nuclear factor-b mediates angiogenesis and metastasis of...

Nuclear Factor-�B Mediates Angiogenesis and Metastasis of HumanBladder Cancer through the Regulation of Interleukin-81

Takashi Karashima, Paul Sweeney,Ashish Kamat, Suyun Huang, Sun J. Kim,Menashe Bar-Eli, David J. McConkey, andColin P. N. Dinney2

Departments of Cancer Biology [T. K., S. H., S. J. K., M. B-E.,D. J. M., C. P. N. D.] and Urology [P. S., A. K., C. P. N. D.], TheUniversity of Texas M. D. Anderson Cancer Center, Houston, Texas77030

ABSTRACTPurpose: Interleukin (IL)-8 is an important mediator of

angiogenesis, tumorigenicity, and metastasis in transitionalcell carcinoma (TCC) of the bladder. Nuclear factor �B(NF-�B)/relA regulates IL-8 expression in several neo-plasms. The purpose of this study was to determine whetherthe organ microenvironment (hypoxia, acidosis) regulatesthe expression of IL-8 in TCC via NF-�B, and whetherinhibition of NF-�B function by mutant I�B-� preventsinduction of IL-8 expression.

Experimental Design: IL-8 mRNA expression and pro-tein production by human TCC cell lines (UM-UC-14,HTB-9, RT-4, KU-7 and 253J B-V) were measured byNorthern blot analysis and ELISA under acidic (pH 7.35–6.0) and hypoxic (1.0% O2) conditions. The involvement ofNF-�B and activator protein 1 in the regulation of IL-8production was evaluated by electrophoretic mobility shiftassay. Furthermore, the tumorigenicity and metastatic po-tential of UM-UC-14 cells were determined after transfec-tion with mutant I�B-�.

Results: We found that acidic and hypoxic conditionsincreased IL-8 mRNA expression and protein production byseveral, but not all, TCC cell lines evaluated. NF-�B, but notactivator protein 1, was inducibly activated in UM-UC-14under both acidic and hypoxic conditions, but not in UM-UC-14 mutant I�B-� transfectants. Tumor growth andlymph node metastasis were inhibited in UM-UC-14 mutantI�B-� transfectants compared with UM-UC-14 controls.This effect was associated with the inhibition of IL-8 pro-duction, cellular proliferation, and angiogenesis.

Conclusions: These results suggest that TCCs of thebladder have heterogenic responses to physicochemicalchanges in the microenvironment and identify NF-�B as apotential molecular target for therapy.

INTRODUCTIONIL-8,3 a member of the superfamily of CXC chemokines,

has a wide range of proinflammatory effects. It was initiallydescribed as a neutrophil and lymphocyte chemoattractant (1, 2)but has subsequently been identified as a proangiogenic agentand a modulator of collagenase secretion (3–10). Recently, ithas been appreciated that IL-8 regulates angiogenesis in a widerange of human malignancies (2, 11–13), including TCC of thebladder (14), and that the level of expression directly correlateswith the metastatic potential of TCC (15) Whether IL-8 acts asan autocrine growth factor or an angiogenic factor in TCC isunclear, but in any event, several biological functions of IL-8are of significance to the pathology and treatment of this disease(13, 15).

Two important promoter regions have been identified forthe transcriptional regulation of IL-8, a distal promoter elementcomposed of an AP-1-binding site and a proximal promoterelement containing binding sites for nuclear factor-IL-6 andNF-�B (16). NF-�B is a dimeric transcription factor composedof five members of the NF-�B/relA family. In nonlymphoidmammalian cells, NF-�B exists predominantly as a heterodimercomposed of RelA (p65) and NF-�B1 [p50 (17, 18)]. Differentneoplasms have variable constitutive and inducible levels ofNF-�B expression (19–21). NF-�B regulates the expression ofproangiogenic molecules, including IL-8 (20), and in turn isregulated by a family of inhibitory proteins, I�Bs, which se-quester NF-�B in the cytosol (21–24). Certain stimuli trigger acascade of events leading to the phosphorylation of I�B-�, itspolyubiquitination, and subsequent degradation by the 26S pro-teasome (25). Thus, NF-�B is liberated, allowing translocationto the nucleus and transcription of target genes including IL-8(26). I�B-� degradation requires phosphorylation of specificserine residues at sites 32 and 36. Substitution of these serineresidues interferes with I�B-� phosphorylation, polyubiquitina-tion, and degradation and therefore inhibits the transcriptionalactivity of NF-�B (27–30). Recently, Huang et al. (31, 32)reported that blockade of NF-�B by mutant I�B-� reducedconstitutive expression of vascular endothelial growth factor

Received 12/9/02; revised 3/12/03; accepted 3/12/03.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by NIH Specialized Programs of Research Excellence Grantin Genitourinary Cancer CA91846 and National Cancer Institute CoreGrant CA16672.2 To whom requests for reprints should be addressed, at Department ofCancer Biology, Box 173, The University of Texas M. D. Anderson CancerCenter, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3250; Fax: (713) 792-8747; E-mail: [email protected].

3 The abbreviations used are: IL, interleukin; TCC, transitional cellcarcinoma; CMEM, complete Eagle’s MEM; NF-�B, nuclear factor �B;TNF, tumor necrosis factor; AP-1, activator protein 1; EMSA, electro-phoretic mobility shift assay; ISH, in situ mRNA hybridization; IHC,immunohistochemistry; PCNA, proliferating cell nuclear antigen;MVD, microvessel density; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase.

2786 Vol. 9, 2786–2797, July 2003 Clinical Cancer Research

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and IL-8 and subsequently decreased angiogenesis, invasion,and metastasis in human ovarian and prostate cancer models.

The regulation of IL-8 by NF-�B in human TCC in re-sponse to physicochemical changes in the microenvironment(acidosis and hypoxia) has not previously been evaluated. Weperformed the studies below to assess the regulation of IL-8 byNF-�B induced by acidosis and hypoxia. We found that NF-�Bhas a central role in regulating the expression of IL-8 in humanTCC.

MATERIALS AND METHODSCell Lines and Culture Conditions. The human bladder

carcinoma cell lines UM-UC-14 (33), HTB-9 (34), RT-4 (35),253J B-V (10, 36), and KU-7 (37) were grown as a monolayerin modified Eagle’s MEM supplemented with 10% fetal bovineserum, vitamins, sodium pyruvate, L-glutamine, nonessentialamino acids, and penicillin-streptomycin (CMEM).

Acidic and Hypoxic Conditions. Cells were plated inculture dishes containing CMEM 48 h before incubation undernormal, acidic, or hypoxic conditions (described below). Whenthe cultures were 70–80% confluent, fresh medium with 1%fetal bovine serum was added, and cultures were incubated for24 h. The dishes were then incubated at 37°C under acidicconditions for 6 h or hypoxic conditions for 12–24 h. Stabili-zation of pH during acidosis was evaluated using three mediaconditions: (a) CMEM (pH 7.35); for acidosis, initial pH (7.0,6.5, 6.0) was adjusted by adding the appropriate amount of 20mM 2-[N-morpholino]ethanesulfonic acid (Fig. 1A); (b) MEMwithout bicarbonate (pH 5.5); for acidosis, initial pH (7.0, 6.5,6.0) was adjusted by adding appropriate amount of 20 mM Tris(hydroxymethyl) aminomethane (Fig. 1B); and (c) mixture ofCMEM/MEM without bicarbonate as buffer medium; for aci-

dosis, an appropriate amount of MEM without bicarbonate (pH5.5) was added to a fixed amount of CMEM (pH 7.35) toachieve initial pH [7.0, 6.5, 6.0 (Fig. 1C)]. During preliminarystudies, we established that maintenance of the pH stability wasoptimized in the CMEM/MEM buffer medium, and thus thiswas used for the experimental studies. For hypoxia experiments,regulation of pH was tested in the three different media de-scribed above. We found that the maintenance of pH stabilitywas optimal in CMEM (Fig. 1D) or in the CMEM/MEM with-out bicarbonate buffered medium (Fig. 1F) but unsatisfactory inthe MEM without bicarbonate alone (Fig. 1E). Thus, the hy-poxia experiments were performed in CMEM at 37°C for 12–24h using 5% CO2-95% air (control) or a hypoxic incubator(Precision Scientific, Winchester, VA) with 1% O2 balancedwith 5% CO2 and nitrogen (hypoxia). For all experiments, thepH of the medium was measured at both the initial and finalpoint of incubation. During hypoxia studies in CMEM, the pHof the medium increased by a maximum of 0.25 unit during theexperiments. During acidosis studies, the pH of the CMEM/MEM buffered medium remained stable except with severeacidosis, during which the pH increased by a maximum of 0.25unit. Thus, the experiments described were performed with pHmonitoring at the start and end of each experiment under verystringent conditions that were achieved using the appropriatelyadjusted medium. Hence, the effects of hypoxia and acidosis oftranscription could be studied independently.

Northern Blot Analysis. Total RNA was extracted di-rectly from each cell line using TRI Reagent (Invitrogen, SanDiego, CA), electrophoresed on 1% denatured formaldehyde-agarose gel, electrotransferred to a Genescreen nylon membrane(DuPont, Boston, MA), and cross-linked with a UV Stratalinker1800 (Stratagene, La Jolla, CA) at 120,000 mJ/cm2. Filters were

Fig. 1 Stabilization of the pH of the medium during acidosis and hypoxia. Preliminary studies were performed to establish optimal mediumconditions to stabilize pH during the hypoxia and acidosis experiments. The pH was measured at the initial and final points in all experiments. Foracidosis, most stable conditions were achieved with CMEM/MEM without bicarbonate buffer medium (C). CMEM or MEM without bicarbonateproduced large variations in pH during the acidosis experiments (A and B). For hypoxia, stable conditions were achieved with CMEM orCMEM/MEM without bicarbonate buffer media (D and F). Large variations in pH occurred with MEM without bicarbonate during hypoxia (E).

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washed, and the membranes were hybridized and probed forIL-8; the presence of GAPDH was used to control for loading.The cDNA probes used in this study were a 0.5-kb EcoRI cDNAfragment corresponding to human IL-8 [a gift of Dr. K. Mat-sushima, Kanazawa, Japan (38)] and a 1.28-kb fragment fromGAPDH for standardizing IL-8 expression level (39). ThecDNA probes were radiolabeled by a random primer techniqueusing a commercial kit (Boehringer Mannheim, Indianapolis,IN) and [�-32P]dCTP (Amersham, Arlington Heights, IL).

IL-8 ELISA. Viable cells (5 � 104) were seeded in96-well plates, and conditioned medium was removed after24 h. The cells were washed and then incubated with 200 �l ofmedium under hypoxic or acidotic conditions as describedabove. For in vivo experiments, blood was collected from thetail vein of each mouse at necropsy. IL-8 protein in cell-freeculture supernatants and in mouse serum was determined usingthe commercial Quantakine ELISA kit (R&D Systems, Minne-apolis, MN). The protein concentration of IL-8 was determinedby comparison of the absorbance with the standard curve. Re-sults were corrected for cell number or tumor weight.

Transfection and Selection of Clones Expressing Mu-tant I�B-�. UM-UC-14 cells were transfected with pLXSN-I�B-�-mutant (pLXSN-I�B-�-M; CLONETECH, Palo Alto,CA) or pLXSN control vector using a lipofection reagent (LifeTechnologies, Inc., Gaithersburg, MD). Mutant I�B-� containsmutations at residues 32 and 36 of the NH2 terminus (S32A andS36A) and a COOH-terminal PEST sequence mutation. Thecultures were placed in a 37°C incubator for 12 h, after whichthe medium was replaced. After 24 h, 800 �g/ml G418 sulfate(Life Technologies, Inc.) was added. The CMEM/G418 mediumwas replaced every 3 days until individual resistant colonieswere isolated and established in culture as individual lines. Allof the lines were maintained in CMEM/G418. The expression ofexogenous pLXSN-I�B-�-M was verified by Western immuno-blot analysis. Two clones (M1 and M2) were selected forin vitro and in vivo studies.

Nuclear and Cell Extracts. Whole-cell lysate was pre-pared in Triton X-100 lysis buffer at 4°C. The supernatants werecleared by centrifugation. For nuclear protein extracts, the cellswere washed with cold PBS and harvested by scraping. Aftercentrifugation, the pellet was resuspended in cytoplasmic lysisbuffer and incubated on ice for 10 min. For nuclear proteinextract from tumor, tissues were mechanically dissociated, ho-mogenized, and resuspended in NP40-based cytoplasmic lysisbuffer and incubated on ice for 20 min. Cell- and tumor-derivedspecimens were centrifuged at 8000 rpm for 2 min at 4°C.Supernatants containing cytoplasmic protein were discarded,and the pellets were resuspended in nuclear lysis buffer for 30min on ice. Protein concentration was measured using the Brad-ford assay.

Western Immunoblot Analysis. Equal amounts of pro-tein were boiled in Laemmli SDS sample buffer, resolved bySDS-PAGE, transferred to nitrocellulose, and probed with rab-bit anti-I�B-� (Santa Cruz Biotechnology, Santa Cruz, CA) orrabbit anti-�-actin (Sigma, St. Louis, MO) at 4°C overnight.After washing, the blots were incubated for 1 h at room tem-perature with horseradish peroxidase-conjugated antirabbit sec-ondary antibody (Amersham). Signals were detected by theenhanced chemiluminescence detection system (Amersham).

The rabbit anti-I�B-� detects both mutant and wild-type I�B-�;however, the former migrates faster in the gel (21).

In Vitro DNA Fragmentation Analysis. UM-UC-14parent cells (UM-UC-14), pLXSN-NEO control vector-trans-fected cells (UM-UC-14-NEO), and mutant I�B-�-transfectedcells (UM-UC-14-M1 and UM-UC-14-M2) were treated withTNF-� for 24 h. Cells were harvested and pelleted by centrif-ugation and resuspended in PBS containing 50 �g/ml propidiumiodide, 0.1% Triton X-100, and 0.1% sodium citrate. Propidiumiodide incorporation as a measure of in vitro DNA fragmenta-tion was measured by fluorescence-activated cell-sorting anal-ysis (37). Cells in the sub-G1 population were assumed to beapoptotic (FACScan; Becton Dickinson, Mountain View, CA).

Electrophoretic Mobility Gel Shift Assay. EMSA wasperformed using nuclear extracts prepared from UM-UC-14cells cultured for various times under normal, acidic, and hy-poxic conditions and from tumor tissue. For EMSA experi-ments, the following double-stranded oligonucleotides wereused: NF-�B; AP-1; and nonspecific oligonucleotide SP-1. Theoligonucleotides were annealed and 5�-end-labeled with[32P]ATP with T4 polynucleotide kinase using standard proce-dures. The binding reaction was carried out by preincubatingnuclear extract protein (5 �g) in 20 ml of HEPES (pH 7.9), 50ml of NaCl, 5% glycerol, 0.1 ml of DTT, and 1 �g ofpoly(deoxyinosinic-deoxycytidylic acid) at room temperaturefor 15 min followed by the addition of the double-stranded[32P]ATP. For labeled competition assays, a 50-fold molarexcess of labeled oligonucleotide was added to the bindingreaction. Where indicated, antibodies to the specific transcrip-tion factors were added for the supershift assays, includinganti-p50 and anti-p65 for NF-�B and anti-jun and anti-fos forAP-1 binding. Samples were loaded onto a 5% polyacrylamidegel. Electrophoresis was performed at room temperature for 3 hat 100 V. The gels were dried and exposed to Kodak film at�70°C

Animals. Male athymic BALB/c nude mice were ob-tained from the Animal Production Area of the National CancerInstitute, Frederick Cancer Research Facility (Frederick, MD).The mice were maintained in a laminar airflow cabinet underpathogen-free conditions and used at 8–12 weeks of age. Allfacilities are approved by the American Association for Accred-itation of Laboratory Animal Care in accordance with the cur-rent regulations and standards of the United States Departmentof Agriculture, the Department of Health and Human Services,and the NIH.

Orthotopic Implantation of Tumor Cells. UM-UC-14,UM-UC-14-NEO, UM-UC-14-M1, and UM-UC-14-M2 cells(60–70% confluent) were prepared for injection as describedpreviously (10). Mice were anesthetized with i.p. sodium pen-tobarbital. For orthotopic implantation, a lower midline incisionwas made, and viable tumor cells were injected into the bladderwall. The formation of a bulla indicated a satisfactory injection.The bladder was returned to the abdominal cavity, and theabdominal wall was closed with a single layer of metal clips.

ISH Analysis. A specific antisense oligonucleotide DNAprobe was designed complementary to the mRNA transcriptsbased on published reports of the cDNA sequences of IL-8 (38,40), and the specificity was confirmed by Northern blot analysis

2788 NF-�B Regulation of Human Bladder Cancer Metastasis



(40). A poly(dT)20 oligonucleotide was used to verify the in-tegrity and lack of degradation of the mRNA in each sample.

ISH was performed as described previously, using theMicroprobe Manual Staining System [Fisher Scientific, Pitts-burgh, PA (40, 41)]. To check the specificity of the hybridiza-tion signal, the following controls were performed: (a) RNasepretreatment of sections; and (b) substitution of a biotin-labeledsense probe for the antisense probe. No hybridization signal wasobserved under either of these conditions. Control for endoge-nous alkaline phosphatase included treatment of the sample inthe absence of the biotinylated probe and the use of chromogenalone (40, 41). The color reaction was quantified with a Zeissphotomicroscope (Carl Zeiss, Thornwood, NY) equipped with athree-chip, charge-coupled device color camera (model DXC-969 MD; Sony Corp., Tokyo, Japan), and the images wereanalyzed using Optimas image analysis software (version 6.2;Media Cybernetics, Silver Spring, MD). The intensity of stain-ing was determined by comparison with the integrated absorb-ance of poly(dT)20. The results were presented as the ratio of the

expression of each gene indexed against controls, which werearbitrarily set at 100 (42).

IHC. For IHC analysis, frozen tissue sections (8 �m)were fixed in cold acetone. Formalin-fixed, paraffin-embeddedspecimens (5 �m) were deparaffinized in xylene and rehydratedin graded ethanol. Antigen retrieval was performed using pepsinfor 12 min. Endogenous peroxidase activity was quenched with3% hydrogen peroxide. Specimens were blocked with 5% nor-mal horse serum plus 1% normal goat serum in PBS. Thesamples were incubated for 18 h at 4°C with one of the follow-ing: (a) a 1:800 rat monoclonal anti-CD31 antibody [Phar-Mingen, San Diego, CA (43)]; (b) a 1:50 dilution of a rabbitpolyclonal anti-IL-8 antibody (Biosource International, Ca-marillo, CA); or (c) a 1:100 dilution of mouse monoclonalanti-PCNA antibody (DAKO, Carpinteria, CA).

The samples were then rinsed four times with PBS beforeincubation with the appropriate secondary antibody: (a) perox-idase-conjugated antirat IgG (H�L; Jackson ImmunoResearchLaboratory, West Grove, PA); (b) antirabbit IgG; (c) F(ab)2

Fig. 2 Bladder cancer cells were incubated in normal and acidic (pH 7.0 to 6.0) medium for 6 h (A) or under control (using nonadjusted pH ofmedium), normoxia, and hypoxia conditions for 12 and 24 h (B). IL-8 protein expression in cell culture supernatants was measured by ELISA assay(bar graph). The concentration of IL-8 was corrected for cell number. IL-8 mRNA expression was measured by Northern blotting. A probe forGAPDH was used as a loading control (data not shown). IL-8 protein and mRNA expression in UM-UC-14, HTB-9, and RT-4 cells was significantlyincreased under acidic conditions (pH 7.0). IL-8 mRNA and protein were undetectable in KU-7 cells. Acidic pH decreased IL-8 protein and mRNAlevels in 253J B-V cells (A). With more severe acidosis (pH 6.7), IL-8 production was decreased in all cells, which may reflect cytotoxicity. TheIL-8 protein and mRNA expression of UM-UC-14 and HTB-9 cells was significantly increased under hypoxia conditions compared with the cellsincubated under either basal or normoxia conditions. KU-7 cells expressed undetectable levels of IL-8. 253J B-V cells constitutively expressed highlevels of IL-8, which were not altered by hypoxia (B).




fragment (Jackson ImmunoResearch Laboratory); (d) antimouseIgG1 (PharMingen); or (e) antimouse IgG (Jackson ImmunoRe-search Laboratory). The specimens were again rinsed with PBS,incubated with diaminobenzidine (Research Genetics), andmounted using universal mount (Research Genetics).

Quantification of IHC for IL-8, MVD, and CellularProliferation. The intensity of IL-8 immunostaining wasquantified in each sample by image analysis using the Optimassoftware program (Bioscan, Edmonds, WA). Five different ar-eas in each sample were evaluated to yield an average meas-urement of intensity of immunostaining. The results were pre-sented as a ratio between the expression by the tumor andnormal mucosa, which was arbitrarily set at 100 (42) MVD wasdetermined by light microscopy after immunostaining of sec-tions with anti-CD31 antibodies according to the procedure ofWeidner et al. (43, 44), and cell proliferation was determined byimmunohistochemical staining of tissue sections with anti-PCNA antibody. Tissue images were recorded using a cooledcharge-coupled device Optronics Tec 470 camera (OptronicsEngineering, Goletha, CA) linked to a computer and digitalprinter (Sony Corp.). The MVD and density of proliferativecells were expressed as the average number of five highest areasidentified within a single �100 field (42).

Statistical Analysis. Tumor weights and staining inten-sities are expressed as median SD. Differences in the numberof blood vessels, proliferative cells, and staining intensity forIL-8 in the bladder tumors between treatment groups wereanalyzed using the Mann-Whitney U test. Incidences of tumors

and metastases were analyzed by the �2 test. P 0.05 wasconsidered significant.

RESULTSHeterogeneity of Induction of IL-8 in Human Bladder

Cancer Cells under Acidic and Hypoxic Conditions. In thepresent study, we used stringent conditions and careful pH

Fig. 3 Electrophoretic gel mobility shiftassay. Nuclear proteins were extracted fromUM-UC-14 cells incubated under normal(pH 7.35) and acidic conditions (pH 7.0 to6.0) for 3 h (A) or under normoxic andhypoxic (1% O2) conditions for 12 h (B).Binding reactions were performed as de-scribed in “Materials and Methods.” Alllanes contain labeled NF-�B probe. Underacidic conditions (pH 7.0; A) and hypoxicconditions (B), enhanced NF-�B protein-DNA binding function accompanied by p50and p65 supershifted bands (but not c-Rel)was observed compared with normal condi-tions.

Fig. 4 Suppression of acidosis- and hypoxia-induced NF-�B proteinbinding and up-regulation of IL-8 mRNA and protein in UM-UC-14cells after transfection with pLXSN-I�B-�-M (mutant). UM-UC-14cells were transfected with mutant I�B-�. After stimulation with 50ng/ml TNF-� for 30 min, cytosolic protein was extracted from UM-UC-14, UM-UC-14-NEO, UM-UC-14-M1, and UM-UC-14-M2 cells.Endogenous I�B-� (top band) and exogenous I�B-�M (bottom band)were detected by Western immunoblotting using rabbit anti-I�B-� poly-clonal antibody. Equal loading was confirmed with anti-�-actin anti-body. Endogenous I�B-� in UM-UC-14 and UM-UC-14-NEO cells wasrapidly degraded after TNF-� stimulation. In contrast, in UM-UC-14-M1 and UM-UC-14-M2 cells, exogenous mutant I�B-� was notdegraded by TNF-� stimulation.




monitoring at the beginning and end of each experiment tocontrol for changes in pH over the course of the experiments toindependently study the effects of hypoxia and acidosis on IL-8transcription. We were able to keep the pH within a range of6.8–7.1 to prevent cytotoxic effects that occur once the pH islowered below 6.7, and we were able to evaluate the effects ofhypoxia on IL-8 expression independent of the confoundingeffects of acidosis that can accompany hypoxia (17). We thendetermined whether IL-8 mRNA expression and protein produc-tion were induced by acidic (Fig. 2A) and hypoxic conditions(Fig. 2B). GAPDH mRNA expression was used as a control forloading (data not shown). Cells were incubated under normal pH(7.35) or acidic pH (7.0, 6.5, 6.0) conditions for 6 h. IL-8 mRNAexpression (Northern blotting) and protein production (ELISA)were significantly up-regulated at pH 7.0 (acidic conditions) inUM-UC-14, HTB-9, and RT-4 cells. IL-8 was not detected inKU-7 cells under normal or acidic conditions. 253J B-V cellsconstitutively expressed high levels of IL-8 under normal con-ditions, which were not up-regulated under acidic conditions.Further reductions in pH to 6.5 and 6.0 suppressed IL-8 in all ofthe cell lines tested (Fig. 2A).

Under conditions of hypoxia, IL-8 mRNA and proteinproduction in UM-UC-14 cells was minimally increased at 12 hbut substantially increased at 24 h compared with normoxicconditions (Fig. 2B). Similar induction of IL-8 was noted inHTB-9 cells under hypoxic conditions. Conversely, hypoxiareduced IL-8 expression in RT-4 cells but had no effect on theconstitutive expression of IL-8 in 253J B-V cells. IL-8 was notdetected in KU-7 cells under normal or hypoxic conditions.

Acidic and Hypoxic Conditions Induce NF-�B in UM-UC-14 Cells. EMSA was performed to evaluate the responseof the transcription-regulatory DNA-binding proteins NF-�B

and AP-1 to acidic and hypoxic conditions. [�-32P]ATP-labeledNF-�B and AP-1 oligonucleotide probes were incubated withnuclear protein extracted from UM-UC-14 cells. Under nor-moxic conditions, low level binding of nuclear protein to NF-�Bwas observed. Acidosis (pH 7.0) increased the binding activityof NF-�B accompanied by p50 and p65 supershift bands thatidentified the NF-�B subunits involved in DNA binding(Fig. 3A). Similarly, during hypoxia, increased binding activityof NF-�B accompanied by p50 and p65 supershift bands wasobserved (Fig. 3B). The DNA binding activity of AP-1 wasunaltered under acidic and hypoxic conditions compared withnormal conditions (data not shown). The highly metastatic 253JB-V cells demonstrate constitutive NF-�B binding activity thatdid not increase upon exposure to acidic or hypoxic conditions(data not shown).

Mutant I�B-� Prevented Induction of NF-�B DNABinding Reaction and IL-8 Expression by Acidosis and Hy-poxia. UM-UC-14 cells were transfected with the pLXSN-I�B-�-M vector. Mutation of Ser32 and Ser36 in I�B-� preventsI��-mediated phosphorylation of the protein, which is requiredfor ubiquitination and degradation by the proteasome. Immuno-blot analysis confirmed that the mutant I�B-� protein expressedby the transfectants was resistant to TNF-induced degradationcompared with UM-UC-14 cells, which are sensitive to rela-tively low doses of TNF-�, as shown in Fig. 4.

We evaluated whether mutant I�B-� suppressed inducibleNF-�B DNA binding under acidic and hypoxic conditions. Cellswere incubated under acidic conditions (pH 7.0) for 6 h or underhypoxic conditions (1% O2) for 24 h. After incubation, nuclearextracts were harvested and examined by EMSA. NF-�B DNA-binding function in UM-UC-14-M1 and UM-UC-14-M2 cellswas suppressed during acidosis and hypoxic conditions com-

Fig. 5 Suppression of acido-sis- and hypoxia-induced up-regulation of IL-8 mRNA andprotein and NF-�B protein-DNA binding in UM-UC-14cells after transfection withpLXSN-I�B-�-M (mutant). To-tal RNA, cell culture superna-tant, and nuclear protein wereextracted from these transfectedand control cells. IL-8 mRNA(bottom panel) and protein(middle panel) and NF-�Bbinding function (top panel) ofUM-UC-14 cells transfectedwith pLXSN-NEO vector wereup-regulated under acidic (pH7.0; A) and hypoxic (B) condi-tions. These effects were notobserved in the I�B-� mutanttransfectants under similar con-ditions.




pared with UM-UC-14 and UM-UC-14-NEO cells. The extentof suppression of NF-�B correlated with the expression level ofexogenous mutant I�B-� (Fig. 5). The presence of endogenousI�B-� in cells expressing the mutant I�B-� protein is consistentwith the presence of a subpopulation of cells not expressing themutant I�B-� protein.

We evaluated whether overexpression of mutant I�B-�blocked IL-8 mRNA expression and protein production inducedby acidic and hypoxic conditions in UM-UC-14-M (UM-UC-14-M1 and UM-UC-14-M2) cells. Under homeostatic condi-tions, the expression of IL-8 by UM-UC-14-M1 and UM-UC-14-M2 cells was equivalent to that of UM-UC-14-NEO controls(Fig. 5). The induction in IL-8 mRNA (Northern blotting) andprotein (ELISA) observed in UM-UC-14 and UM-UC-14-NEOcells under acidic (Fig. 5A) and hypoxic conditions (Fig. 5B)was not observed in UM-UC-14-M1 and UM-UC-14-M2 cellsunder similar conditions.

Inhibition of Tumorigenicity and Metastasis of Ortho-topic Xenografts Expressing Mutant I�B-�. We implantedUM-UC-14, UM-UC-14-NEO, UM-UC-14-M1, and UM-UC-14-M2 cells into the bladder wall of athymic nude mice. Eightweeks after tumor implantation, mice were killed, bladder tumorswere removed and weighed, and retroperitoneal lymph nodes wereassessed for metastasis (Table 1). Tumors in the UM-UC-14-M1and UM-UC-14-M2 cells were smaller than those in the UM-UC-14 cells (UM-UC-14-M1, P 0.005; UM-UC-14-M2, P 0.05) or UM-UC-14-NEO cells (UM-UC-14-M1, P 0.005; UM-UC-14-M2, P 0.05). In keeping with the expression pattern ofmutant I�B, the UM-UC-14-M1 tumors were significantly smallerthan the UM-UC-14-M2 tumors (P 0.05). The incidence oflymph node metastasis in UM-UC-14-M1 tumors was significantlylower compared with that for either UM-UC-14 or UM-UC-14-NEO tumors (P 0.05). The incidence of lymph node metastasisin UM-UC-14-M2 tumors was lower than that in controls, but thedifference was not statistically significant.

Inhibition of NF-�B-DNA Binding, Down-Regulation ofIL-8, MVD, and Cell Proliferation by Mutant I�B-� Trans-fection in UM-UC-14 Orthotopic Tumors. NF-�B-DNAbinding was suppressed in nuclear extracts prepared from invivo tumors derived from UM-UC-14-M1 and UM-UC-14-M2cells compared with the tumors derived from UM-UC-14-NEOcells (Fig. 6). The degree of suppression of NF-�B DNA bind-ing activity was proportionate to the expression of mutant I�Bby the UM-UC-14-M1 and UM-UC-14-M2 cells.

IL-8 mRNA expression was determined by ISH using anantisense oligonucleotide probe. IL-8 protein expression, cellproliferation, and MVD were determined by IHC using anti-IL-8, anti-PCNA, and anti-CD31 antibodies, respectively(Table 2 and Fig. 7). IL-8 mRNA and protein expression, cellproliferation, and MVD were significantly lower within UM-UC-14-M1 tumors than in either the UM-UC-14 or UM-UC-14-NEO tumors (P 0.05). Similar differences in thesefactors were observed between UM-UC-14-M2 and UM-UC-14 and UM-UC-14-NEO tumors. However the differ-ences in the expression of IL-8 protein and MVD betweenUM-UC-14-M2 and UM-UC-14-NEO tumors were not sta-tistically significant.

DISCUSSIONPreviously, we demonstrated that the level of IL-8 expres-

sion correlated with the metastatic potential of human TCC (15).In this study, we report for the first time that conditions in thehost microenvironment (acidosis and hypoxia) alter IL-8 expres-sion by human TCC via NF-�B. We studied a panel of humanTCC cells that differed in their metastatic potential and discov-ered a heterogenic response in NF-�B activation and IL-8 ex-

Table 1 Tumorigenicity of UM-UC-14 cells growing orthotopicallyin athymic nude mice after transfection with mutant I�B�

Mice were implanted with 5 � 105 UM-UC-14, UM-UC-14-NEO,UM-UC-14-M1, and UM-UC-14-M2 cells. All mice were killed 8weeks after implantation of tumor cells. UM-UC-14-M1 tumors weresignificantly smaller (P 0.005) and had fewer lymph node metastases(P 0.05) compared with either UM-UC-14 or UM-UC-14-NEOtumors. UM-UC-14-M2 tumors were significantly smaller than eitherUM-UC-14 (P 0.05) or UM-UC-14-NEO (P 0.05), and the inci-dence of lymph node metastasis was reduced. The UM-UC-14-M1tumors were significantly smaller than UM-UC14-M2 tumors (P 0.05). This is one representative experiment of two.

Group Tumorigenicity

Tumor weight Lymphnode

metastasisMedian (mg) Range

UM-UC-14 10/10 141 45–236 5/10UM-UC-14-NEO 10/10 113 49–210 4/10UM-UC-14-M1 10/10 43a 30–57 0/10b

UM-UC-14-M2 10/10 57c,d 38–164 1/10a P 0.005 compared with UM-UC-14 and UM-UC-14-NEO.b P 0.05 compared with UM-UC-14 and UM-UC-14-NEO (�2

test).c P 0.05 compared with UM-UC-14 and UM-UC-14-M1d P 0.05 compared with UM-UC-14-NEO (Mann-Whitney U

test).

Fig. 6 In vivo suppression of NF-�B DNA binding in nuclear extractsfrom tumors derived from UM-UC-14-M1 and UM-UC-14-M2 cells(containing mutant I�B) compared with tumors derived from controls(UM-UC-14-NEO cells). The degree of suppression of NF-�B DNAbinding activity was proportionate to the expression of mutant I�B bythe UM-UC-14-M1 and UM-UC-14-M2 cells; more NF-�B DNA bind-ing activity exists in the UM-UC-14-M2 cells. Each lane representsnuclear extracts derived from a single tumor.




pression in response to hypoxia and acidosis. We observed thathighly metastatic TCC constitutively expressed high levels ofNF-�B and IL-8, whereas less aggressive TCC cells expressedlower constitutive levels of NF-�B activity that were inducibleafter exposure to hypoxia or acidosis and led to the up-regula-tion of IL-8 expression. Blockade of NF-�B by mutant I�B-�prevented the induction of IL-8 and resulted in inhibition ofangiogenesis and metastasis in human TCC xenografts growingorthotopically in the bladder of nude mice. These experimentsidentify NF-�B as a promising target for novel therapeuticstrategies being designed by us for the treatment of advancedhuman bladder cancer.

Human tumors, including TCCs, are heterogeneously ox-ygenated due to functional and structural abnormalities of thevasculature. As a consequence, hypoxia is a common feature ofbladder cancer, and this hypoxia in turn increases anaerobicmetabolism and leads to increased production of acidic metab-olites (46–53). The resulting low extracellular pH in associationwith hypoxia can promote malignant progression by altering theexpression of IL-8 and many other genes, including cell cycle-regulatory proteins, metabolic enzymes, metastasis-regulatoryproteins, transcription factors, and angiogenesis-regulatory pro-teins (45, 54–56).

In clinical studies, high IL-8 expression has been reportedto correlate with clinical stage and grade in human tumors(57–60). In the present study, we demonstrated that TCCs ofdifferent phenotypes have heterogenous expression patterns ofIL-8 expression under control conditions and variable responsesto environmental stress. Neither IL-8 mRNA nor protein wasdetectable in KU-7 cells (superficial papillary TCC cells) undercontrol conditions, and neither was induced by hypoxia oracidosis. The well-differentiated grade 1 TCC cells (RT-4) andgrade 2 TCC cells (HTB-9) expressed low and moderate levelsof IL-8, respectively, under homeostatic conditions. In both celllines, IL-8 expression was inducible under stressful conditions(hypoxia or acidosis). In contrast, 253J B-V cells (high-gradeTCC) constitutively expressed high levels of IL-8 and NF-�Bactivity, which did not increase under acidic or hypoxic condi-tions. Paradoxically, under homeostatic conditions, UM-UC-14cells (high-grade TCC) expressed low levels of IL-8 that were

inducible by acidosis and hypoxia due to an increase in NF-�Bbinding activity. Although UM-UC-14 cells were derived froma high-grade TCC, they demonstrate morphological and histo-logical features characteristic of low-grade TCC and have lowermetastatic potential in comparison with the highly metastatic253J B-V cells (10). Thus, these data suggest that the hetero-geneity of constitutive NF-�B activity and induction observed inthis study might correlate with the histological grade of TCCcells: well- to moderately differentiated TCC cells may expresslow basal NF-�B activity and IL-8 expression (which is induc-ible in response to appropriate stimuli); whereas poorly differ-entiated TCC cells may express IL-8 constitutively due to con-stitutively active NF-�B. Interestingly, these observations are incontrast to findings in human melanoma cells, in which IL-8mRNA and protein are inducible in the highly aggressive andmetastatic cells, but not in the poorly aggressive cells (61).

The IL-8 promoter region –133 to –70 contains bindingsites for NF-�B, AP-1, and nuclear factor-IL-6 and is requiredfor IL-8 transcription. IL-8 transcription is regulated by cyto-kines, growth factors, irradiation, cytotoxic agents, nitric oxide,and physical changes in the microenvironment such as acidosisand hypoxia. In melanoma (62), ovarian tumors (63, 64), andpancreatic tumors (25), constitutive or inducible expression ofIL-8 is controlled via NF-�B and/or AP-1. However, the relativeimportance of these transcription elements is stimulus specificand cell type specific (16). In the present study, we demon-strated that the induction of IL-8 in TCC is regulated throughNF-�B, but not AP-1: specifically, the dimer of p50 and p65regulated IL-8 expression during acidosis and hypoxia. Underbasal conditions, NF-�B is sequestered in the cytoplasm viaassociation with I�B, which prevents NF-�B translocation to thenuclei. The I�B family consists of seven I�Bs, I�B-�-�-�,Bcl-3, p100, and p105 (65). These I�Bs preferentially associatewith NF-�B/Rel family protein dimers. Specifically, I�B-� pre-dominantly associates with p65/p50 heterodimers. In responseto a wide variety of extracellular and intracellular signals in-cluding acidosis and hypoxia, I�Bs are phosphorylated by I�Bkinase and subsequently polyubiquitinated and proteolyticallydegraded. This liberates NF-�B, allowing it to translocate to thenucleus and function as a transcription factor. Recently, detailed

Table 2 In vivo IL-8 mRNA and protein expression, cell proliferation, and MVD of UM-UC-14 cells growing orthotopically in athymic nudemice after transfection with mutant I�B�

Group IL-8 mRNA indexa IL-8 protein indexbProliferation indexc

(mean SD)MVDd

(mean SD)

UM-UC-14 100 100 142 45 57 9UM-UC-14-NEO 92 89 118 26 55 10UM-UC-14-M1 61e,f 63e,f 68 15e,f 30 2e,f

UM-UC-14-M2 64e,f 70e 81 25e,f 41 8e

a The intensity of the cytoplasmic color reaction obtained by ISH was quantified using image analysis and compared with the maximal intensityof the poly (dT)20 color reaction in each sample. The results are presented for each cell type indexed to the controls, which were arbitrarily definedas 100.

b The intensity of the cytoplasmic immunostaining was quantified using image analysis in five different areas of each sample to yield an averagemeasurement that is indexed to the control tumors, which were arbitrarily defined as 100.

c The density of cell proliferation was measured by IHC with anti-PCNA antibodies and is expressed as the average of the five highest areasidentified within a single �100 field.

d MVD was expressed as an average number of CD31-positive cells in the five highest areas identified within a single �100 field.e P 0.05 compared with UM-UC-14.f P 0.05 compared with UM-UC-14-NEO.




structures and functions of I�B-� have been elucidated. I�B-�is a cytoplasmic protein distinguished by the presence of sixankyrin-like repeats (30, 66–68). Phosphorylation of serine 32and serine 36 residues on the NH2 terminus in response tocertain stimuli, including TNF-�, IL-1, and 12-O-tetradecan-ylphorbol-13-acetate, precedes its degradation and dissociationfrom NF-�B (69–71). One of the striking structural features ofI�B-� is the presence of a COOH-terminal region that is rich inthe amino acids proline, glutamic acid, serine, and threonine[PEST (72)]. The presence of such PEST sequences in manyother proteins predicts rapid protein turnover (72). The PESTsequence of I�B-� lies downstream of the sixth ankyrin, begin-ning at amino acid 281. There are phosphoacceptor sites in thePEST region of I�B-�, with serines 283, 289, and 293 andthreonines 291 and 299 all reported to be at least partiallyphosphorylated (73–76). It has been suggested that constitutivephosphorylation of COOH-terminal phosphoacceptors is neces-sary for efficient turnover of free I�B-� in unstimulated cells(77). For the experiments described above, we chose thepLXSN-I�B-�-M (mutant), which contains substitutions at theserine 32 and serine 36 residues in the NH2-terminal and dele-tion of the PEST sequence in the COOH-terminal. Transfectionof ovarian and prostate cancer cells with similar mutants ofI�B-� decreased tumorigenicity by reducing the expression ofproangiogenic molecules (IL-8 and vascular endothelial growthfactor) that are regulated by NF-�B (31, 32). In a murine tumor

model with murine lung alveolar cells expressing dominantnegative I�B-�, the incidence of metastasis was reduced viainhibitory effects on matrix metalloproteinase 9, urinary plas-minogen activator, and heparanase production independent ofprimary tumor size (59).

In the current study, mutant I�B-�-transfection of UM-UC-14 TCC cells prevented the rapid TNF-�-induced degrada-tion of I�B-� (Fig. 4A) and increased TNF-�-induced DNAfragmentation (Fig. 4B). TNF-� induces apoptosis by activatingcaspases 8 and 10, but it can also induce the expression ofinhibitors of apoptosis via NF-�B (78, 79). The TNF-�-inducedDNA fragmentation in UM-UC-14-M1 and UM-UC-14-M2cells was greatly increased compared with UM-UC-14 or UM-UC-14-NEO cells, suggesting a relative excess of proapoptoticstimuli over antiapoptotic stimuli.

During hypoxia and acidosis, DNA binding by NF-�B inthe mutant I�B-�-transfected cells (UM-UC-14-M1 and UM-UC-14-M2) was inhibited, and therefore IL-8 expression in vitrowas reduced. Similarly, DNA binding by NF-�B was inhibitedin vivo in the mutant I�B-�-transfected cells that produced lessIL-8 in vivo. Furthermore, the growth and metastatic potential ofmutant I�B-�-transfected cells in vivo was reduced proportion-ate to the level of expression mutant I�B-�. In keeping with thepreviously described antiangiogenic effects of IL-8, MVD wasreduced in tumors arising from mutant I�B-�-transfected cellscompared with controls. Other studies have demonstrated that

Fig. 7 IL-8 mRNA expression, IL-8 protein expression, cell proliferation, and MVD in orthotopic xenografts. Tumors from I�B-�-M-transfectedcells expressed lower IL-8 mRNA and protein and demonstrated reduced proliferation and decreased neovascularization compared with controls(UM-UC-14-P or NEO).




inhibition of NF-�B function by the proteasome inhibitor PS-341 reduces tumor growth and enhances chemo- and radiosen-sitivity by inhibiting chemotherapy- and radiotherapy-inducedNF-�B activation in colorectal (80, 81), squamous cell (82), andpancreatic cancers (83). In our studies, the reduction in lymphnode metastasis correlated with the degree of inhibition ofNF-�B function in the transfected cell lines UM-UC-14-M1 andUM-UC-14-M2. This finding, coupled to the known IL-8 tran-scription-regulatory activity of NF-�B, supports a role for IL-8as a mediator of metastasis in bladder cancer. We and othershave previously reported proposed this role for IL-8 in bladdercancer (15), melanoma (4), and prostate cancer (13). Consider-ing the present data, blockade of NF-�B translocation via inhi-bition of the NF-�B-I�B complex dissociation has potential as atherapeutic intervention in bladder cancer due to its inhibitoryeffects on IL-8 expression and subsequent inhibition of angio-genesis, tumor growth, and metastasis (15). We are planningadditional studies to evaluate the potential of mutant I�B-� as anovel gene therapy strategy in combination with chemotherapyand radiotherapy.

In summary, our experiments demonstrate heterogeneity inconstitutive and inducible NF-�B activity in human TCC. More-over, blockade of NF-�B by mutant I�B-� inhibits IL-8 induc-tion by acidosis and hypoxia and inhibits angiogenesis, growth,and metastasis of human TCC growing within the bladder wallof athymic nude mice.

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2003;9:2786-2797. Clin Cancer Res Takashi Karashima, Paul Sweeney, Ashish Kamat, et al. Interleukin-8Human Bladder Cancer through the Regulation of

B Mediates Angiogenesis and Metastasis ofκNuclear Factor-

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