}B{alpha} phosphorylation and degradation and subsequently to a reduction of p65 and p50 nuclear translocation in both MDA-MB-231 breast cancer and C8161.9 melanoma cells suggests that constitutive and TNF-{alpha}–induced NF-{kappa}B suppression by BRMS1 could be through the classic NF-{kappa}B IKK pathway. To more directly assess the role of BRMS1 in the inhibition of NF-{kappa}B signaling, we activated NF-{kappa}B through overexpressing IKKß. NF-{kappa}B activation through IKKß overexpression resulted in increased uPA expression in both parental and BRMS1-expressing cells, supporting a role for BRMS1 in the upstream inhibition of NF-{kappa}B signaling. BRMS1 is reported to be a nuclear protein, and this result suggests that BRMS1 may have additional cellular functions to those described here.

The AP-1 transcription factor has also been reported to regulate the expression of uPA and cell migration of MDA-MB-231 cells (16). Phosphatidylinositol 3-kinase has been shown to regulate expression of uPA through the activation of both AP-1 and NF-{kappa}B transcription factors (16). However, we found that BRMS1 expression did not affect AP-1 activity in either MDA-MB-231 or C8161.9 cells, implying that NF-{kappa}B and not AP-1 is the major transcription factor responsible for uPA expression in MDA-MB-231 and C8161.9 cells.

NF-{kappa}B, like many other inducible transcription factors, regulates gene expression at least in part through histone acetylation-deacetylation reactions (47). The p65/RelA subunit of NF-{kappa}B has been shown to activate transcription by interacting alternatively with multiple coactivators or corepressor complexes. Among the corepressors, three members of the HDAC class I family of proteins (HDAC1, HDAC2, and HDAC3) modulate NF-{kappa}B transcriptional activity (48–50). TNF-{alpha}–induced, NF-{kappa}B–dependent gene expression has been reported repressed by HDAC1 and HDAC2 through a direct association between HDAC1 and the p65 subunit of NF-{kappa}B (48). HDAC2 seems to exert its influence through its interaction with HDAC1 (48). Recent studies show that BRMS1 interacts with retinoblastoma binding protein-1 and a complex that includes at least seven members of the mammalian Sin3-HDAC complex, including HDAC1 and HDAC2 (3). These data would suggest that BRMS1 could modulate dissociation of NF-{kappa}B from its consensus DNA sequence in part through its interaction with HDAC1 and HDAC2. However, our data support an upstream role for BRMS1 in NF-{kappa}B signaling by inhibiting I{kappa}B{alpha} phosphorylation/degradation. Further studies will be needed to determine whether the effects of BRMS1 on NF-{kappa}B signaling and histone acetylation-deacetylation reactions are related and how these two pathways may interact to ensure the ability of BRMS1 to suppress metastasis.

We observed a strong correlation between expression of BRMS1 and uPA, a key mediator of ECM remodeling, in both breast cancer and melanoma cells. uPA stimulates the conversion of inactive plasminogen to the broad-spectrum serine protease plasmin (4, 5). This in turn mediates cellular invasion by degrading members of the matrix proteins, such as fibronectin, collagen, and laminin, and by activating MMPs, including MMP2, MMP3, and MMP9 (6–8). Through this action, uPA may promote tumor cell migration and invasion. Although the BRMS1-expressing cells exhibit significantly lower uPA activity, we have shown previously that they do not show significantly reduced invasive abilities (1, 51). This is probably due to redundancies of proteolytic activities of many different proteinases in cancer cells, and it is important to note that uPA and other proteinases likely play a role in many different stages of metastasis besides invasion (52). Proteinases, including uPA, are produced by both tumor and stromal cells. Depending on the tumor, the proportions produced by each compartment vary. Our study does not address modifications in stromal production of uPA but only that produced directly by the breast cancer cells. Modification of uPA production by stromal cells in a BRMS1-expressing microenvironment is possible but has not been measured. The data presented here do not delimit the exact step(s) in the metastatic cascade affected by BRMS1 or uPA. Nonetheless, the roles of both molecules are important in metastasis and our data suggest that they converge to some extent.

Elevated expression of uPA has been reported in colon (53), lung (54), prostate (55), and breast (56) cancers, and several studies have implicated uPA in cancer metastasis, particularly that of the breast (10–14, 57). Using a commercial cDNA dot blot assay, we observed a general inverse correlation between BRMS1 and uPA expression in breast tumor samples with a decrease in BRMS1 mRNA levels seen in 90% of the breast cancers compared with their corresponding normal tissues (P < 0.0001). Conversely, 88% of the breast tumor samples showed higher uPA mRNA levels compared with their corresponding normal tissues (P < 0.01).

This general inverse correlation between BRMS1 and uPA expression both in vitro and in vivo in breast cancer cells implies that an important mechanism of BRMS1-mediated metastasis suppression in breast cancer results from its inhibition of NF-{kappa}B-mediated uPA expression. The resulting reduction in uPA expression would be expected in turn to lead to reduced ECM remodeling and a lower metastatic potential. Despite this observation, we observed no consistent reduction in expression of BRMS1 in any other cancers (melanomas were not represented on the cDNA dot blots). This suggests that the effect of BRMS1 on suppression of metastasis may be cancer specific. That we observed a general inverse correlation between BRMS1 and uPA expression in the majority of primary breast tumors suggests that BRMS1 may also play a role in the growth of primary breast cancer, although in vivo tumorigenicity assays showed a limited effect of BRMS1 expression on primary tumor growth. Further studies will be needed to determine the effect of reduced BRMS1 expression, if any, on primary breast tumor growth.

In summary, we showed a correlation between BRMS1 expression and inhibition of both constitutive and TNF-{alpha}–induced NF-{kappa}B activity in both highly invasive MDA-MB-231 breast cancer and C8161.9 melanoma cell lines, which seems to be through the inhibition of I{kappa}B{alpha} phosphorylation and degradation. We also showed an inverse correlation between BRMS1 expression and TNF-{alpha}–induced activation of the NF-{kappa}B–dependent gene uPA expression. These studies suggest that at least some of the ability of BRMS1 to suppress breast cancer and possibly melanoma metastasis may be due to its ability to inhibit NF-{kappa}B–dependent regulation of genes important in the metastatic process, including uPA. Although the mechanism of inhibition of NF-{kappa}B activation has not been determined, our data suggest that this inhibition may involve the classic IKK pathway.


   Acknowledgments
 
Grant support: Lerner Foundation (G. Casey) and NIH grants RO-1 CA100748 (N. Sizemore) and P50-CA89019 (D.R. Welch).

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 8/30/04. Revised 1/13/05. Accepted 2/17/05.


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 Results
 Discussion
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D. G. Hicks, B. J. Yoder, S. Short, S. Tarr, N. Prescott, J. P. Crowe, A. E. Dawson, G. T. Budd, S. Sizemore, M. Cicek, et al.
Loss of Breast Cancer Metastasis Suppressor 1 Protein Expression Predicts Reduced Disease-Free Survival in Subsets of Breast Cancer Patients.
Clin. Cancer Res., November 15, 2006; 12(22): 6702 - 6708.
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Z. Zhang, H. Yamashita, T. Toyama, Y. Yamamoto, T. Kawasoe, and H. Iwase
Reduced expression of the breast cancer metastasis suppressor 1 mRNA is correlated with poor progress in breast cancer.
Clin. Cancer Res., November 1, 2006; 12(21): 6410 - 6414.
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A. Albini, F. Tosetti, R. Benelli, and D. M. Noonan
Tumor Inflammatory Angiogenesis and Its Chemoprevention
Cancer Res., December 1, 2005; 65(23): 10637 - 10641.
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