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Clinical Cancer Research Vol. 6, 2573-2581, June 2000
© 2000 American Association for Cancer Research

Cancer Biology, Immunology, Cytokines

Nuclear Factor-{kappa}B Activity Correlates with Growth, Angiogenesis, and Metastasis of Human Melanoma Cells in Nude Mice1

Suyun Huang, Ariel DeGuzman, Corazon D. Bucana and Isaiah J. Fidler2

Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of this study was to determine the role of nuclear factor (NF)-{kappa}B/relA activity in the induction of angiogenesis and production of metastasis by human melanoma cells. Highly metastatic melanoma variant cells expressed high levels of constitutive NF-{kappa}B activity. Transfection of highly metastatic human melanoma variant cells with a dominant-negative mutant inhibitor of nuclear factor-{kappa}B{alpha} (I{kappa}ß{alpha}) expression vector (I{kappa}ß{alpha}M) decreased the level of constitutive NF-{kappa}B activity, inhibited s.c. tumor growth, and prevented lung metastasis in nude mice. Furthermore, the slow-growing s.c. tumors formed by the I{kappa}B{alpha}M-transfected cells exhibited a decrease in microvessel density (angiogenesis), which correlated with a decrease in the level of interleukin-8 expression. Collectively, these results demonstrate that NF-{kappa}B/relA activity significantly contributes to tumorigenicity, angiogenesis, and metastasis of human melanoma cells implanted in nude mice.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite improvements in early diagnosis and treatment of cutaneous melanoma, many patients still die from distant metastasis (1 , 2) . The pathogenesis of cancer metastasis consists of a series of sequential and selective steps, which include growth, angiogenesis, detachment, invasion, intravasation, survival in the circulation, adhesion to endothelial cells, and growth in distant organs (2 , 3) . Like primary neoplasms, the progressive growth of metastasis is dependent on adequate vasculature, i.e., angiogenesis (4 , 5) . In fact, the progression of neoplasms from benign to malignant often is associated with a switch to an "angiogenic phenotype," representing an increase in proangiogenic molecules produced by the tumor cells and organ-specific environments (6 , 7) . Melanoma cells secrete a variety of proangiogenic molecules, including basic fibroblast growth factor (8) , vascular endothelial growth factor (9 , 10) , and IL-83 (11) . Recent studies have demonstrated that the pleiotropic transcription factor NF-{kappa}B/relA (p50/p65) plays an important role in the control of cell proliferation and apoptosis, and hence, oncogenesis (12, 13, 14, 15, 16) . NF-{kappa}B/relA has also been shown to regulate the expression of proangiogenic molecules such as IL-8 and MMP-9 (17, 18, 19, 20) ; however, whether this transcription factor directly regulates angiogenesis and metastasis of human melanoma remains unknown.

NF-{kappa}B is an inducible dimeric transcription factor that belongs to the Rel/NF-{kappa}B family of transcription factors whose prototype in most nonlymphoid cells is a heterodimer composed of the RelA (p65) and NF-{kappa}B1 (p50) subunits (16 , 21) . NF-{kappa}B complexes typically are retained in the cytoplasm by inhibitory I{kappa}B proteins, including I{kappa}B{alpha}. Upon stimulation, I{kappa}B{alpha} is rapidly phosphorylated and degraded via the ubiquitin-proteasome pathway, permitting activation and nuclear import of NF-{kappa}B. Substitutions for serines 32 and 36 by alanine residues protect I{kappa}B{alpha} from ubiquitination and proteasome-mediated proteolysis (22, 23, 24) . Therefore, such I{kappa}ß{alpha} mutants can function as dominant-negative inhibitors of NF-{kappa}B activation. Indeed, a dominant-negative I{kappa}ß{alpha} mutant (I{kappa}ß{alpha}M) has been a powerful tool with which to study NF-{kappa}B function in cytokine activation, cell survival, apoptosis, and tumor growth (25, 26, 27, 28) .

Although high expression of Rel/NF-{kappa}B has been demonstrated in several different tumors (15 , 29, 30, 31, 32) , whether the constitutive expression of NF-{kappa}B/relA is relevant to the progression and metastasis of human melanomas is unknown. In this study, we demonstrate that metastatic human melanoma cells express higher levels of NF-{kappa}B activity than their nonmetastatic counterparts. Moreover, I{kappa}ß{alpha}M transfection, which inhibits NF-{kappa}B activity (25) , suppressed tumor growth and metastasis of metastatic human melanoma cells in vivo. The inhibition of tumor growth and abrogation of metastasis by I{kappa}ß{alpha}M correlated with decreased vascularization of melanoma in nude mice, which was at least partially due to decreased IL-8 expression. Collectively, these results suggest that NF-{kappa}B/relA is constitutively activated in human malignant melanoma and that blocking of NF-{kappa}B/relA activity suppresses IL-8 expression and hence angiogenesis and metastasis of human melanoma.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Reagents.
The A375P (low metastatic) human melanoma cell line was originally established in culture from a lymph node metastasis of a 54-year-old woman with melanoma (33) . The A375SM metastatic line was established from lung metastases produced by the A375P cells growing s.c. in nude mice (34) . Low metastatic A375-C5 and highly metastatic A375-C28 clonal lines were derived from A375P by a limited dilution technique (34) . All cell lines were maintained in culture (5% CO2 and 95% air at 37°C) as adherent monolayers in MEM supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, and vitamin solution (Flow Laboratories, Rockville, MD). All cultures were free of Mycoplasma and pathogenic murine viruses. All reagents used in tissue culture were free of endotoxin as determined by Limulus amebocyte lysate assay (sensitivity limit, 0.125 ng/ml) purchased from Associates of Cape Cod (Falmouth, MA).

Animals.
Male athymic BALB/c nude mice were purchased from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). The mice were housed in laminar flow cabinets under specific pathogen-free conditions and used at 8 weeks of age. Animals were maintained according to institutional regulations in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with present regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and NIH.

ELISA for Human IL-8 Expression.
The level of IL-8 protein in culture supernatants was determined by a quantitative ELISA kit (Quantikine IL-8 ELISA kit; R&D Systems, Minneapolis, MN). The absorbance of the samples was compared with the standard curve (35) .

Northern Blot Analysis.
Cellular mRNA was extracted from melanoma cells by the FastTrack mRNA isolation kit (Invitrogen Co., San Diego, CA). The mRNA (2 µg) was separated electrophoretically on 1% denaturing formaldehyde agarose gels, transferred to a GeneScreen nylon membrane (DuPont Co., Boston, MA) in 20x SSC, and cross-linked with a UV-Stratalinker 1800 (Stratagene, La Jolla, CA). The cDNA probe used in the analysis was a 0.5-kb EcoRI cDNA fragment corresponding to human IL-8 (35) . The cDNA probes were labeled with [32P]deoxycytidine triphosphate by a random labeling kit (Boehringer Mannheim Biochemicals, Indianapolis, IN). The equivalence of mRNA sample loading was monitored by hybridizing the same membrane filter with a human ß-actin cDNA probe (35) . IL-8 mRNA expression was quantitated in the linear range by a PhosphorImager with the ImageQuant software program (Molecular Dynamics, Sunnyvale, CA). Measurement of samples was calculated from the ratio between the areas of the IL-8-specific mRNA and the ß-actin transcripts (35) .

Promoter Reporters and Dual Luciferase Assays.
Luciferase reporters driven by either two-copy wild-type (2x NF-{kappa}B-Luc) or mutant (2x NF-{kappa}B-mt-Luc) NF-{kappa}B-responsive elements (36 , 37) and IL-8 promoter -133 and its NF-{kappa}B-mutant, -133-NF-{kappa}B-mt IL-8 (19) , were used in this study. Melanoma cells (2 x 105) growing in 10-cm tissue culture dishes were transfected with the indicated reporter plasmids by the Lipofectin reagent (Life Technologies, Inc., Gaithersburg, MD). Normalization of transfection efficiency was done by cotransfection with a pB-Actin-RL reporter containing a full-length renilla luciferase gene (Promega, Madison, WI) under the control of a human ß-actin promoter (38) . Six h after transfection, the medium was replaced with serum-containing medium, and the cells were then incubated for an additional 48 h at 37°C. The cells were then washed with PBS and harvested in passive lysis buffer (Promega). Firefly luciferase and renilla luciferase activities were quantified using the dual luciferase assay system (Promega). Specific IL-8 promoter activity and NF-{kappa}B activity were calculated as described previously (38) .

Stable Transfection of Melanoma Cells with I{kappa}B{alpha}M and Control Vector.
The cDNA plasmid pLXSN-I{kappa}B{alpha}M contains mutations at S32 and S36 of the NH2 terminus and a COOH-terminal PEST sequence mutation (25) . The pLXSN vector contains the neo resistance gene (39) . A375SM cells (5 x 106) were transfected with 15 µl of lipofectin reagent (Life Technologies) and 4 µg of pLXSN-I{kappa}B{alpha}M expression vector or control pLXSN vector. Transfections were carried out according to the manufacturer’s instructions. Six h after transfection, the medium was changed to serum-containing medium, and the cells were incubated for another 48 h at 37°C. Cells were then selected with standard medium containing G418 at 600 µg/ml Fourteen days later, neo-resistant colonies were isolated by trypsinization and established as subcultures. The expression of exogenous I{kappa}B{alpha}M was verified by Western blot analysis.

Western Blot Analysis.
Control and transfected melanoma cells (2.5 x 106 in 10 ml of completed MEM) were seeded in 100-mm Petri dishes and incubated overnight. The cells were scraped into PBS, washed in 4°C PBS containing 5 mM EDTA, and pelleted. The pellets were placed into 100-µl Triton lysis buffer [150 mM NaCl, 25 mM Tris (pH 7.5), 1% (w/v) Triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml trypsin inhibitor, 20 mM leupeptin, 0.15 units/ml aprotinin] on ice for 20 min. The soluble protein in the lysates was separated by a 20-min centrifugation at 14,000 rpm at 4°C. The protein concentration was assayed by the Bio-Rad protein assay reagent (Bio-Rad Laboratories) and stored at -70°C. Before loading, protein samples were boiled in a sample buffer [62.5 mM Tris-HCl (pH 6.8), 10% (w/v) glycerol, 100 mM DTT, 2.3% SDS, 0.002% bromphenol blue] for 5 min. Thirty-µl samples were loaded and separated on 10% SDS-PAGE by electrophoresis. Proteins in the gels were electrophoretically transferred onto Immobilon-P Transfer Membrane (Millipore) at 4°C. The membranes were washed in blocking buffer [TBS; 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5% fat-free milk] for 2 h at room temperature with shaking and then rinsed once briefly with TTBS (99.9% TBS, 0.1% Tween 20). The membranes were incubated with a polyclonal rabbit antihuman and antimouse I{kappa}B{alpha} (C-21; Santa Cruz Biotechnology) overnight at 4°C, washed three times with TTBS at room temperature, and then incubated with a second antibody [antirabbit IgG, horseradish peroxidase-linked F(ab')2 fragment from a donkey; Amersham, Arlington Heights, IL] for 1 h at room temperature with shaking. The membranes were rinsed twice and washed three times with TTBS with shaking. The probe proteins were detected with the Amersham ECL system according to the manufacturer’s instructions.

EMSA.
Nuclear protein extracts were prepared according to Dignam et al. (40) . Extracts were assayed for protein content by the Bio-Rad protein assay reagent (Bio-Rad Laboratories) and stored at -70°C. Double-stranded DNA oligonucleotide probes for wild-type NF-{kappa}B and mutant NF-{kappa}B and were purchased commercially (Promega and Santa Cruz Biotechnology). The oligonucleotide sequences used were as follows: NF-{kappa}B, 5'-AGTTGAGGGACTTTCCCAGGC-3'; NF-{kappa}B mutant, 5'-AGTTGAGGCGACCTTTAAAAGGC-3'; TFIID, 5'-GCAGAGCATATAAGGTGAGGTAGGA-3'. EMSAwas performed as described previously with minor modifications (38) . EMSA reactions were performed in a 25-µl volume with 1 µg of poly(dI-dC), 2 µg of BSA, 4.6 mM MgCl2, 63 mM KCl, 1 mM DTT, and 12% glycerol in 20 mM HEPES buffer (pH 7.9). Five µg of nuclear extract protein and 30,000 cpm of end-labeled double-stranded DNA probe were added to the mixture. The binding reaction was allowed to proceed for 25 min at 22°C. For unlabeled and mutant probe competition, extracts were incubated with 10–50-fold molar excess of each probe before the addition of labeled probe. For supershift reactions, extracts were preincubated with anti-p65 or anti-p50 antibody (Calbiochem, San Diego, CA) for 45 min on ice. Protein-DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel. The gels were dried and exposed to X-ray film at -80°C overnight.

In Vitro Growth Assay.
Tumor cells were plated at a density of 3 x 104 cells per 38-mm2 well in 96-well plates. After 72 h, cell number was determined by the MTT assay (Sigma Chemical Co., St. Louis, MO). After the cells were incubated for 2 h in medium containing MTT at 0.42 mg/ml, the medium was removed, and the cells were lysed in DMSO. The conversion of MTT to formazan by metabolically viable cells (41) was monitored by a 96-well microtiter plate reader at 570 nm (Dynatech, Inc., Chantilly, VA). The percentage of cytostasis was calculated by the formula: Cytostasis (%) = [1 - (B/A)] x 100, where A is the absorbance of parental cells, and B is the absorbance of the transfected cells incubated in medium. Cell proliferation was also measured by [3H]thymidine incorporation. Briefly, tumor cells (3 x 104 cells per 38-mm2 well) were seeded in a 96-well plate, incubated at 37°C for 60 h, and pulse labeled by the addition of 0.1 µCi/well [3H]thymidine. Twelve h later, free [3H]thymidine was removed. The adherent cells were lysed by 0.1 N NaOH, and the radioactivity (cpm) was monitored in a beta counter.

In Vivo Tumor Growth and Metastasis.
For all in vivo experiments, tumor cells in their exponential growth phase were harvested after a brief exposure to 0.25% trypsin-0.02% EDTA solution (w/v). The flask was tapped to dislodge the cells, MEM was added, and the cell suspension was pipetted to obtain a single-cell suspension. The cells were washed, resuspended in Ca2+- and Mg2+-free HBSS, and diluted to the desired cell number/inoculum. Cell viability was determined by trypan blue exclusion, and only single-cell suspensions of >95% viability were used to determine tumorigenic and metastatic potential in nude mice. To produce tumors, 1 x 106 cells suspended in 0.1 ml of HBSS were injected s.c. in the flanks of nude mice (n = 5). Tumor take and size were monitored three times per week. Tumors exceeding 3 mm in diameter were recorded as positive. To produce experimental lung metastasis, 5 x 105 viable tumor cells suspended in 0.2 ml of HBSS were injected into the lateral tail veins of unanesthetized mice. The mice were killed 8 week later, and the lungs were removed, washed, and fixed in Bouin’s solution to differentiate the neoplastic lesions from the organ parenchyma. The lung nodules were counted with the aid of a dissecting microscope.

Immunohistochemistry.
s.c. tumors harvested at autopsy were placed into OCT compound (Miles Laboratories, Elkhart, IN) to be snap-frozen in liquid nitrogen. Frozen tissue sections (5 µm thick) were fixed with cold acetone and transferred to PBS. Endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in PBS for 12 min. The samples were incubated for 20 min at room temperature with a protein-blocking solution consisting of PBS (pH 7.5) containing 5% normal horse serum and 1% normal goat serum. Excess blocking solution was drained, and the samples were incubated for 18 h at 4°C with a 1:50 dilution of rabbit polyclonal anti-IL-8 antibody (Biosource International, Camarillo, CA). The samples were then rinsed and incubated for 1 h at room temperature with peroxidase-conjugated antirabbit IgG. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine (Research Genetics, Huntsville, AL). The sections were washed three times with distilled water, counterstained with Gills hematoxylin (Sigma), and then washed once with distilled water and once with PBS. The slides were mounted with a Universal mount (Research Genetics) and examined in a bright-field microscope. A positive reaction was indicated by a reddish-brown precipitate in the cytoplasm. Nonspecific IgG was used for negative controls. The average measurement was derived from the intensity of staining quantitated in five different areas of each sample by an image analyzer using the Optimas software program (Bioscan, Edmonds, WA).

Quantitation of MVD.
s.c. tumors produced by control and I{kappa}B{alpha}M-transfected cells were embedded in OCT compound and frozen, sectioned, fixed, stained with antibodies to CD31/PECAM-1 (42) , and examined using a bright-field microscope. A positive reaction was indicated by a reddish-brown precipitate in the cytoplasm. Nonspecific IgG was used for negative controls. Areas containing the highest number of capillaries and small venules were identified by scanning tumor sections at low powers (x40). Images of 10 (x100) fields per sample were digitized and stored for further analysis. The number of blood vessels was counted in each field of each sample. The total area of tumors in each field was determined with an Optimas software program (43) . MVD was calculated as the mean number of blood vessels (CD31/PECAM-1+ cells) in a 1-mm2 area of tumors. On the basis of criteria described by Weidner et al. (44) , vessel lumens were not required for a structure to be classified as a vessel. All vessel counts were performed on coded samples by two investigators.

Statistics.
The significance of the in vitro results was determined by Student’s t test (two-tailed); the significance of the in vivo metastasis results was determined by the Mann-Whitney U test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Metastatic Human Melanoma Cells Have High Constitutive NF-{kappa}B/relA Activities.
To provide direct evidence that NF-{kappa}B/relA was constitutively activated in the human melanoma cells, a 2x NF-{kappa}B-Luc reporter or a 2x NF-{kappa}B-mut-luc reporter (36 , 37) were transfected into the low metastatic A375P and A375-C5 cells and high metastatic A375SM and A375-C28 cells. As shown in Fig. 1ACitation , constitutive NF-{kappa}B promoter activity was significantly higher in the metastatic cells. To confirm that the highly metastatic A375 cells had high constitutive NF-{kappa}B activities, EMSA was performed using nuclear proteins. Double-stranded NF-{kappa}B oligonucleotides were used as probes. As shown in Fig. 1BCitation , all of the melanoma cell lines expressed NF-{kappa}B binding activity, and significantly higher NF-{kappa}B binding activity was detected in the highly metastatic A375SM and A375-C28 cells than in low metastatic A375P and A375-C5 cells (Fig. 1B)Citation .



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  		Fig. 1. Constitutive NF-{kappa}B activities in human melanoma cells. A, constitutive NF-{kappa}B promoter activity. The NF-{kappa}B-wt ({blacksquare}) and NF-{kappa}B-mt ({square}) luciferase reporters were transfected with an internal control, pBActin-RL, into A375P (P), A375-C5 (C5), A375SM (SM), and A375-C28 (28) cells. Specific NF-{kappa}B promoter activity was determined as described in "Materials and Methods." Bars, SD. B, constitutive NF-{kappa}B binding activities. Nuclear protein was extracted from A375P (Lane 1), A375-C5 (Lane 2), A375SM (Lane 3), and A375-C28 (Lane 4) cells, and EMSA was performed as described previously. As a control, TFIID oligonucleotides were used to monitor equal nuclear protein extract loading. C, characterization of NF-{kappa}B complexes. Nuclear protein was extracted from A375P (Lane 2) and A375SM (Lanes 3–10) melanoma cells, and EMSA was performed as above. Lane 1 contains only oligonucleotide probes. Unlabeled cold wild-type NF-{kappa}B oligonucleotides were added to the reaction mixture (1:10 in Lane 4, 1:25 in Lane 5, and 1:50 in Lane 6). Unlabeled cold mutant NF-{kappa}B oligonucleotides (1:50; Lane 7) and cold TFIID oligonucleotides (1:50; Lane 8) were added to the reaction mixture. Specific anti-p50 (Lane 9) and anti-p65 (Lane 10) antibodies were added to the reaction mixture. This is one representative experiment of three.

 
To further characterize the components of NF-{kappa}B complexes, nuclear protein was extracted from A375P and A375SM melanoma cells and EMSA was performed as described above. Unlabeled cold wild-type NF-{kappa}B oligonucleotides (Fig. 1CCitation , 1:10 in Lane 4, 1:25 in Lane 5, 1:50 in Lane 6), unlabeled cold mutant NF-{kappa}B (Citation , 1:50 in Lane 7) and TFIID (Citation , 1:50 in Lane 8) oligonucleotides, and specific anti-p50 (Citation , Lane 9) and anti-p65 (Citation , Lane 10) antibodies were added to the reaction. As shown in Citation , NF-{kappa}B complexes contained both p50 and p65 components.

Down-Regulation of Constitutive NF-{kappa}B/relA Activity and IL-8 Promoter Activity in Metastatic Human Melanoma Cells Transfected with an I{kappa}ß{alpha}M Expression Vector.
To determine the effect of decreasing the constitutive NF-{kappa}B activities on tumor growth, angiogenesis, and metastasis, we transfected the metastatic A375SM cells with the I{kappa}B{alpha}M expression vector. The expression of exogenous I{kappa}B was confirmed by Western blot analysis (Fig. 2A)Citation . The constitutive NF-{kappa}B activities were determined as described above using both the promoter assay and EMSA. I{kappa}ß{alpha}M significantly suppressed constitutive NF-{kappa}B binding activity (Fig. 2B)Citation , constitutive IL-8 promoter activities (Fig. 2C)Citation , and NF-{kappa}B reporter activity (Fig. 2D)Citation .



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  		Fig. 2. NF-{kappa}B binding and IL-8 promoter activities in A375SM cells transfected with pLXSN-I{kappa}B{alpha}M. A, exogenous I{kappa}B{alpha}M expression. Cytosolic protein was extracted from A375SM cells (SM), A375SM cells transfected with pLXSN (Neo), and A375SM cells transfected with pLXSN-I{kappa}ß{alpha}M (I{kappa}M.1, I{kappa}M.2, and I{kappa}M.3). Western blot was performed using specific anti-I{kappa}M antibody. Endogenous I{kappa}ß{alpha} and exogenous I{kappa}ß{alpha}M were detected (indicated by arrows). Equal loading was monitored by hybridizing the filter with an anti-ß-actin antibody. B, NF-{kappa}B binding activities. Nuclear protein was extracted from A375SM (SM), A375SM transfected with pLXSN (Neo), and A375SM transfected with pLXSN-I{kappa}ß{alpha}M (I{kappa}M.1, I{kappa}M.2, and I{kappa}M.3) cells. EMSA was performed as described. C, IL-8 promoter activities. -133-Luc ({blacksquare}) and its NF-{kappa}B mutant (-133-NF-{kappa}B-mt; {square}) were transfected into the variant cells. Constitutive NF-{kappa}B and IL-8 promoter activities were determined by the dual luciferase assay kit. Bars, SD; *, P < 0.001. D, 2x NF-{kappa}B-Luc reporter activities. Luciferase reporters driven by either wild-type (2x NF-{kappa}B-wt-luc; {blacksquare}) or mutant (2 x NF-{kappa}B-mt-luc; {square}) NF-{kappa}B response elements were transfected into the cell variants. *, P < 0.001. Data from one representative experiment of three; bars, SD.

 
Suppression of Tumorigenicity and Metastasis in Human Melanoma Cells Transfected with I{kappa}B Dominant-Negative Expression Vector.
Parental A375SM cells, A375SM-Neo, A375SM-I{kappa}M.1, A375SM-I{kappa}M.2, and A375SM-I{kappa}M.3 melanoma cells were injected s.c. into nude mice (n = 5). All control cells produced large tumors, whereas the A375SM-I{kappa}M.1, A375SM-I{kappa}M.2, and A375SM-I{kappa}M.3 cells did not (Fig. 3)Citation . Sixty days after i.v. injection of tumor cells (5 x 105/mouse), control A375SM and A375SM-Neo cells produced many experimental lung metastases, whereas the A375SM-I{kappa}M.1, A375SM-I{kappa}M.2, and A375SM-I{kappa}{alpha}M.3 cell lines produced very few (Table 1)Citation , demonstrating that decreased constitutive NF-{kappa}B/relA activities led to suppression of tumor formation and metastasis.



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  		Fig. 3. Growth of melanoma cells transfected with I{kappa}ß{alpha}M expression vectors. A375SM (•), A375SM-Neo ({blacksquare}), A375SM-I{kappa}M.1 ({triangleup}), A375SM-I{kappa}M.2 ({circ}), and A375SM-I{kappa}M.3 ({square}) cells (1 x 106 cells/mouse) were injected s.c. into groups of nude mice (n = 5). The diameter of the s.c. tumors was determined every 5 days. Note that I{kappa}B{alpha}M-transfected cells produced smaller s.c. tumors than control A375SM cells. Data from one representative experiment of two; bars, SD.

 

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  		Table 1 Suppression of metastasis by dominant-negative I{kappa}B{alpha}M expression vector in human melanoma

A375SM control, A375SM-Neo, A375SM-I{kappa}M.1, A375SM-I{kappa}M.2, and A375SM-I{kappa}M.3 cells (5 x 105 cells/mouse) were injected i.v. into groups of nude mice (n = 10). Experimental lung metastasis was determined 60 days after tumor inoculation as stated in "Material and Methods."

 
In Vitro Growth of Human Melanoma Cells Transfected with I{kappa}B Dominant-Negative Expression Vector.
The decrease in tumor growth of A375SM cells transfected with I{kappa}B{alpha}M could have been due to a decrease in proliferation caused by decreased expression of growth factors including IL-8 (11) or to an increase in apoptosis caused by inhibition of NF-{kappa}B (25 , 26 , 45) . To distinguish between these possibilities, A375SM cells, A375SM-Neo, and A375 I{kappa}B{alpha}M-transfected cells were seeded into 96-well plates (5 x 103 cells/well) for 48 h. In vitro cell growth and proliferation was measured by MTT assay and [3H]thymidine incorporation. The growth rate of all of the lines was very similar (Fig. 4)Citation .



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  		Fig. 4. In vitro growth of I{kappa}B{alpha}M-transfected melanoma cells. A, MTT assay. Cells were cultured for 48 h. The number of metabolically active cells was determined by the MTT assay. B, uptake of [3H]thymidine. Cells were plated in 96-well plates for 24 h, at which point 0.5 µCi/ml [3H]thymidine was added, and [3H]thymidine incorporation was measured 12 h later. This is one representative experiment of three; bars, SD.

 
Decreased IL-8 Expression and MVD in I{kappa}B{alpha}M-transfected Human Melanoma Tumors.
As shown above, the in vivo suppression of tumor growth and metastasis by cells transfected with I{kappa}B{alpha}M did not directly correlate with the in vitro growth. In this set of experiments, we determined whether decreased NF-{kappa}B activity and the consequent decrease in IL-8 production led to suppression of angiogenesis. Previous studies have indicated that IL-8 acts as a direct or indirect angiogenic factor (46, 47, 48, 49) . Consistent with these findings, constitutive IL-8 expression was significantly decreased at both the mRNA (Fig. 5A)Citation and protein (Fig. 5B)Citation levels in culture. A375SM, A375SM-Neo, A375SM-I{kappa}M.1, A375SM-I{kappa}M.2, and A375SM-I{kappa}M.3 cells were then injected into the subcutis of nude mice. Thirty-five to 60 days after injection, the tumors were resected and processed for immunohistochemical analyses of vascular formation using anti-CD31 antibodies. As shown in Fig. 6Citation , tumors formed by control vector-transfected cells (Fig. 6A)Citation were highly vascularized, whereas the tumors formed by I{kappa}B{alpha}M-transfected cells (Fig. 6B)Citation had a 50.17–72.92% decrease in vascular density (Table 2)Citation . Consistent with the alteration of vascular density, high IL-8 expression was found in tumor lesions formed by control tumor cells (Fig. 6C)Citation , whereas much lower IL-8 expression was found in tumor lesions formed by I{kappa}B{alpha}M-transfected melanoma cells (Fig. 6D)Citation .



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  		Fig. 5. Expression of IL-8 in highly metastatic human melanoma cells transfected with pLXSN-I{kappa}ß{alpha}M. A, Northern blot. mRNA was extracted from A375SM (SM) cells, A375SM cells transfected with pLXSN (Neo), and A375SM cells transfected with pLXSN-I{kappa}ß{alpha}M (I{kappa}M.1, I{kappa}M.2, and I{kappa}M.3). Quantitative analysis was performed by densitometry, standardized to ß-actin, and expressed as fold difference from the untreated controls (numbers in italics). B, culture supernatants were collected 48 h after medium change, and IL-8 protein in the supernatants was determined by ELISA and expressed as ng/ml. The IL-8 concentrations in the supernatant were standardized by the cell number (*, P < 0.01). This is one representative experiment of two; bars, SD.

 


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  		Fig. 6. Tumor MVD and IL-8 expression in s.c. I{kappa}ß{alpha}M- or Neo-transfected A375SM melanomas. Cells were injected s.c. into groups of nude mice (n = 10). Thirty to 60 days later, the resulting s.c. tumors with similar size were resected and processed for immunohistochemical analysis. Blood vessels were counted using a light microscope after immune staining of sections with anti-CD31 antibodies (A and B). IL-8 staining (C and D) was performed as described in "Materials and Methods."

 

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  		Table 2 Suppression of angiogenesis by dominant-negative I{kappa}B{alpha}M expression vector in human melanoma

A375SM control, A375SM-Neo, A375SM-I{kappa}M.1, A375SM-I{kappa}M.2, and A375SM-I{kappa}M.3 cells (1 x 106 cells/mouse) were injected s.c. into groups of nude mice (n = 5). Thirty-five days after tumor injection, the resulting tumors were removed and processed for immunohistochemical analysis. Vessels from these tumors were counted under a light microscope after immune staining of sections with anti-CD31 antibodies as stated in "Material and Methods."

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study shows that human melanoma cells with high metastatic potential express high levels of constitutive NF-{kappa}B/relA activity. The data also provide the first direct evidence for the essential role of NF-{kappa}B/relA in angiogenesis, growth, and metastasis of human melanoma. This conclusion is based on the results showing that enforced expression of phosphorylated mutant I{kappa}B{alpha}, which suppresses NF-{kappa}B/relA activities, was associated with decreasing angiogenesis, tumorigenicity, and metastasis in part through down-regulation of IL-8 expression.

Although constitutive activation of NF-{kappa}B/Rel family members has been reported in several cancers (15 , 29, 30, 31, 32) , our data demonstrate that NF-{kappa}B activity differs between metastatic and nonmetastatic human melanoma cells. Specifically, the metastatic ability of human A375 melanoma cells correlated with constitutive NF-{kappa}B/relA activity. To provide direct evidence for the contribution of NF-{kappa}B/relA to metastasis of human melanoma, we stably transfected the highly metastatic A375SM cells with I{kappa}B{alpha}M. I{kappa}B{alpha}M-expressing vector blocked the NF-{kappa}B/relA activity and abrogated the ability of these cells to form lung metastases in nude mice. Therefore, overexpression of NF-{kappa}B/relA activity could play an important role in the progression of melanoma from the nonmetastatic to the metastatic phenotype.

The mechanisms accounting for overexpression of NF-{kappa}B/relA in malignant melanoma and other types of tumor cells are unknown at present. The interaction between host environment and tumor cells could play a role in overexpression of NF-{kappa}B/relA in malignant cells because the development of cancer metastasis is determined by the interaction of tumor cells with their immediate environment, including tissue- or organ-specific cytokines (3 , 50) . A diversity of stimuli can activate NF-{kappa}B. For example, many inflammatory signals (21) , hypoxia (51) , and oncogenic proteins such as mutated Ras (52) can regulate NF-{kappa}B activity.

The activity of NF-{kappa}B can protect tumor cells from apoptosis by mediating cellular survival responses (25 , 26 , 45) . A super-repressor form of I{kappa}ß{alpha} (I{kappa}ß{alpha}-SR) has been shown to block Bcr-Abl-mediated transformation (27) , and expression of a dominant-negative mutant I{kappa}ß{alpha} of NF-{kappa}B inhibits proinflammatory cytokine expression, cell survival, and tumor growth of head and neck squamous carcinomas (28) . Moreover, NF-{kappa}B activity is required for oncogenic Ras-induced transformation, which likely occurs through the inhibition of transformation-associated apoptosis (52) . In the present study, no discernible differences for the in vitro growth were found between I{kappa}ß{alpha}M-transfected and control cells, but whether the transfected cells are more sensitive to in vivo induction of apoptosis is unclear and is under investigation.

The inhibition of tumor growth and metastasis by inhibition of NF-{kappa}B/relA could have resulted from events other than the promotion of apoptosis. For example, cell treatment with antisense for p65 has been shown to inhibit adhesion and growth of tumor cells both in vitro and in vivo (53) , and NF-{kappa}B can regulate urokinase-type plasminogen activator and MMP-9 expression, which promote tumor growth and metastasis (17 , 18 , 54) . Our results showing that NF-{kappa}B activity directly correlated with in vivo angiogenesis provide an additional mechanism for NF-{kappa}B-mediated promotion of tumor growth and metastasis and are consistent with recent findings suggesting that NF-{kappa}B plays a role in retinal neovascularization in a murine ischemic retinopathy model (55 , 56) and in tumor necrosis factor-dependent or oxidative stress-induced tubular morphogenesis of endothelial cells growing in culture (57 , 58) .

IL-8, which is regulated by NF-{kappa}B (19 , 20) , plays a major role in melanoma progression and metastasis (11 , 35 , 50 , 59) . Our present study shows that although I{kappa}ß{alpha}M-transfected cells secreted decreased levels of IL-8, their growth rate in vitro was not changed, suggesting that IL-8 per se is not the critical autocrine growth factor for this metastatic melanoma, a finding consistent with a previous report showing that IL-8 was an autocrine growth factor for parental A375 melanoma cells but not for the metastatic A375SM cells (59) . A large body of recent data also suggests that IL-8 can serve as a proangiogenic factor (46, 47, 48, 49) by a mechanism that may involve interaction with IL-8 receptor on endothelial cells (60) . In our study, IL-8 expression was down-regulated in I{kappa}B{alpha}M-transfected cells, which was correlated with decreased NF-{kappa}B activity. Because the NF-{kappa}B activity, IL-8 expression, MVD, and tumor growth were well correlated in the tumors produced by the A375SM, A375-Neo, and A375SM-I{kappa}ß{alpha}M cells, our data provide direct evidence that NF-{kappa}B activation plays an important role in tumor angiogenesis, at least in part via production of IL-8. Whether NF-{kappa}B up-regulates angiogenesis by activation of other proangiogenetic molecules, such as MMP-9, vascular endothelial growth factor, and basic fibroblast growth factor, is under active investigation.

In summary, metastatic human melanoma cells constitutively express a high level of NF-{kappa}B activity, which can provide a growth advantage by multiple mechanisms, including increased angiogenesis through overexpression of IL-8. These data suggest that targeting NF-{kappa}B can be a potential approach to controlling angiogenesis and metastasis of human melanoma.


   ACKNOWLEDGMENTS
 
We thank Dr. P. Chiao (Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX) for the generous gift of pLXSN-I{kappa}ß{alpha}M, Dr. B. Su (Department of Immunology, The University of Texas M. D. Anderson Cancer Center, Houston, TX) for 2x NF-{kappa}B reporter constructs, Walter Pagel for critical editorial comments, and Lola López for expert assistance in the preparation of this manuscript.


   FOOTNOTES
 
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.

1 This work was supported in part by Cancer Center Support Core Grant CA16672 and Grant R35-CA42107 (to I. J. F.) from the National Cancer Institute, NIH. Back

2 To whom requests for reprints should be addressed, at Department of Cancer Biology-173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-8577; Fax: (713) 792-8747; E-mail: ifidler{at}notes.mdacc.tmc.edu Back

3 The abbreviations used are: IL-8, interleukin-8; NF, nuclear factor; MMP, matrix metalloproteinase; I{kappa}B, inhibitor of nuclear factor-{kappa}B; TBS, Tris-buffered saline; TTBS, TBS plus Tween 20; EMSA, electrophoretic mobility gel shift assay; TFIID, transcription factor IID; MTT, dimethylthiazole diphenyltetrazolium bromide; MVD, microvessel density. Back

Received 1/27/00; revised 3/13/00; accepted 3/16/00.


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 RESULTS
 DISCUSSION
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