Constitutive NF- KB Activation in Colorectal Carcinoma ...Constitutive NF- KB Activation in Colorectal Carcinoma Plays a Key Role in Angiogenesis, PromotingTumor Growth Kei Sakamoto,1,2

Constitutive NF-KB Activation in Colorectal Carcinoma Plays a KeyRole in Angiogenesis, PromotingTumor GrowthKei Sakamoto,1,2 Shin Maeda,1,2 Yohko Hikiba,1Hayato Nakagawa,2 Yoku Hayakawa,2

Wataru Shibata,1,2 Ayako Yanai,1Keiji Ogura,2 and Masao Omata2

Abstract Purpose: Nuclear factor nB (NF-nB) is an important transcription factor in various biologicalprocesses. Constitutive NF-nB activation has been noted in many tumors, including colorectalcancers. However, the precise role of this activation in colorectal cancer is unclear.Experimental Design: Constitutive NF-nB activation was evaluated in colorectal cancer tissuesand cell lines. To inhibit NF-nB activation, we established cancer cells with stable knockdownof InB kinase g (NF-nB essential modulator), which is the regulatory subunit of the InB kinasecomplex, by RNA interference. Cell growth and apoptosis were evaluated in wild-type cells(WT) and knocked-down cells (KD). Microarray and protein array analysis were also done. Todetermine involvement of angiogenesis, human umbilical vein endothelial cells were used. By s.c.transplantation of the cells into nude mice, tumor sizes, vascularity, and chemodrug sensitivitywere analyzed.Results: Constitutive NF-nB activation was observed in 40% of colorectal cancer tissues and67% of cell lines. Cell proliferation was not different between WT and KD in vitro, whereasapoptosis mediated by tumor necrosis factor-a and 5-fluorouracil were increased in KD. Severalangiogenic chemokines were decreased in KD. Human umbilical vein endothelial cells incubatedin WT supernatant showed more branch points than in KD, suggesting that constitutive NF-nBactivation was involved in angiogenesis. Subcutaneous tumor expansion was suppressed to23% in KD, and vessels were also decreased. By 5-fluoruracil treatment, tumor expansion wassuppressed to a greater extent in KD (to 6%) than inWT (to 50%).Conclusion: NF-nB inhibition may represent a potent treatment modality in colorectal cancer,especially in cases with constitutive NF-nB activation.

Colorectal cancer is the second leading cause of cancer-relateddeath in industrialized nations (1). The development ofcolorectal cancer results from the sequential accumulation ofactivating mutations in oncogenes, such as ras , and mutations,truncations, or deletions in the coding sequences of severaltumor suppressor genes, including p53 and adenomatouspolyposis coli (APC ; ref. 2).Over the last decade, there has been a great deal of progress in

the development of new therapies for the treatment of colorectal

cancer. The cytotoxic chemotherapy drug 5-fluorouracil (FU)was reformulated, and two new drugs, oxaliplatin andirinotecan, have been investigated as adjunctive therapies. Inaddition, targeted therapies, including monoclonal antibodiesagainst vascular endothelial growth factor (VEGF; bevacizumab)and the epidermal growth factor receptor (cetuximab), arenow standard treatments for metastatic colorectal carcinoma(3, 4). However, many cases show tolerance of such treatments(3, 4). Therefore, it is necessary to develop new approachesto replace or complement current therapies.Nuclear factor nB (NF-nB) transcription factors are key

regulators of innate immune responses, inflammation, and cellsurvival (5), and are assembled by dimerization of two ofthe five subunits, p65 (RelA), c-Rel, RelB, p50/NF-nB1, andp52/NF-nB2. Without stimulation, most NF-nB dimers arebound to specific inhibitory proteins, InBs, in the cytoplasm.Many proinflammatory stimuli, such as tumor necrosis

factor-a (TNFa), lipopolysaccharide, and various drugs, canactivate NF-nB, primarily through InB kinase (IKK)-dependentphosphorylation and degradation of the InB inhibitoryproteins. Most stimulation activates the IKK complex, whichconsists of two catalytic subunits, IKKa and IKKh, and aregulatory component, IKKg/NF-nB essential modulator(NEMO). Typically, IKK activation occurs primarily throughIKKh, the absence of which increases susceptibility to TNFa-mediated apoptosis (6). The IKK-dependent phosphorylation

Human Cancer Biology

Authors’Affiliations: 1Division of Gastroenterology, Institute for Adult Diseases,Asahi Life Foundation, 1-6-1 Marunouchi, Chiyoda-ku, and 2Department ofGastroenterology, University of Tokyo, 7-3-1Hongo, Bunkyo-ku,Tokyo, JapanReceived 5/27/08; revised 12/24/08; accepted 12/30/08; published OnlineFirst3/10/09.Grant support: S. Maeda and M. Omata were supported by grants-in-aid from theMinistry of Education, Culture, Sports, Science, and Technology of Japan(#17209026 and #19390205).The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.Note: Supplementary data for this article are available at Clinical Cancer ResearchOnline (http://clincancerres.aacrjournals.org/).Requests for reprints: Shin Maeda, Division of Gastroenterology, Institutefor Adult Diseases, Asahi Life Foundation, 1-6-1 Marunouchi, Chiyoda-ku, Tokyo100-0005, Japan. Phone: 81-3-3201-6781; Fax: 81-3-3201-6881; E-mail:[email protected].

F2009 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-08-1383

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of InBs can lead to ubiquitination and subsequent degradation.NF-nB, free of InB, translocates to the nucleus, where it activatesthe transcription of target genes, including cytokines, chemo-kines, and antiapoptotic genes (7).Many recent studies have indicated the constitutive activation

of NF-nB in various malignant cells, such as lymphomas,leukemias, breast cancers, melanomas, pancreatic cancers, andcolorectal cancers (8–10). Many of these reports suggested thatconstitutively active NF-nB is associated with harmful features oftumor cells, such as antiapoptotic effects, cell growth, and meta-stasis (8, 11). However, tumor cells with constitutively activeNF-nB have not been studied in detail. In addition, the effects ofsuch constitutive activation in vivo are not well understood.As NF-nB activation contributes to antiapoptotic effects, cell

growth, metastasis, and chemokine secretion, the inhibition ofNF-nB activation may be useful in antitumor therapy. Indeed,there have been some reports of NF-nB inhibition as a methodof tumor therapy (12), but these are as yet insufficient toconsider the use of a NF-nB inhibitor as a first-line therapy forcancer.The present study was designed to investigate the nature of

constitutively active NF-nB in vitro and in vivo, using colorectalcarcinoma cell lines. To inhibit NF-nB activation, we knockeddown IKKg, a key element in NF-nB activation, by smallinterfering RNA (siRNA), and established stable knockdowncell lines. In vivo, tumor expansion was strongly suppressed inknockdown cells and tumor vascularity was also suppressed.The results suggest that NF-nB inhibition may be an attractivetarget for the treatment of colorectal cancer.

Materials and Methods

Cell lines. The human colorectal carcinoma cell lines weremaintained as instructed by the American Type Culture Collection.Human umbilical vein endothelial cells (HUVEC) were purchased

from PromoCell and were maintained according to the manufacturer’sinstructions.

Reagents. Human TNFa was purchased from R&D Systems. FU waspurchased from Wako Pure Chemical Industries, Ltd. The polyclonal

anti-phospho-InBa(Ser32), anti-phospho-NFnB-p65(Ser536), anti-phospho-Jun NH2-terminal protein kinase (JNK; Thr183/Tyr185), and

anti-phospho-p38 (Thr180/Tyr182) antibodies were purchased from

Cell Signaling Technology. The anti–von Willebrand factor antibodywas from DAKO. The other antibodies were obtained from Santa Cruz

Biotechnology.Animals. Six-week-old male BALB/cAJcl-nu/nu (nude mice) were

purchased from CLEA Japan, Inc. The animals were maintained under

standard laboratory conditions at room temperature with relativehumidity of 55% F 5% (mean F SD) and a 12/12-h light/dark cycle.

This study was approved by the Ethics Committee of the Institute forAdult Diseases, Asahi Life Foundation. Animal experiments were

conducted in accordance with the Guidelines for the Care and Use of

Laboratory Animals of the Institute for Adult Diseases, Asahi LifeFoundation, according to NIH guidelines.

Establishment of IKKg knockdown cells. IKKg siRNA plasmid and

negative control plasmid were purchased from IMGENEX Corporation.Transfection was done using the FuGENE HD Transfection Reagent

(Roche Diagnostics). To establish stable knockdown cell lines, wetransfected siRNA plasmids into DLD-1 and LOVO cells, and cultured

the cells in the presence of 100 Ag/mL geneticin.

Microarray analysis. RNA was extracted from DLD-1, LOVO, andknockdown cells using Isogen (Wako). The samples were treated with

DNase for 1 h, and then purified using an RNA purification kit(Qiagen). We used the Agilent Human Whole Genome Array (Agilent

Technologies) containing 60-mer DNA probes synthesized in situ in a

44-k format. Chips were scanned by Takara Bio Inc. according to themanufacturer’s hybridization protocol.

Quantitative real-time PCR, immunoblotting, and electrophoretic

mobility shift assay. These procedures were conducted as described

previously (13).Cell cycle analysis by flow cytometry. The proportions of cells in

G0-G1, S, and G2-M were determined by flow cytometric analysis ofDNA content. Briefly, cells obtained by trypsinization were washed withPBS, and suspended in 70% ethanol. Then cells were washed againwith PBS, and incubated in propidium iodide solution (50 mg/mL in

PBS) for 1 h. Then cells were analyzed with fluorescence-activatedcell sorting.Chemokine array, human interleukin-8 (CXCL8), and monocyte

chemotactic protein-1 (CCL2) ELISA. Culture supernatants wereassayed using a Human Chemokine Antibody Array (RayBiotech,Inc.). The supernatants were assayed by ELISA (R&D Systems Inc.).Surgically resected human colorectal carcinoma and neighboringnormal colon tissue, stored at -70jC, were minced in ice-cold bufferconsisting of 50 mmol/L Tris-HCl (pH 7.4), 1% (v/v) Triton X-100,150 mmol/L NaCl, 1 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonylfluoride (TNET buffer), 0.1 mmol/L Na3VO4 supplemented withComplete Mini EDTA-free (Roche Diagnostics). The lysates werecentrifuged (10,000 g , 5 min, 4jC) and the supernatants were usedfor analyses.Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphos-

phate nick-end labeling assay. Cells were plated in chamber slides andincubated for 24 h, and then stimulated with TNFa (30 ng/mL) or FU(15 Amol/L) for 24 h. Apoptosis was analyzed using an Apoalert DNAFragmentation Assay kit (Takara Bio Inc.). Apoptosis in tumors wasanalyzed using paraffin-embedded sections as described above.Measuring DNA fragments. Cells were plated in 12-well plates

(5 � 104 cells/well), incubated for 24 h, and then stimulated with TNFa(30 ng/mL) or FU (15 Amol/L) for 24 h. The supernatants wereharvested, and DNA fragments were measured using a Cell DeathDetection ELISA plus kit (F. Hoffmann-La Roche Ltd.).RNA interference. The interleukin 8 (IL-8) siRNA was purchased

from Qiagen. The monocyte chemotactic protein-1 (MCP-1) siRNA was

Translational Relevance

There has been a great deal of progress in the develop-ment of new therapies for the treatment of patients withcolorectal cancer. However, colorectal cancer is still thesecond leading cause of cancer-related death in industrial-ized nations. Therefore, it is necessary to develop newapproaches to replace or complement current therapies.

Nuclear factor nB (NF-nB) is an important transcriptionalfactor that controls various biological processes. Consti-tutive NF-nB activation has been noted in many tumorsincluding colorectal cancer. However, the exact role of theactivation in colorectal cancer is still unclear.

In the present study, we showed that constitutive activa-tion of NF-nB was frequently observed in colorectal cancer.Cancer cells with stable knockdown of InB kinase g wereestablished by using RNA interference and found tumorexpansion was strongly suppressed in knocked-downcells and tumor vascularity was also suppressed. With thecombination of 5-fluorouracil, tumor expansion was nearlystopped. We propose that NF-nB inhibition may representa potent treatment modality in colorectal cancer, especiallyin cases with constitutive NF-nB activation.

Constitutive NF-kBActivation in Colorectal Carcinoma

www.aacrjournals.org Clin Cancer Res 2009;15(7) April 1, 20092249



Fig. 1. Detection of constitutively active NF-nB incolorectal carcinomas and establishment of IKKgknock-down cell lines. A, colon cancer tissues werestained using an anti-p65 antibody. A representativesample of constitutively activated NF-nB (�400; leftpanel) and a negative sample (�400; right panel).The stained nuclei were recognized as dark brownspots, and pointed by arrows. B, detection ofconstitutively active NF-nB in colorectal cancercell lines by electrophoretic mobility shift assay.Nuclear protein recovery was determined byimmunoblotting withTF2D. To determine NF-nBband, supershift by anti-p65 antibody and anti-p50antibody, and loss of the band by cold probe wereconfirmed. C, IKKg was stably knocked down inDLD-1and LOVO by siRNA. Cell lysates of each cellline were gel-separated and immunoblotted withantibodies to the indicated proteins. NF-nBactivation was determined in wild-type cells (WT)and knock-down cells (KD) by electrophoreticmobility shift assay. As a control, we transfectednegative control plasmid to DLD-1and LOVO(DLD-1Emp and LOVO Emp). D, cell growth wasmeasured by counting the number of cells. Cell cyclewas analyzed by flow cytometry with propidiumiodide staining.





purchased from GE Healthcare. These siRNAs were transfected withLipofectamine 2000 (Invitrogen Life Technologies) into the cells. After24 h of transfection, we changed the medium, cultured cells for24 h more, and measured the concentration of IL-8 and MCP-1 byELISA.

In vitro angiogenesis assay. Transformation of HUVEC was assessedusing a Fibrin Gel In vitro Angiogenesis Assay Kit (ChemiconInternational, Inc.), in accordance with the manufacturer’s instructions.HUVECs were incubated in the test medium for 72 h, and branchpoints in several random views (200 � magnification) were countedand the values averaged.

In vivo angiogenesis assay. A DIVAA assay (Trevigen) was done inaccordance with the manufacturer’s instructions. Briefly, angioreactors

(silicone cylinders closed at one end) were filled with 20 AL ofTrevigen’s basement membrane extract premixed with supernatant ofDLD-1 or LOVO. They were then implanted s.c. into 6-wk-old nudemice. Fourteen days after implantation, the angioreactors were collectedand processed. To quantify the invasion of endothelial cells into theangioreactors, they were labeled with FITC-lectin, and the fluorescentsignals were determined.

In vivo evaluation in human tumor xenograft models. Nude micewere implanted s.c. with 1 � 106 cells of the LOVO human colorectalcarcinoma cell line, and tumor size was measured. For FU treatment,the method has been described previously (14). Briefly, nude mice wereimplanted s.c. with LOVO, and when tumors reached a diameter ofapproximately 5 mm, the animals were pair-matched into treatment

Fig. 2. IncreasedTNFa-induced apoptosis and JNK activation in IKKg knockdown cells. A,WT, Emp, and KD were incubated withTNFa (30 ng/mL) for 24 h and wereanalyzed by 46-diamidino-2-phenylindole (DAPI) and TUNEL staining, and percentages of apoptotic cells are shown. B, free DNA levels in the supernatant of WT, Emp, andKD after 24-h incubation with 30 ng/mL TNFa were measured. C, cells were lysed at the indicated times afterTNFa treatment, and were gel-separated and immunoblottedwith antibodies to the indicated proteins. D, antiapoptosis-related gene expression in WTand KD of LOVO was determined by real-time PCR. GAPDH RNA was used fornormalization.





and control groups (day 1). Each group consisted of eight tumors. FUwas administered i.p. at the dose of 100 mg/kg on days 1, 12, and 19.The control group received only PBS, the vehicle used for FU.Immunohistochemistry and in vivo TUNEL assay. The tumors were

removed and fixed in 10% formalin, embedded in paraffin, sectioned,and stained with H&E. For immunohistochemistry, sections weredeparaffinized, rehydrated, treated with 3% H2O2 in PBS, andincubated overnight at 4jC with primary antibodies. The binding ofthe primary antibody was detected with biotin-labeled antirabbit IgG orantimouse IgG antibodies (Vector Laboratories), followed by strepta-vidin-horseradish peroxidase reaction, visualization with 3,3¶-diamino-benzidine (Sigma-Aldrich), and counterstaining with hematoxylin. Toevaluate apoptosis, a terminal deoxynucleotidyl transferase–mediateddeoxyuridine triphosphate nick-end labeling (TUNEL) assay was done,in accordance with the manufacturer’s instructions (ApopTag kit;Intergen Company).Statistical methods. Statistical analysis was done using Student’s

t-test and two-sided or one-way ANOVA with Dunnett’s multiplecomparison. Differences were deemed statistically significant atP < 0.05.

Results

Constitutive activation of NF-kB in colorectal carcinoma. Todetermine whether NF-nB was constitutively active in humancolorectal carcinoma tissues, a tissue array was stained

immunohistochemically with an anti-p65 antibody. NF-nBactivation was determined when p65 nuclear staining wasobserved in >50% of the cancer cells in the carcinoma tissues(Fig. 1A and Supplemental Fig. S1); activation was observed in40% (35 of 88) of colorectal carcinomas.We also examined whether NF-nB was constitutively active in

colorectal carcinoma cell lines by electrophoretic mobility shiftassay (Fig. 1B). Constitutive NF-nB activation was observed in6 of 9 cell lines (DLD-1, HCT15, HCT116, HT29, LOVO, andSW620), among which DLD-1 and LOVO showed relativelystrong activity. Total p65 levels were slightly different amongcells, but were not correlated with the levels of NF-nB activity(Supplemental Fig. S2). These results suggest that constitutiveactivation of NF-nB occurs frequently in colorectal carcinoma.To explore the role of constitutive NF-nB activation in

colorectal carcinomas, we established cancer cells with stableknock-down of IKKg (NEMO), a regulatory subunit of the IKKcomplex, using siRNA, in DLD-1 and LOVO cells. As a control,we transfected negative control plasmid to DLD-1 and LOVO(DLD-1 Emp and LOVO Emp). IKKg was successfully knockeddown, but there were no differences in IKKa or IKKh amongwild-type cells (WT), Emp, and knock-down cells (KD; Fig. 1C).Phosphorylated p65 levels were decreased in KD as comparedwith WT and Emp, whereas p65 levels were unchanged

Table 1. Microarray analysis was done on RNA samples from DLD-1-WT, DLD-1-KD, LOVO-WT, and LOVO-KD

Gene type Accession no. Gene symbol Fold change inDLD-1 (Log10)

Fold change inLOVO (Log10)

Chemokines NM_001511 CXCL1 -1.07 -2.56NM_002089 CXCL2 -0.721 -1.63NM_002090 CXCL3 -0.636 -1.77NM_000584 IL8 (CXCL8) -0.695 -1.93NM_004591 CCL20 -0.808 -1.78

Cytokine NM_002341 LTB -0.923 -1.39Cytokine receptor NM_000640 IL13RA2 0 -0.655Apoptosis NM_006290 TNFAIP3 (A20) -0.326 -0.831

NM_001165 BIRC3 (IAP-2) -0.155 -0.714Drug resistance NM_014331 SLC7A11 -0.539 -1.06

NM_000767 CYP2B6 -0.178 -0.815NM_017460 CYP3A4 0 -1.22NM_000777 CYP3A5 0 -1.47NM_000765 CYP3A7 -0.00538 -1.21NM_023944 CYP4F12 0.0532 -0.871NM_014331 SLC7A11 -0.539 -1.06NM_000691 ALDH3A1 -0.533 -0.724NM_020299 AKR1B10 -0.605 -0.564NM_052816 TRIM31 -0.976 -0.784NM_005764 PDZK1IP1 -0.749 -0.905NM_001072 UGT1A6 -0.696 -0.503

metastasis NM_000201 ICAM1 -0.045 -1.17AF100640 Homo sapiens metastasis

related protein (MB2)-0.991 -1.78

Tumor growth NM_001432 EREG -0.208 -0.641NM_000909 NPY1R 0 -1

Tumor marker NM_001712 CEACAM1 (CEA) 0.0037 -1NM_001815 CEACAM3 (CEA) 0 -1.07NM_006890 CEACAM7 (CEA) -0.088 -0.799

Oncogene NM_020387 RAB25 0.0385 -0.886NM_020630 RET 0 -0.819

Ubiquitination NM_006398 UBD -0.507 -1.33Cytoskeleton BC022483 ARHGAP29 (Rho GTP ase) -0.747 -0.833

NOTE: Those genes that were suppressed about 30% or less (<10-0.5) at least in one KD were considered to be NFnB-regulated genes. TheGenBank accession number, the common name, the fold change are shown.





(Fig. 1C). We also examined NF-nB activation by electropho-retic mobility shift assay, and binding activities were markedlydecreased in KD (Fig. 1C).To evaluate the effects of constitutive NF-nB activation on cell

growth, we measured cell numbers at several time points andshowed that cell growth did not differ among WT, Emp, andKD in either DLD-1 or LOVO (Fig. 1D). Cell-cycle of WTand KD was determined by flow cytometry, and G2/G1 ratio didnot change in WT versus KD (Fig. 1D). Cell cycle regulators inWT and KD were also analyzed; the levels of cyclin A, cyclin D1,and cyclin E were not different between WT and KD (data notshown).Increased TNFa-induced apoptosis and JNK activation in IKKg

knock-down cells. It has been reported that blocking NF-nBsignals reduces survival ability against TNFa stimulation. Wetested TNFa-induced apoptosis in WT, Emp, and KD. Cells wereincubated for 24 hours with or without TNFa (30 ng/mL), andTUNEL assay was then done. TNFa induced more apoptosisin KD than in WT in both DLD-1 and LOVO (Fig. 2A andSupplemental Fig. S3). The incubated supernatant was sampledto determine the free DNA levels as an indicator of apoptosis byELISA. The free DNA level was increased to a greater extent inKD than in WT by TNFa treatment (Fig. 2B).It has been suggested that NF-nB activation may prevent

TNFa-induced apoptosis by attenuating the activation of JNK(15, 16). TNFa-induced JNK activation was weak in WT andEmp, whereas the activity was strongly induced in KD. Theseresults indicate that one of the mechanisms by whichconstitutive NF-nB activation prevents TNFa-induced apoptosisin WT and Emp is through attenuation of JNK activation.Activation of p38 was also weak in WT and Emp as comparedwith KD. On the other hand, InBa was constitutively activatedwith or without TNFa stimulation in WT and Emp, whereasconstitutive activation was suppressed in KD, as expected(Fig. 2C).Chemokines were secreted from cells with constitutively active

NF-kB. To investigate the target genes of constitutively activeNF-nB in DLD-1 and LOVO cells, transcription profiles weredetermined by microarray analysis. In DLD-1 cells, 234 of43,376 genes were down-regulated in KD as compared withWT. In LOVO cells, 693 of 43,376 genes were down-regulated.The down-regulated genes included those for immuneresponses, antiapoptosis, and signal transduction; representa-tive genes are shown in Table 1. We did real-time PCR analysisof the genes related to antiapoptotic effects. The levels of A20and cIAP2 RNA expression were decreased in LOVO-KD inaccordance with the results of microarray analysis, whereasCYLD and cIAP1 expression were unchanged (Fig. 2D). Severalof the genes that were down-regulated were chemokines; theywere strongly suppressed by inhibition of NF-nB activity. Thus,we next analyzed chemokines secreted into the supernatant ofWT, Emp, and KD using a chemokine protein array. IL-8 andGro in DLD-1 cells, and IL-8, MCP-1, Gro, and Groa in LOVOcells were highly secreted in WT and Emp relative to KD celllines (Fig. 3A). These results suggest that human colorectalcarcinomas with constitutively active NF-nB secrete suchchemokines. Among these chemokines, we measured IL-8 andMCP-1 in 20 advanced cancer tissue samples and surroundingnormal tissue samples. Seven tumors showed high levels ofIL-8 (>500 pg/mg) and six showed high levels of MCP-1(>80 pg/mg). Interestingly, the tissues showing high levels of

IL-8 coincided almost completely with those showing elevationof MCP-1. None of the normal tissues showed high levels ofIL-8 or MCP-1 (Fig. 3B).Next, IL-8 and MCP-1 were measured in supernatants of

colorectal carcinoma cell lines. High levels of IL-8 (>100 pg/mL)were detected in 4 of 9 samples, and high levels of MCP-1(>8 pg/mL) were detected in 4 of 9 samples. Especially, DLD-1,LOVO, and SW620 secreted high IL-8 and high MCP-1, andall of the cell lines with high IL-8 showed constitutive NF-nBactivation in Fig. 1B (Fig. 3C). These results suggested thatconstitutive IL-8 and MCP-1 secretion are strongly related toconstitutive NF-nB activation. As in the case of NF-nB activation,IL-8 secretion in the supernatant of KD was markedly sup-pressed relative to WT and Emp especially in LOVO (Fig. 3D).MCP-1 secretion was also strongly suppressed in KD of LOVOand decreased to about 62% in KD of DLD-1 (Fig. 3D).Angiogenic effects of tumor cells with constitutively active

NF-kB. As previous studies have indicated that chemokines,including IL-8 and MCP-1, are angiogenic factors (17, 18), weevaluated the angiogenic effects of the supernatants of WTand KD. HUVECs were incubated for 72 hours in thesupernatants of WT and KD. Although many branch pointswere formed in HUVECs incubated in WT supernatant,HUVECs incubated in KD supernatant showed fewer branchpoints (Fig. 4A). These results indicated that certain factorsderived from cells with constitutively activated NF-nB inducedangiogenic effects in HUVECs.Next, to evaluate whether the angiogenic effect was depen-

dent on IL-8 and/or MCP-1, WT were knocked down in IL-8and/or MCP-1 by siRNA, and HUVECs were incubated in thesupernatants of these cells. Branch points in HUVECs incubatedin each supernatant were counted. Although the angiogeniceffect of KD supernatant showed a marked reduction relative toWT and Emp, HUVECs incubated in knockdown IL-8 and/orMCP-1 supernatants showed relatively reduced effects (Fig. 4Band C). These results suggested that certain factors in additionto IL-8 and/or MCP-1 may also be involved in the angiogeniceffects of constitutive NF-nB activation.To confirm these observations in vivo, semiclosed silicone

cylinders (angioreactors) with supernatants of WT and KD wereimplanted s.c. into mice for 14 days. This assay enabledcomparison of the potencies of angiogenic factors or inhibitors.As expected, the KD supernatant induced markedly fewer bloodvessels than that of WT (Fig. 4D).Involvement of constitutively active NF-kB in tumor growth

in vivo. To investigate the role of constitutively active NF-nBin tumor growth in vivo , 1.0 � 106 LOVO-WT and LOVO-KDcells were implanted s.c. into nude mice and the sizes of theresulting tumors were measured. The volumes of LOVO-KDtumors were markedly smaller than those of LOVO-WT tumors(25% on day 52; Fig. 5A). Each tumor was stained immuno-histochemically with an antibody to von Willebrand factor, amarker of vascular endothelial cells. The numbers of bloodvessels in LOVO-KD tumors were markedly lower than those inWT tumors (Fig. 5B). In addition, LOVO-WT tumors containedmore proliferating cells, as determined by proliferating cellnuclear antigen staining, than LOVO-KD tumors (Fig. 5B),whereas both cells showed the same proliferative capacityin vitro (Fig. 1D). We also detected apoptosis by the TUNELassay and showed that LOVO-KD tumors contained moreapoptotic cells than LOVO-WT tumors (Fig. 5B). We confirmed





Fig. 3. Chemokines were secreted from cells with constitutively active NF-nB. A, chemokines secreted into the supernatant of WTand KD were analyzed using a proteinarray. Red, IL-8; blue, MCP-1; green, Gro; brown, Groa. B, IL-8 levels in human colorectal carcinomas and normal tissue surrounding the tumor were measured by IL-8 ELISA(*,19,076 pg/mg; **,12,593 pg/mg; ***, 4,656 pg/mg). MCP-1levels were measured in the same samples as IL-8 ELISA by MCP-1ELISA (b, 345.4 pg/mg; bb, 200.4 pg/mg;bbb, 399.1pg/mL). C, IL-8 levels in the supernatants of colorectal cancer cell lines were measured by IL-8 ELISA (*, 823.4 pg/mL; **, 8,757.5 pg/mL). MCP-1level wasmeasured in the same samples as IL-8 ELISA by MCP-1ELISA (b, 115.7 pg/mL). D, IL-8 and MCP-1levels in the supernatants of DLD-1-WT, DLD-1-Emp, DLD-1-KD,LOVO-WT, LOVO-Emp, and LOVO-KD.





that LOVO-WT tumor cells showed constitutive NF-nB activa-tion by p65 nuclear staining and phosphorylated p65, whereasnuclei of LOVO-KD tumor cells showed less staining (Fig. 5Band Supplemental Fig. S4).Decreased sensitivity to FU treatment in tumor cells with

constitutive NF-kB activation. FU has been a mainstay ofchemotherapy for colorectal cancer. To investigate the role ofconstitutive NF-nB activation in FU treatment, LOVO-WT,LOVO-Emp, and LOVO-KD were treated with FU and apoptoticcell death was evaluated using the TUNEL assay. As in the caseof TNFa, KD were more sensitive to FU treatment than wereWT and Emp (Supplemental Fig. S5 and Fig. 5C). We alsoconfirmed this by measurement of the free DNA level, anindicator of apoptosis, by ELISA (data not shown). Theseresults suggest that constitutive NF-nB activation was involvedin resistance to chemotherapy, such as with FU.

Next, LOVO-WT, Emp, and KD were implanted s.c. into nudemice, and when the tumor diameter reached 5 mm, 100 mg/kgof FU was administered i.p. The toxicity of the therapy, such asdiarrhea, or therapy-related death was not observed. Obviousbody weight loss was not observed in each group. After 35 days,the volumes of the LOVO-WT tumors with FU were about50% of those without FU. FU administration in KD-implantedmice was very effective. KD tumors with FU showed almostno growth during the observation period, and the volume wasabout 23% of the LOVO-KD without FU and about 6% ofthe LOVO-WT tumors without FU (Fig. 5D). Tumor growth ofWT and Emp was almost same. Levels of IL-8 in xenografttumors and serum of tumor-bearing mice were measured.IL-8 levels were higher in WT tumors and serum of WT-inplanted mice than in KD tumors and serum of KD-implantedmice (Supplemental Fig. S6). DLD-1-WT and KD were also

Fig. 4. Angiogenic effects of tumor cellswith constitutively active NF-nB.A, angiogenesis was evaluated byincubating HUVECs in the supernatants ofWTand KD (�400). HUVECs wereincubated for 72 h in the supernatants ofDLD-1-WT, DLD-1-Emp, DLD-1-KD,LOVO-WT, LOVO-Emp, and LOVO-KD.B, C, IL-8 and/or MCP-1were knockeddown by siRNA in DLD-1and LOVO, andHUVECs were incubated in the supernatant.To evaluate angiogenesis, the number ofbranch points was counted. Results areaverages F S.E. Asterisks, significantdifference observed by two-sided ANOVAwith Dunnett’s multiple comparison.D, semiclosed silicone cylinders(angioreactors) with the supernatantsof WTand KD were implanted s.c. intomice for 14 d. Tubes were harvested and thenumbers of blood vessels were counted.Results are averages F S.E. Asterisks,P < 0.05 by Student’s t-test.





implanted s.c. into nude mice, and when the tumor diameterreached 5 mm, 100 mg/kg of FU was administered i.p. The

growth curves of DLD-1 cells were similar to that of LOVO cells

(Supplemental Fig. S7).

Discussion

It has been reported that constitutive activation of NF-nB isfrequently observed in tumors, including leukemias and

Fig. 5. Involvement of constitutively active NF-nB in tumor growth in vivo and decreased sensitivity against FU treatment in tumor cells with constitutive NF-nB activation.Five mice were used for each group. A, LOVO-WT/-KD were transplanted s.c. into BALB/c nude mice, and tumor volume was measured. B,WTand KD tumors were stainedimmunohistochemically with an anti ^ von Willebrand factor antibody (�200), an anti ^ proliferating cell nuclear antigen antibody (�600), and an anti-p65 antibody(�600). In anti ^ von Willebrand factor antibody ^ stained praparat, tumor vascularity was estimated and the arrows indicate blood vessels.WTand KD tumors were alsoanalyzed byTUNEL (green) and DAPI (blue) staining (�400).The stained p65 nuclei were recognized as dark brown spots, and pointed by arrows. C, LOVO-WT/-Emp/-KDwere treated with FU for 24 h and apoptotic cell death was evaluated byTUNEL and DAPI staining, and percentages of apoptotic cells are shown. D, LOVO-WT, -Emp, and-KD were implanted s.c. into BALB/c nude mice; 100 mg/kg of FU was administered i.p. when the tumor diameter became 5 mm, and tumor volume was later measured.





lymphomas, as well as solid tumors, such as prostate,colorectal, and pancreatic carcinomas (8, 19, 20). Thisactivation in tumor cells leads to overexpression of a varietyof NF-nB target genes, conferring growth advantages, such asantiapoptosis- and cell cycle–promoting genes. In addition,constitutive activation of NF-nB is associated with strongresistance to chemotherapy and radiotherapy (21).We showed that secretion of angiogenic factors and

antiapoptotic effects may be important mechanisms of tumor-mediated constitutive NF-nB activity. VEGF is believed to beone of the most important factors in tumor angiogenesis (22).It has been reported that VEGF is constitutively secreted fromcertain tumor types and that its expression can be inducedunder hypoxic conditions dependent on hypoxia-induciblefactor (23). Anti-VEGF therapy for colorectal cancer has beenused clinically, but it does not show long-term effects (24, 25).In advanced colon cancer, tumor angiogenesis is thought tobe the most important factor for tumor growth. In additionto VEGF, many other factors involved in angiogenesis havebeen reported (22). However, the effects of these factors onangiogenesis remain unknown in vivo .Here, we propose that NF-nB inhibition may represent a

potent antiangiogenic treatment modality in colorectal cancer,especially in cases with constitutive NF-nB activation. Micro-array and protein array analyses showed that the levels ofseveral chemokines associated with angiogenesis were increasedin cells with constitutive NF-nB activation (26–28). However,there have been few studies examining the significance of suchchemokine secretion in clinical cases. Using surgically resectedspecimens of colorectal carcinomas and adjacent normaltissues, IL-8 and MCP-1 levels were shown to be significantlyincreased in >30% of tumor specimens. In addition, specimenswith high IL-8 levels and with high MCP-1 levels tended tocoincide, as seen in colorectal carcinoma cell lines. Although wehave not yet analyzed other candidates, such as CXCL1 orCXCL2 (29), it seems that there are no unique factors amongNF-nB–regulated genes involved in angiogenesis. However,inhibition of NF-nB, a master regulator of these candidates,is sufficient to inhibit angiogenesis mediated by tumor cells.In this regard, NF-nB inhibition may be a potent form ofantiangiogenic therapy for colorectal cancer.Second, NF-nB activation is also a master regulatory step in

antiapoptosis. Several mechanisms have been reported regard-ing this antiapoptotic effect of NF-nB activation (30). NF-nBexerts its prosurvival activity primarily through the induction oftarget genes, the products of which inhibit components ofthe apoptotic machinery. These include FLIP, Bcl-XL, cellularinhibitor of apoptosis (c-IAP), and X chromosome-linkedinhibitor of apoptosis (XIAP; refs. 30, 31). FLIP inhibitsapoptosis by interfering with caspase-8 activation. c-IAP andXIAP bind directly to and inhibit effector caspases. In addition,decreased NF-nB activity and elevated JNK activity have beenshown to promote cell death through ROS accumulation(15, 16, 32). In this study, we showed that constitutiveactivation of NF-nB promoted antiapoptotic effects againstTNFa and FU in tumor cells. Microarray and real-time PCRanalyses indicated that the levels of expression of apoptosis-related genes, such as A20 and cIAP2 , were decreased byinhibition of constitutive NF-nB activation, whereas apoptosis-related genes, such as cIAP1 and CYLD , known to be regulatedby NF-nB activation, were apparently unaffected. It remains to

be determined whether gene expression regulated by NF-nBactivity differs between inducible and constitutive activation.We also showed that constitutive activation of NF-nB promotedthe antiapoptotic function against FU in vivo . This increasedsensitivity of FU treatment by inhibition of NF-nB activationhas been reported in many cancer cell types, suggesting that theeffect is not only in cells with constitutively active NF-nBactivity, but also in those with FU-inducible NF-nB activation(33). These results suggest that combination therapy with FUand an inhibitor of NF-nB should be effective in tumors bothwith and without constitutively active NF-nB.We have not analyzed the mechanism(s) leading to

constitutive activation of NF-nB. The fundamental reason forthe constitutive activation of NF-nB in tumors remains unclear.However, in hematopoietic tumor cells, such as BCL10 andMALT1, NF-nB is activated and has been implicated in thepathogenesis of mucosa-associated lymphoid tissue lympho-mas (34). In multiple myeloma, a majority of myelomasamples and cell lines had elevated NF-nB target geneexpression, associated with genetic or epigenetic alterations inNIK, TRAF3, CYLD, CD40, NFKB1, or NFKB2 (35, 36). CYLDwas originally identified as a tumor suppressor that is mutatedin familial cylindromatosis and plays a role in NF-nBregulation. Hellerbrand et al. reported that CYLD was down-regulated or lost in all tumor cell lines investigated as comparedwith primary human colonic epithelial cells (37). In addition,Zhang et al. reported that CYLD-deficient mice were moresusceptible to induction of colonic inflammation and showed amarked increase in the incidence of tumors as compared withcontrols in a colitis-associated cancer model (38). We analyzedCYLD mRNA and protein expression, but found no differencebetween WT and KD (data not shown). In addition, it hasbeen reported that basal levels of NF-nB activity were notdifferent between wild-type and CYLD-deficient cells.The application of an inhibitor of NF-nB may prove useful as

anticancer therapy. Given the antiapoptotic effect of NF-nB, itsinhibition should result in apoptosis. Furthermore, constitutiveNF-nB activation is involved in tumor angiogenesis, assuggested by this study. Currently, a great deal of researcheffort is being devoted to the development of IKKh/NF-nBinhibitors (39). Recently, to test NF-nB inhibitors for potentialclinical applicability, we showed that the NEMO-bindingdomain peptide, an amino-terminal a-helical region of NEMO,associated with a carboxyl-terminal segment of IKKa andIKKh (40) and has been shown to block the association ofNEMO with IKKh and to inhibit NF-nB activity and reducedinflammatory injury (41) and the incidence of colitis-associatedcancer (data not shown) in mice. These results indicate thatIKKh-targeted NF-nB blockade, using the NEMO-bindingdomain peptide, may be a reasonable therapeutic approachfor inflammatory bowel disease and colitis-associated cancer. Inaddition to the NEMO-binding domain peptide, as describedabove, several NF-nB inhibitors, such as proteasome inhibitors,BAY11, the soy isoflavone genistein, parthenolide, and dehy-droxymethylepoxyquinomicin, have been developed. It hasalso been shown that steroids and nonsteroidal anti-inflam-matory drugs block NF-nB (42). However, few cell types willundergo apoptosis solely via NF-nB inhibition, and it is likelythat a NF-nB inhibitor will be effective only in combinationwith other anticancer agents or radiotherapy. As activation ofNF-nB by some agents or stimuli may mediate apoptosis under





certain conditions, it will be necessary to investigate thefunction of NF-nB, proapoptotic or antiapoptotic, before theuse of inhibitors against such tumors.In summary, constitutive activation of NF-nB is frequently

observed in colon cancer, and plays a key role in angiogenesisand antiapoptosis, promoting tumor growth. Molecular tar-geted therapy against NF-nB activation should be effective incolorectal carcinomas, especially those with constitutive NF-nBactivation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are grateful to Drs.Takaaki SanoI andTeiji MotojimaII (Divisions of IPathologyand IIAbdominal Surgery, Motojima General Hospital, Gumma, Japan) for providingus with the specimen of colorectal carcinoma, and to MitsukoTsubouchi for technicalassistance.

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2009;15:2248-2258. Clin Cancer Res Kei Sakamoto, Shin Maeda, Yohko Hikiba, et al. a Key Role in Angiogenesis, Promoting Tumor Growth

B Activation in Colorectal Carcinoma PlaysκConstitutive NF-

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Constitutive NF- KB Activation in Colorectal Carcinoma ...Constitutive NF- KB Activation in Colorectal Carcinoma Plays a Key Role in Angiogenesis, PromotingTumor Growth Kei Sakamoto,1,2