April 2010
Volume 51, Issue 4
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Anatomy and Pathology/Oncology  |   April 2010
Characterizing the Involvement of the Nuclear Factor-kappa B (NFκB) Transcription Factor in Uveal Melanoma
Author Affiliations & Notes
  • Rinat Dror
    From the Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
  • Michal Lederman
    From the Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
  • Kazuo Umezawa
    the Department of Applied Chemistry, Keio University, Yokohama, Japan; and
  • Vivian Barak
    the Immunology Laboratory for Tumor Diagnosis, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
  • Jacob Pe'er
    From the Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
  • Itay Chowers
    From the Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
  • Corresponding author: Itay Chowers, Department of Ophthalmology, Hadassah-Hebrew University Medical Center, PO Box 12000, Jerusalem, Israel, 91120; chowers@hadassah.org.il
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1811-1816. doi:10.1167/iovs.09-3392
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      Rinat Dror, Michal Lederman, Kazuo Umezawa, Vivian Barak, Jacob Pe'er, Itay Chowers; Characterizing the Involvement of the Nuclear Factor-kappa B (NFκB) Transcription Factor in Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2010;51(4):1811-1816. doi: 10.1167/iovs.09-3392.

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Abstract

Purpose.: To examine the involvement of nuclear factor-kappa B (NFκB) pathways in uveal melanoma (UM) and to assess their potential as a therapeutic target for metastatic UM.

Methods.: Samples from primary (n = 7) and metastatic (n = 7) UM were evaluated for NFκB transcription factor family expression by quantitative PCR (QPCR), immunofluorescent staining, and Western blot analysis. The effect of two NFκB inhibitors, DHMEQ and BMS-345541, on two cell lines derived from UM liver metastases was assessed. Cell proliferation was examined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, methylene blue assay, and immunostaining for Ki-67. Apoptosis was assessed by immunostaining for activated caspase 3.

Results.: NFκB1, NFκB2, RelA, RelB, and NIK were expressed in primary UM and in its liver metastases. NFκB2, RelB, and NIK showed significantly higher mRNA levels in metastases from UM compared with primary tumors (3.4-fold, P = 0.03; 3.6-fold, P = 0.05; 3.5-fold, P = 0.03; respectively). NFκB2 protein activation was 3.9-fold higher in metastases (P = 0.03). NFκB inhibition reduced metastatic cell proliferation by 9.2-fold and 1.9-fold according to Ki67 staining (P = 0.04) and methylene blue assay (P = 6 × 10−7), respectively. Both NFκB inhibitors achieved dose-dependent reductions of UM cell proliferation in both cell lines (P < 0.001). NFκB inhibition resulted in a 6.3-fold increase of apoptosis (P = 7 × 10−7).

Conclusions.: These data indicate that the NFκB1 and NFκB2 pathways are active in both primary and metastatic UM and that these pathways regulate metastatic cell proliferation and apoptosis. The role of NFκB as a therapeutic target for UM should be further evaluated.

Metastases develop in approximately 50% of patients with uveal melanoma (UM), the most common primary eye cancer in adults. In 90% of those patients, metastases spread to multiple organs including the liver, whereas in 40% of the patients metastases are found only in the liver. 13 Despite the application of current therapies, the average survival time after diagnosis of metastases is in the range of 2 to 14 months. 1,2,4  
We have previously demonstrated that UM liver metastases have a different gene expression pattern than UM primary tumors, including upregulation of the transcription factor nuclear factor-kappa B2 (NFκB2). 5 The NFκB transcription factor family includes the genes RelA (p65), RelB, c-Rel, NFκB1 (p105/p50), and NFκB2 (p100/p52), which form various heterodimers and homodimers. 6 There are two main signal transduction pathways that activate NFκB. In the canonical pathway, NFκB forms a cytoplasmic inactive complex with a member of the IκB inhibitory protein family. After stimulation there is ubiquitination and proteolysis of IκB, which then leaves NFκB free to translocate to the nucleus and promote gene transcription. In the noncanonical pathway, stimulation leads to the cleavage of cytoplasmic NFκB2 (p100) to p52, which creates a dimer with RelB and promotes gene transcription. 69 Another important protein for the activation of both NFκB pathways is the NFκB-inducing kinase (NIK). 10  
NFκB regulates the transcription of genes that are involved in apoptosis, proliferation, angiogenesis, immune response, cell invasion, and cell adhesion. 8,11,12 It is known to be constitutively activated and involved in the development of different malignancies, 11,13,14 including carcinoma of the liver 15 and retinoblastoma. 16 Given that understanding the molecular pathways that underlie UM metastases growth may enable identification of new therapeutic targets for this condition and given the role of NFκB in a variety of malignancies, we examined the potential role of NFκB transcription factor in the development of UM and its metastases. 
Methods
Tissues and Patients
Primary UM tumor samples (n = 7), liver metastases from UM samples (n = 7), and normal choroid samples (n = 2) were included in the study. All patients were treated at the Hadassah-Hebrew University Medical Center in Jerusalem, Israel. The study was approved by the Institutional Ethics Committee and adhered to the tenets of the Declaration of Helsinki. Additional information on patients included in the study is presented in Supplementary Table S1
Quantitative Real-Time RT-PCR
Quantitative real-time RT-PCR (QPCR) was performed on RNA from seven primary UM samples and seven UM liver metastasis samples, as we have recently described. 5 Briefly, RNA was extracted by reagent (TRIzol; Sigma-Aldrich Corp., St. Louis, MO). One microgram of RNA was reverse transcribed with a first-strand synthesis kit (Reverse-iT; ABgene, Epsom, UK) and was used as the template for QPCR. Reactions were performed in triplicate using SYBR green PCR Mix (Applied Biosystems, Foster City, CA) and the following primers: NFκB1 forward 5′-ACAGCAGATGGCCCATACCT-3′, NFκB1 reverse 5′-CATACATAACGGAAACGAAATCCTCT-3′; RelA forward 5′-GCTGACTGATAGCCTGCTCCA-3′, RelA reverse 5′-CATCCACAGTTTCCAGAACCTG-3′; RelB forward 5′-ACCGCCAGATTGCCATTGTGTTC-3′, RelB reverse 5′-AGTGTGGGGGCCGTAGGGTCGTAG-3′; NIK forward 5′-GAAGAAACAGAGCTCCGTCTACAAG-3′, NIK reverse 5′-CATTCAGGATCTCCCACTTTCC-3′; GAPDH forward 5′-TAGCCAAATTCGTTGTCATACC-3′, GAPDH reverse 5′-CTGACTTCAACAGCGACACC-3′; primer assay (QuantiTect; Qiagen, Valencia, CA) QT00012404 for NFκB2 and QT00000721 for TBP. Assays were performed on a real-time PCR system (7900 HT Fast; Applied Biosystems). Results were quantified (ABI Prism 7900 SDS software; Applied Biosystems), and measurements of the GAPDH or TBP gene were used for normalization. 
Protein Extraction
Fresh tumor tissue was frozen and stored at −80°C until use. For extraction of proteins, tissue samples were placed in lysis buffer (1% deionized Triton X-100 and 0.1% sodium azide in 50 mM Tris-HCl, pH 7.5, incubated with Chelex-100 for 24 hours at room temperature) and 0.1% phenylmethylsulfonyl-fluoride. Samples were homogenized mechanically, vortexed, and placed in an ultrasonic cell disruptor (Microson; Misonix, Farmingdale, NY) at 10 W for 1 minute. After 30 minutes incubation at 4°C, samples were centrifuged at 3000 rpm for 15 minutes, and supernatant was collected and analyzed for total protein. Total protein content was estimated with a protein assay kit (BCATM; Pierce, Rockford, IL). Results were read using a microplate reader (MR 5000; Dynatech Laboratories, Chantilly, VA) at 570 nm. 
Western Blot Analysis
Protein extract samples, mixed (1:2) with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) containing 5% β-mercaptoethanol, were boiled for 5 minutes and separated by 10% SDS-acrylamide gel. Fifty micrograms of each sample was loaded in each lane, together with standard marker (Precision Plus Protein; Bio-Rad). Samples were electrotransferred to a PVDF membrane (Bio-Rad) using a mini transblot system (Bio-Rad). The membrane was placed in blocking solution containing 5% skim milk (Difco; Becton-Dickinson, Franklin Lakes, NJ) for 20 minutes and was incubated overnight at 4°C with the indicated antibody. Antibodies used include anti-NFκB2 (Cell Signaling Technology Inc., Danvers, MA), phosphorylated-p65 (P-p65; Cell Signaling), both diluted 1:1000, RelA (p65; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:500, and β-actin (Cell Signaling Technology) diluted 1:5000. After incubation with primary antibody, the membrane was washed with TBST (3 × 5 minutes) and incubated with anti–rabbit IgG secondary antibody conjugated to horseradish peroxidase (DakoCytomation, Glostrup, Denmark) diluted 1:100 for 1 hour at room temperature. The membrane was washed with TBST (3 × 5 minutes), and immunoreactive bands were visualized by chemiluminescence (EZ-ECL; Biological Industries, Kibbutz Beit Ha'Emek, Israel). Then the membranes were exposed to light film (medical x-ray film; Fuji, Tokyo, Japan) and developed by a processor (X-OMAT 2000; Kodak, Rochester, NY). Band intensity was quantified with personal computer software (DNR BIS 303; DNR Bio-Imaging Systems, Jerusalem, Israel). P-p65 quantification was based on calculation of the P-p65/p65 band density ratios in primary UM (n = 4) and liver metastases samples (n = 4); p52 quantification was performed after normalization according to β-actin levels in primary UM (n = 5) and liver metastases (n = 5). 
Cell Culture
Two cell lines derived from liver metastases of UM patients (M15 and M85) were kindly provided by Michal Lotem (Hadassah-Hebrew University Medical Center, Jerusalem, Israel). Cells were cultured in RPMI 1640 (Sigma-Aldrich) and DMEM (Sigma-Aldrich) supplemented with 1% penicillin-streptomycin (Gibco-Invitrogen, Carlsbad, CA), 1% of 200 mM l-glutamine (Gibco), and 10% FBS (Gibco). Cell cultures were incubated at 37°C and 8% CO2 in a humidified incubator. 
NFκB Inhibition
Two NFκB inhibitors were used for this study. Dehydroxymethylepoxyquinomicin (DHMEQ), an NFκB inhibitor that blocks the nuclear translocation of NFκB, 17,18 was synthesized by Kazuo Umezawa (Keio University, Yokohama, Japan). DHMEQ was dissolved with dimethylsulfoxide (DMSO; Fisher Scientific, Pittsburgh, PA) and used for experiments at concentrations of 2.5, 7.5, and 15 μg/mL medium. The second inhibitor used was BMS-345541 (Sigma-Aldrich). This compound inhibits IκB kinase protein and can therefore be used as an inhibitor of the NFκB pathway. 19,20 BMS was dissolved in DMSO and used for experiments at concentrations of 1.25, 2.5 and 3.75 μg/mL medium. During the experiment, medium was removed from the cell cultures daily, and fresh medium containing the inhibitor was added. 
Cell Viability Assay
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MTT was performed at days 1 to 5 after the addition of an NFκB inhibitor to the culture. Medium was removed, and 300 μL of 0.3 mg/mL MTT (Sigma-Aldrich) was added to each well (in six-well plates). The cells were incubated with MTT for 1 hour at 37°C, then MTT was removed and 300 μL DMSO was added to each well. Quantification was performed using a spectrophotometer at a wavelength of 570 (UV-2100; Unic, Shanghai, China). 
Methylene Blue Analysis
Cells were fixed with 0.5% glutaraldehyde for 10 minutes, washed with double-distilled water (DDW), and incubated with 0.1 M borate buffer (pH 8.5) for 1 hour at 37°C. After washes, cells were stained with 1% methylene blue (Sigma-Aldrich) in 0.1 M borate buffer (pH 8.5) for 1 hour at room temperature. Plates were then extensively washed with DDW to remove excess dye and dried. The dye taken up by cells was eluded in 0.1 M HCl for 1 hour at 37°C and read at 620 nm by a spectrophotometer. 
Immunohistochemistry Analysis
Immunohistochemistry was performed on the M15 cell line after 30 minutes incubation with 100% cold methanol (Mallinckrodt Chemicals, Hazelwood, MO) followed by PBS washes and 8 minutes incubation with 1% triton (J.T. Baker, Phillipsburg, NJ) at room temperature in a humidified chamber. Cells were then incubated with mouse monoclonal anti–Ki67 antibody (1:100; DakoCytomation) overnight at 4°C. After PBS washes, slides were incubated for 30 minutes with universal immunoperoxidase polymer (DakoCytomation). Cells were then washed with PBS and incubated (AEC; DakoCytomation) for 20 minutes, followed by counterstaining with hematoxylin (Sigma-Aldrich). For negative control, primary antibody was substituted with nonimmune buffer. Stained sections from three independent experiments were evaluated under high magnification (200–400×). The ratio of mean positive cell count to all cell count was calculated in 10 random high-power fields from each slide. The percentage of mean positive cells was then compared using t-test. 
Immunofluorescence Analysis
Immunofluorescence was performed on the M15 cell line and on 6-μm–thick frozen sections from primary UM and from liver metastases of UM using NFκB p100/p52 antibody (1:50; Abcam), RelA (p65) antibody (1:100; NeoMarkers), and cleaved caspase 3 antibody (1:100; Cell Signaling). 
Sections from UM were incubated for 15 minutes with cold acetone (Bio Laboratory, Jerusalem, Israel). M15 cell fixation was performed by 100% cold methanol for 30 minutes. Sections and cells were then washed in PBS (3 × 5 minutes) and incubated with 3% goat serum (Biological Industries) and 0.1% triton-X in PBS containing 1% bovine serum albumin (PBS-BSA), for 30 minutes. Sections and cells were then incubated overnight at 4°C with the primary antibody, washed with PBS (3 × 5 minutes), and incubated with Cy3-conjugated goat anti-rabbit antibodies (1:200; Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature. After washes with PBS, slides were mounted with mounting medium containing DAPI (UltraCruz; Santa Cruz Biotechnology). Negative controls were run using the same protocol while replacing primary antibody with nonimmune buffer. Stained sections were evaluated under high magnification (400×). For cleaved caspase 3, the ratio of mean positive cell count to total cell count was calculated in 10 random high-power fields of four independent experiments. 
Results
NFκB Expression and Activation in Primary UM and in Its Liver Metastases
We have previously shown that NFκB2 mRNA levels are higher in liver metastases from uveal melanoma than in primary uveal melanoma. 5 To further assess NFκB family gene expression in uveal melanoma, we performed QPCR analysis for NFκB1, RelA, NFκB2, RelB, and NIK. All five genes were expressed in UM, and the mRNA levels of NFκB2, RelB, and NIK were significantly higher in metastases samples than in the primary tumors (P = 0.03, 0.05 and 0.03, respectively; t-test; Fig. 1). mRNA from each of these genes was also detected in the two samples of normal choroid, but its level was lower than the levels detected in primary and metastatic tissue. 
Figure 1.
 
QPCR analysis of RelA, RelB, NFκB1, NFκB2, and NIK in primary tumors and in liver metastases from uveal melanoma. RQ, relative quantification of gene expression. Error bars, mean ± SD. All five genes were expressed in each of the samples. *RelB, NFκB2, and NIK show higher expression in metastases than in primary samples (P = 0.03, P = 0.05, and P = 0.03, respectively; t-test).
Figure 1.
 
QPCR analysis of RelA, RelB, NFκB1, NFκB2, and NIK in primary tumors and in liver metastases from uveal melanoma. RQ, relative quantification of gene expression. Error bars, mean ± SD. All five genes were expressed in each of the samples. *RelB, NFκB2, and NIK show higher expression in metastases than in primary samples (P = 0.03, P = 0.05, and P = 0.03, respectively; t-test).
Protein expression of RelA and NFκB2 was assessed by immunofluorescence. Both RelA and NFκB2 were expressed in primary UM tumors and in liver metastases samples. Staining was present in the cytoplasm and in the nucleus (Figs. 2A, 2B). The activation of NFκB in the canonical and noncanonical pathways was further assessed by Western blot analysis. NFκB2 activation was assessed by identifying the p52 form, indicating cleavage of p100. p52 was identified in both primary UM and in its metastases. p52 levels in metastases samples were higher than in primary tumors (P = 0.03; t-test) indicating higher activation of the noncanonical pathway in metastases. RelA (p65) activation was assessed by identifying the phosphorylated form P-p65. P-p65 was identified in both primary and metastatic samples, and there was a nonsignificant trend toward a higher P-p65/p65 ratio in metastatic samples (Fig. 3). 
Figure 2.
 
Immunofluorescence staining for RelA (A, C) and NFκB2 (B, D) in tissues of liver metastases from uveal melanoma (A, B) and in uveal melanoma cell lines (C, D). RelA and NFκB2 are labeled in red; nuclei were counterstained with DAPI (blue). Both RelA and NFκB2 were expressed in the cytoplasm and in the nucleus of tumor cells (400× magnification).
Figure 2.
 
Immunofluorescence staining for RelA (A, C) and NFκB2 (B, D) in tissues of liver metastases from uveal melanoma (A, B) and in uveal melanoma cell lines (C, D). RelA and NFκB2 are labeled in red; nuclei were counterstained with DAPI (blue). Both RelA and NFκB2 were expressed in the cytoplasm and in the nucleus of tumor cells (400× magnification).
Figure 3.
 
Quantification of NFκB1 and NFκB2 proteins in uveal melanoma. (A) Western blot analysis for p100, p52, p65, and P-p65 in lysates from primary uveal melanoma (P) and liver metastases samples (M). (B) Quantification of blots according to band densities (error bars, mean ± SD) demonstrated a 3.9-fold increase of p52 levels in metastases compared with the primary tumor (*P =0.03; t-test).
Figure 3.
 
Quantification of NFκB1 and NFκB2 proteins in uveal melanoma. (A) Western blot analysis for p100, p52, p65, and P-p65 in lysates from primary uveal melanoma (P) and liver metastases samples (M). (B) Quantification of blots according to band densities (error bars, mean ± SD) demonstrated a 3.9-fold increase of p52 levels in metastases compared with the primary tumor (*P =0.03; t-test).
NFκB Protein Expression in Cell Lines from UM Metastases
Immunofluorescent staining demonstrated NFκB2 and RelA expression in the nucleus and cytoplasm in two cell lines derived from metastases of UM (M15 and M85; Figs. 2C, 2D). 
Cell Proliferation Following NFκB Inhibition In Vitro
Effect of inhibition of NFκB using DHMEQ and BMS-345541 was tested on two cell lines, M15 and M85. We first validated that DHMEQ inhibits nuclear translocation of NFκB in uveal melanoma cells (Fig. 4). There was a dose-dependent effect of DHMEQ (2.5, 7.5, and 15 μg/mL medium) on UM cell proliferation in culture, as estimated by MTT assay. Cell proliferation was significantly lower at each inhibitor concentration compared with control cells from day 2 through day 6 (P < 0.001; t-test; Fig. 5). Methylene blue analysis was performed on M15 cells after treatment with 15 μg/mL DHMEQ in comparison with control cells. The results validated findings obtained with MTT and demonstrated a 1.9-fold decrease of cell proliferation (P = 6 × 10−7; t-test). 
Figure 4.
 
DHMEQ inhibits the translocation of NFκB heterodimers to the nucleus. Immunofluorescence staining for RelA in (A) control cells and (B) cells treated with 15 μg/mL DHMEQ for 4 days. RelA is labeled in red; nuclei were counterstained with DAPI (blue).
Figure 4.
 
DHMEQ inhibits the translocation of NFκB heterodimers to the nucleus. Immunofluorescence staining for RelA in (A) control cells and (B) cells treated with 15 μg/mL DHMEQ for 4 days. RelA is labeled in red; nuclei were counterstained with DAPI (blue).
Figure 5.
 
NFκB inhibition in vitro reduces uveal melanoma metastatic cell proliferation as measured by MTT assay. (A, B) NFκB inhibition by DHMEQ (2.5, 7.5, and 15 μg/mL medium) in M15 (A) and M85 (B) cell lines. (C, D) NFκB inhibition by BMS-345541 (1.25, 2.5, and 3.75 μg/mL medium) in M15 (C) and M85 (D) cell lines. Each point represents the average MTT score (±SD). The proliferation of cells treated by DHMEQ and BMS differed from control cell proliferation during days 2 to 6 (P < 0.001 for each time point; t-test).
Figure 5.
 
NFκB inhibition in vitro reduces uveal melanoma metastatic cell proliferation as measured by MTT assay. (A, B) NFκB inhibition by DHMEQ (2.5, 7.5, and 15 μg/mL medium) in M15 (A) and M85 (B) cell lines. (C, D) NFκB inhibition by BMS-345541 (1.25, 2.5, and 3.75 μg/mL medium) in M15 (C) and M85 (D) cell lines. Each point represents the average MTT score (±SD). The proliferation of cells treated by DHMEQ and BMS differed from control cell proliferation during days 2 to 6 (P < 0.001 for each time point; t-test).
BMS-345541, which inhibits NFκB activation, was also tested on M15 and M85 UM cell lines at concentrations of 1.25, 2.5, and 3.75 μg/mL medium for 4 days. There was a dose-dependent decrease in the number of viable cells in treatment compared with control groups in both cell lines on each of the days tested according to MTT assay (P < 0.001; t-test; Fig. 5). 
We further assessed the effect of inhibition of NFκB with 15 μg/mL DHMEQ and 2.5 μg/mL BMS on cell proliferation by immunohistochemistry staining for Ki67, a cell marker for proliferation. A higher number of Ki67-positive cells were present in control cells than cells treated with DHMEQ or BMS for 4 days (9.2-fold; P = 0.04 and 2.1-fold, P = 6 × 10−14; respectively; t-test; Fig. 6). 
Figure 6.
 
Immunohistochemistry for Ki67 (A–C) and activated caspase 3 (D–F) in uveal melanoma cell line. (A, D) Control cells treated with DMSO (magnification, 400×). (B, E) Cells treated with 15 μg/mL DHMEQ for 4 days. (C) Quantification of cells positively stained for Ki67 (error bars, mean ± SD) demonstrated a 9.2-fold lower score in cells treated with 15 μg/mL DHMEQ (*P = 0.04; t-test). (F) Quantification of activated caspase 3 staining showing a higher percentage (6.3-fold) of stained cells in cultures treated with DHMEQ (*P = 7 × 10−7; t-test).
Figure 6.
 
Immunohistochemistry for Ki67 (A–C) and activated caspase 3 (D–F) in uveal melanoma cell line. (A, D) Control cells treated with DMSO (magnification, 400×). (B, E) Cells treated with 15 μg/mL DHMEQ for 4 days. (C) Quantification of cells positively stained for Ki67 (error bars, mean ± SD) demonstrated a 9.2-fold lower score in cells treated with 15 μg/mL DHMEQ (*P = 0.04; t-test). (F) Quantification of activated caspase 3 staining showing a higher percentage (6.3-fold) of stained cells in cultures treated with DHMEQ (*P = 7 × 10−7; t-test).
Apoptosis Following NFκB Inhibition In Vitro
The effect of inhibition of NFκB on apoptosis was analyzed by immunofluorescent staining for cleaved caspase 3 in M15 cells treated with 15 μg/mL DHMEQ for 4 days. A 6.3-fold increase in cells showing caspase 3 activation was found after treatment with DHMEQ compared with controls (P = 7 × 10−7; t-test; Fig. 6). 
Discussion
NFκB transcription factor is constitutively activated and involved in the development of a variety of malignancies, among them carcinoma of the breast, colon cancer, skin melanoma, lymphoma, neuroblastoma, and others, 11,1315,21,22 but to date NFκB involvement in UM has not been characterized. 
We found that NFκB transcription factor family genes, including RelA, NFκB1, RelB, NFκB2, and NIK, are expressed in primary UM and in liver metastases from UM. RelB, NFκB2, and NIK also showed higher expression in liver metastases than in primary tumors. Thus, activation of both the canonical and the noncanonical pathways is present in primary and metastatic UM, with increased activation of the noncanonical pathway in metastases compared with the primary tumor. 
Several mechanisms that underlie NFκB pathway activation in cancer have been described, 23 among them activation by the inflammatory microenvironment of the tumor and genetic aberrations in genes encoding proteins controlling NFκB activation such as NIK and IKK and in regulators of the pathway such as cIAP1/2 and TRAF3, which are crucial for the control of NIK activation. NFκB gene rearrangement, amplification, and overexpression have also been associated with activation of the pathway in cancer. It is still unclear what causes NFκB activation in uveal melanoma. The presence of an inflammatory microenvironment in uveal melanoma has been established 2428 and may potentially account for activation of the pathway in this tumor. Yet, further research is necessary to evaluate the presence of genetic aberrations in genes involved in the control of the pathway. 
Although NFκB is also expressed in noncancerous cells, the contribution of NFκB activation to the progression of several malignancies makes it a potential therapeutic target for cancer. Conceivably, enhanced activation of the pathway in cancer compared with normal tissue will provide a sufficient therapeutic window for such a strategy. Multiple inhibitors of NFκB have been developed, affecting different levels of the NFκB signal transduction pathway. 2932 We evaluated the feasibility of such a strategy for the treatment of metastases from UM. The two inhibitors of NFκB that we have tested act through different mechanisms. DHMEQ inhibits NFκB nuclear translocation, 17,18 whereas BMS-345541 inhibits IKK and, thereby, NFκB activation. 19,20 Both inhibitors were demonstrated to be tolerated by mice in concentrations equivalent to the ones we have used in culture. 19,20,3336 NFκB inhibition by both compounds resulted in reduced UM liver metastatic cell proliferation in the two cell lines we evaluated and in increased apoptosis. 
NFκB regulates more than 100 different genes involved in several important physiological processes, such as inflammation and immune response, cell growth, apoptosis, angiogenesis, cell invasion, and cell adhesion. 8,11,37 Altered NFκB levels and function may modulate the expression of genes regulated by this transcription factor, thereby influencing processes important for tumor progression and metastasis development. 
The feasibility of targeting molecules that play a key role in the growth of a tumor as a therapeutic approach has been demonstrated for the treatment of several cancers. For example, application of the drug imatinib, a tyrosine kinase inhibitor, is useful in patients with chronic myeloid leukemia. 38 Further studies are required to explore whether NFκB may serve as a therapeutic target for metastases from UM. 
Supplementary Materials
Footnotes
 Supported by a Clinical-Research Career Development Award from the Israel Cancer Research Fund (IC) and by a research grant from the Israel Cancer Organization (IC).
Footnotes
 Disclosure: R. Dror, None; M. Lederman, None; K. Umezawa, None; V. Barak, None; J. Pe'er, None; I. Chowers, None
The authors thank Yinon Ben Neriah and Eli Pikarsky for helpful discussions and technical support. 
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Figure 1.
 
QPCR analysis of RelA, RelB, NFκB1, NFκB2, and NIK in primary tumors and in liver metastases from uveal melanoma. RQ, relative quantification of gene expression. Error bars, mean ± SD. All five genes were expressed in each of the samples. *RelB, NFκB2, and NIK show higher expression in metastases than in primary samples (P = 0.03, P = 0.05, and P = 0.03, respectively; t-test).
Figure 1.
 
QPCR analysis of RelA, RelB, NFκB1, NFκB2, and NIK in primary tumors and in liver metastases from uveal melanoma. RQ, relative quantification of gene expression. Error bars, mean ± SD. All five genes were expressed in each of the samples. *RelB, NFκB2, and NIK show higher expression in metastases than in primary samples (P = 0.03, P = 0.05, and P = 0.03, respectively; t-test).
Figure 2.
 
Immunofluorescence staining for RelA (A, C) and NFκB2 (B, D) in tissues of liver metastases from uveal melanoma (A, B) and in uveal melanoma cell lines (C, D). RelA and NFκB2 are labeled in red; nuclei were counterstained with DAPI (blue). Both RelA and NFκB2 were expressed in the cytoplasm and in the nucleus of tumor cells (400× magnification).
Figure 2.
 
Immunofluorescence staining for RelA (A, C) and NFκB2 (B, D) in tissues of liver metastases from uveal melanoma (A, B) and in uveal melanoma cell lines (C, D). RelA and NFκB2 are labeled in red; nuclei were counterstained with DAPI (blue). Both RelA and NFκB2 were expressed in the cytoplasm and in the nucleus of tumor cells (400× magnification).
Figure 3.
 
Quantification of NFκB1 and NFκB2 proteins in uveal melanoma. (A) Western blot analysis for p100, p52, p65, and P-p65 in lysates from primary uveal melanoma (P) and liver metastases samples (M). (B) Quantification of blots according to band densities (error bars, mean ± SD) demonstrated a 3.9-fold increase of p52 levels in metastases compared with the primary tumor (*P =0.03; t-test).
Figure 3.
 
Quantification of NFκB1 and NFκB2 proteins in uveal melanoma. (A) Western blot analysis for p100, p52, p65, and P-p65 in lysates from primary uveal melanoma (P) and liver metastases samples (M). (B) Quantification of blots according to band densities (error bars, mean ± SD) demonstrated a 3.9-fold increase of p52 levels in metastases compared with the primary tumor (*P =0.03; t-test).
Figure 4.
 
DHMEQ inhibits the translocation of NFκB heterodimers to the nucleus. Immunofluorescence staining for RelA in (A) control cells and (B) cells treated with 15 μg/mL DHMEQ for 4 days. RelA is labeled in red; nuclei were counterstained with DAPI (blue).
Figure 4.
 
DHMEQ inhibits the translocation of NFκB heterodimers to the nucleus. Immunofluorescence staining for RelA in (A) control cells and (B) cells treated with 15 μg/mL DHMEQ for 4 days. RelA is labeled in red; nuclei were counterstained with DAPI (blue).
Figure 5.
 
NFκB inhibition in vitro reduces uveal melanoma metastatic cell proliferation as measured by MTT assay. (A, B) NFκB inhibition by DHMEQ (2.5, 7.5, and 15 μg/mL medium) in M15 (A) and M85 (B) cell lines. (C, D) NFκB inhibition by BMS-345541 (1.25, 2.5, and 3.75 μg/mL medium) in M15 (C) and M85 (D) cell lines. Each point represents the average MTT score (±SD). The proliferation of cells treated by DHMEQ and BMS differed from control cell proliferation during days 2 to 6 (P < 0.001 for each time point; t-test).
Figure 5.
 
NFκB inhibition in vitro reduces uveal melanoma metastatic cell proliferation as measured by MTT assay. (A, B) NFκB inhibition by DHMEQ (2.5, 7.5, and 15 μg/mL medium) in M15 (A) and M85 (B) cell lines. (C, D) NFκB inhibition by BMS-345541 (1.25, 2.5, and 3.75 μg/mL medium) in M15 (C) and M85 (D) cell lines. Each point represents the average MTT score (±SD). The proliferation of cells treated by DHMEQ and BMS differed from control cell proliferation during days 2 to 6 (P < 0.001 for each time point; t-test).
Figure 6.
 
Immunohistochemistry for Ki67 (A–C) and activated caspase 3 (D–F) in uveal melanoma cell line. (A, D) Control cells treated with DMSO (magnification, 400×). (B, E) Cells treated with 15 μg/mL DHMEQ for 4 days. (C) Quantification of cells positively stained for Ki67 (error bars, mean ± SD) demonstrated a 9.2-fold lower score in cells treated with 15 μg/mL DHMEQ (*P = 0.04; t-test). (F) Quantification of activated caspase 3 staining showing a higher percentage (6.3-fold) of stained cells in cultures treated with DHMEQ (*P = 7 × 10−7; t-test).
Figure 6.
 
Immunohistochemistry for Ki67 (A–C) and activated caspase 3 (D–F) in uveal melanoma cell line. (A, D) Control cells treated with DMSO (magnification, 400×). (B, E) Cells treated with 15 μg/mL DHMEQ for 4 days. (C) Quantification of cells positively stained for Ki67 (error bars, mean ± SD) demonstrated a 9.2-fold lower score in cells treated with 15 μg/mL DHMEQ (*P = 0.04; t-test). (F) Quantification of activated caspase 3 staining showing a higher percentage (6.3-fold) of stained cells in cultures treated with DHMEQ (*P = 7 × 10−7; t-test).
Supplementary Table S1
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