Roles of Akt/PKB and IKK Complex in Constitutive Induction ofNF-�B in Hepatocellular Carcinomas of Transforming Growth

Factor �/c-myc Transgenic Mice

VALENTINA FACTOR,1 AUNDREA L. OLIVER,2 GANESH R. PANTA,3 SNORRI S. THORGEIRSSON,1 GAIL E. SONENSHEIN,2

AND MARCELLO ARSURA2,3

NF-�B regulates liver cell death during development, regen-eration, and neoplastic transformation. For example, we showedthat oncogenic Ras- or Raf-mediated transformation of rat liverepithelial cells (RLEs) led to altered NF-�B regulation throughIKK complex activation, which rendered these cells more resis-tant to TGF-�1-induced apoptosis. Thus, based on these find-ings, we sought to determine whether NF-�B could also be in-volved in tumor growth of liver cells in vivo. Hepatocellularcarcinomas (HCCs) derived from bitransgenic mice harboringTGF-� and c-myc transgenes targeted specifically to the liverwere compared with HCCs from c-myc single transgenic mice.Tumors from bitransgenic mice are characterized by a higherfrequency of appearance, lower apoptotic index, and a higherrate of cell proliferation. Here we show that NF-�B is activated inHCCs of double TGF-�/c-myc transgenic mice, but not of c-mycsingle transgenic mice, suggesting that TGF-� mediates induc-tion of NF-�B. Activation of the IKK complex was observed inthe HCCs of double TGF-�/c-myc transgenic mice, implicatingthis pathway in NF-�B induction. Lastly, activation of the Akt/protein kinase B (PKB), which has recently been implicated inNF-�B activation by PDGF, TNF-�, and Ras, was also observed.Importantly, human HCC cell lines similarly displayed NF-�Bactivation. Thus, these studies elucidate an anti-apoptotic mech-anism by a TGF-�-Akt/PKB-IKK pathway, which likely contrib-utes to survival and proliferation, thereby accelerating c-myc-induced liver neoplastic development in vivo. (HEPATOLOGY

2001;34:32-41.)

NF-�B was first identified as a nuclear factor specific to Bcells that bound to the B site of the � light chain gene en-hancer.1 NF-�B is now known to be a family of dimeric tran-scription factors with subunits that contain an amino terminalstretch of approximately 300 amino acids termed the Rel ho-mology domain (RHD) because it shares homology with thev-Rel oncoprotein.2 The RHD is involved in DNA binding anddimerization of the various subunits.3 Classical NF-�B is com-posed of a p50 (NF-�B1) and a p65 (RelA) subunit,4,5 and isubiquitously expressed. In non-B cells, NF-�B is sequesteredin the cytoplasm by specific inhibitory proteins termedI�Bs,6,7 of which I�B-� is the most well characterized.

NF-�B can be activated by many inducers including pro-inflammatory cytokines, phorbol esters, bacterial endotoxins,and oxidative stress. Recently the Akt/protein kinase B (PKB)kinase has been shown to play a role in this signaling in re-sponse to TNF-� or PDGF signalling.8,9 The most well-char-acterized pathway leading to NF-�B activation involves theIKK complex, which includes 2 catalytic kinase subunitsIKK-1 (IKK-�) and IKK-2 (IKK-�),10-13 and a third regulatorysubunit termed IKK-� or NEMO.14,15 Knock-out studies haveunderscored the nonoverlapping functions of IKK-1 andIKK-2 during vertebrate limb development and immune re-sponse, respectively.16 Following signal-induced IKK activa-tion, sequestered NF-�B/I�B-� is recruited to the IKK com-plex where the I�B-� is phosphorylated on serine residues 32and 36. As a consequence of phosphorylation, I�B-� protein israpidly ubiquitinated and degraded through the proteasomepathway,17-19 allowing for migration of NF-�B to the nucleus.

Recently, NF-�B/Rel factors have been strongly implicatedin the regulation of malignant cell growth, survival, and trans-formed phenotype.20,21 As we first showed, TGF-�1–medi-ated cell killing of murine B cell lymphomas is mediated viainhibition of NF-�B, and direct repression of this activity canlead to death of these cells.22,23 Furthermore, we found thathuman and rodent breast cancer cells are typified by aberrantNF-�B/Rel expression, and inhibition of this activity can sim-ilarly lead to cell death.24 To date, constitutive activation ofNF-�B has been observed in a variety of tumors includingHodgkin’s and T-cell lymphomas,25,26 melanomas,27 breastand pancreatic adenocarcinomas,24,28-30 and primary adult T-cell leukemias.31 Inhibition of NF-�B activity potentiated cellkilling of human breast cancer and fibrosarcoma cell lines byTNF-�, ionizing radiation, and daunorubicin32-35 and led tosensitization of tumors to TNF-�- and to the chemotherapeu-tic compound CPT-11–mediated cell killing in vivo.36 Simi-larly, expression of a phosphorylation mutant I�B-� inhibited

Abbreviations: PKB, protein kinase B; RLE, rat liver epithelial; TGF, transforminggrowth factor; HCC, hepatocellular carcinoma; NF-�B, nuclear factor �B; I�B, inhibitor�B; IKK, I�B kinase; PDGF, platelet-derived growth factor; TNF, tumor necrosis factor;RHD, Rel homology domain; EMSA, electrophoretic mobility shift assay; URE, upstreamregulatory region; EGFR, epidermal growth factor receptor; DMBA, 7,12-dimethylbenz(�)anthracene.

From the 1Laboratory of Experimental Carcinogenesis, Division of Basic Sciences,National Cancer Institute, Bethesda, MD; the 2Department of Biochemistry, Boston Uni-versity School of Medicine, Boston, MA; the 3Department of Pharmacology, University ofTennessee College of Medicine, Memphis, TN.

Received October 2, 2000; accepted April 2, 2001.Supported by grants from the Charlotte Geyer Foundation (M.A.), from the ACS

IRG-72-001-24 (M.A.), and from the NIH grants CA36355 (G.E.S.) and CA78616(M.A.).

V. Factor and A. L. Oliver equally contributed to this publication. G. E. Sonensheinand M. Arsura share the last co-authorship.

Address reprint requests to: Marcello Arsura, Ph.D., Department of Pharmacology,University of Tennessee College of Medicine, 874 Union Avenue, Memphis TN 38163.E-mail: marsura@utmem.edu; fax: 901-448-7206.

Copyright © 2001 by the American Association for the Study of Liver Diseases.0270-9139/01/3401-0007$35.00/0doi:10.1053/jhep.2001.25270

32

the growth in vivo of human head and neck squamous cellcarcinoma.37 More recently, we found oncogenic Ras or Raftransformation of rat liver epithelial cells (RLEs) inducedNF-�B binding and activity, which prevented TGF-�1-medi-ated apoptosis.38 We showed that the translocation in the Ras-and Raf-transformed RLEs is mediated through constitutiveIKK activation. Of note, ectopic expression of dominant neg-ative forms of IKK-1 or -2 not only restored TGF-�1-mediatedcell killing but also reduced focus forming activity of Rastransformed RLEs.38

Although the expression of NF-�B has been well character-ized in a variety of tumor models, the mechanism(s) leading toNF-�B activation during cancer progression have only begunto be elucidated. Thus, based on the findings with Ras- andRaf-transformed RLEs, here we have explored the possibilitythat IKKs might also play a role in vivo during liver tumorformation through NF-�B activation. To this purpose, we an-alyzed NF-�B activity in human hepatocellular carcinoma(HCC) cell lines and in solid HCCs derived from transgenicmice co-expressing TGF-� and c-myc in the liver.39 Previ-ously, these double transgenic mice displayed a dramatic ac-celeration of neoplastic development compared with the longlatency of those from single c-myc transgenic mice.39 Thiseffect correlated with enhanced cell survival of HCCs suggest-ing a role for TGF-� as an anti-apoptotic factor for neoplasticcells.40 Here, we show that hepatic cell lines derived fromhuman HCCs, and HCCs from double c-myc and TGF-� butnot single c-myc transgenic mice express constitutive NF-�Bnuclear translocation, and implicate Akt and the IKK complexin this aberrant activation. These findings indicate the TGF-�-Akt-IKK pathway induces the prosurvival/neoplastic factorNF-�B in HCCs.

MATERIALS AND METHODS

Mice. Male TGF-�/c-myc double transgenic and wild type micewere generated by crossing homozygous MT/TGF-�41 and Alb/c-myc39 single transgenic mice in (CD1 X B6CBA) F1 background andhoused as described previously.42 Normal livers were obtained from10-week-old mice of the same background as for the double trans-genic lines. Dysplastic livers were obtained from 11-week-old TGF-�/c-myc transgenic mice before foci development. The animal studyprotocols were conducted according to National Institutes of Healthguidelines for animal care. Mice had free access to standard rodentchow and water.

Cell Culture. Human HCC cell lines were obtained from ATCC,Qidong Liver Cancer Institute, China, and Dr. C. Harris (NCI, NIH).The biological and cytogenetic characteristics of these HCC lineswere described previously.43-46 Cells were cultivated in Dulbecco’smodified Eagle’s medium (DMEM)/F12 medium supplemented with10% fetal bovine serum. Human breast cancer Hs578T cells werepropagated in DMEM with 10% heat-inactivated fetal bovine serum(FBS) (Gibco/BRL, Gaithersburg, MD), 4.5 g/L glucose, 10 �g/mL ofinsulin, 100 units/mL penicillin, 100 �g/mL streptomycin (all fromSigma Chemical Co., St. Louis, MO). Jurkat and 293T cell lines weregrown in RPMI 1640 medium supplemented with 10% FBS and an-tibiotics as described earlier. The murine B cell lymphoma WEHI 231cells were maintained in DMEM supplemented as described previ-ously.24 F22-Ras RLEs were maintained in F-12 Nutrient Mixture(Ham) medium (Gibco/BRL), supplemented with, 50 units/mL pen-icillin, 50 �g/mL streptomycin.

Electrophoretic Mobile Shift Assay (EMSA). Nuclear extracts wereprepared from livers by a modification of a protocol described pre-viously.24 Briefly, frozen tissues were pulverized in a stainless steeltissue or ceramic mortar, resuspended in 1 mL of cold homogeniza-tion buffer (10 mmol/L KCl, 10 mmol/L Hepes, pH 7.9, 0.1 mmol/L

EDTA, 0.1 mmol/L EGTA, 50 mmol/L sucrose) containing 0.5mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF),10 �g/mL leupeptin and 5 �g/mL aprotinin. After 5 minutes of in-cubation on ice, the cells were dounced until cell lysis occurred. KClconcentration was adjusted to 100 mmol/L, and extracts were cen-trifuged 4,000 rpm for 5 minutes at 4°C. Nuclei were then resus-pended in 2 packed nuclear volumes of extraction buffer (10 mmol/LHepes pH 7.9, 400 mmol/L NaCl, 0.1 mmol/L EDTA, 0.1 mmol/LEGTA, 20% glycerol) plus protease inhibitors as described earlier,and incubated on ice for 30 minutes. Protein concentration wasdetermined using the Bio-Rad protein assay, following the manufac-turer’s directions (Bio-Rad Laboratories, Hercules, CA). The se-quences of the URE-�B-, Octamer-1 (Oct-1), PU.1-, and TCF-1-con-taining oligonucleotides are as follows:URE-�B: 5�-AAGTCCGGGTTTTCCCCAACC-3�;Oct-1: 5�-TGTCGAATGCAAATCACTAGAA-3�;PU.1: 5�-GATCTACTTCTGCTTTTG-3�;TCF-1: 5�-GGGAGACTGAGAACAAAGCGCTCTCACAC.

Oligonucleotides were end-labeled with Klenow and [�-32P]dNTPs, as previously described.22 EMSA was performed using ap-proximately 2 ng of labeled oligonucleotide (20,000 dpm), 5 �g ofnuclear extract, 5 �L of sample buffer (10 mmol/L HEPES, 4 mmol/LDTT, 0.5% Triton X-100, and 2.5% glycerol), 0.5 �g poly dI-dC asnonspecific competitor, and adjusted to 100 mmol/L with KCl in afinal volume of 25 �L. This mixture was incubated at 25°C for 30minutes. Complexes were resolved in a 4.5% polyacrylamide gelusing 0.5� TBE running buffer (90 mmol/L Tris, 90 mmol/L boricacid, 2 mmol/L EDTA pH 8.0). For antibody supershift analysis, thebinding reaction was performed in the absence of the probe, theappropriate antibody was added, and the mixture was incubated for10 minutes at 25°C. The probe was then added, and the reactionincubated an additional 30 minutes at 25°C and the complexes re-solved by gel electrophoresis, as described earlier. Antibodies usedinclude: anti-RelA subunit sc-372X, anti-p50 subunit sc-114, andc-Rel subunit sc-70 from Santa Cruz Biotechnology, Inc. (SantaCruz, CA). For I�B-� blocking experiments, 100 ng I�B-�-Glutathi-one S-transferase (GST) fusion protein was added to the bindingreaction as described for antibody supershift analysis.

Immunoblot Analysis. For isolation of whole-cell extracts (WCEs),pulverized tissues were resuspended in cold PD buffer (40 mmol/LTris pH 8, 500 mmol/L NaCl, 6 mmol/L EDTA, 6 mmol/L EGTA, 10mmol/L glycerophosphate, 10 mmol/L NaF, 10 mmol/L PNPP [p-nitrophenyl phosphate], 300 �mol/L Na3V04, 1 mmol/L benzami-dine, 2 �mol/L PMSF, 1 mmol/L DTT, 1 �g/mL leupeptin, 10 �g/mLaprotinin, 1 �g/mL pepstatin, and 0.5% NonidetP-40 [NP-40]) andsheared through a syringe needle (25 G). Extracts were then clearedby centrifugation at 40,000 rpm for 30 minutes at 4°C. Samples(20-40 �g) were subjected to electrophoresis on a 10% polyacryl-amide (PAGE)-SDS gel, transferred to PVDF membrane (Millipore,Bedford, MA), and immunoblotting performed as previously de-scribed.22 The antibodies preparation for p65 (sc-372), I�B-� (sc-371), IKK-1 (sc-7182), IKK-2 (sc-7607) were purchased from SantaCruz Biotechnology, Inc. The antibodies for CKII� and CKII� werepurchased from Stressgen (Victoria, British Columbia, Canada) andfrom Transduction Laboratories (Lexington, KY).

IKK and Akt Kinase Assay. For the IKK kinase assay, WCEs (80 �g)were immunoprecipitated using protein A-Sepharose, as describedpreviously.47 For Akt kinase assay, WCEs (80 �g) were immunopre-cipitated with an agarose conjugated anti-Akt antibody (9279, NewEngland Biolabs, Beverly, MA) at 4°C overnight. Kinase assay wasdone in kinase buffer C (20 mmol/L Hepes pH 7.7, 2 mmol/L MgCl2,10 mmol/L �-glycerophosphate, 10 mmol/L NaF, 10 mmol/L PNPP,300 mmol/L Na3VO4, 1 mmol/L benzamidine, 2 mmol/L PMSF, 10�g/mL aprotinin, 1 �g/mL leupeptin, 1 �g/mL pepstatin, 1 mmol/LDTT) at 30°C for 45 minutes in the presence of 10 �mol/L adenosinetriphosphate (ATP), 3 �Ci [�-32P] ATP, and 1 �g of the indicatedsubstrate. The kinase reaction was stopped by addition of 4� SDS-PAGE sample buffer, subjected to SDS-PAGE analysis and visualizedby autoradiography.

HEPATOLOGY Vol. 34, No. 1, 2001 FACTOR ET AL. 33

CKII Kinase Assay. For evaluation of I�B-� phosphorylation di-rected by CKII, 10 �g WCEs prepared using PD buffer were dilutedto 10 �L final volume with the same buffer. Following addition of 15�L buffer D (100 mmol/L Tris pH 8.0, 100 mmol/L NaCl, 50 mmol/LKCl 20 mmol/L MgCl2, 100 �mol/L Na3VO4, 10 �Ci [�-32P] GTP),reactions were incubated at 30°C for 10 minutes in the presence of200 ng wtI�B-�-GST or �2I�B-�-GST as substrate. The �2IkB-GST-� substrate, kindly provided by J. Hiscott (Institut Lady Davisde Recherches Medicales, Montreal, Quebec), carries a deletion of aa.269-317 at the C-terminal PEST domain of I�B-�.48 The kinase re-action was stopped, and the products processed as described earlier.

RESULTS

Elevated NF-�B Binding Activity in c-myc/TGF-�-Induced Dys-plastic Livers and HCCs. To begin to assess the role of NF-�B inliver tumor growth, we compared the NF-�B binding levels inmultiple c-myc/TGF-�–induced dysplastic livers and HCCs,and normal liver tissue as control. Nuclear extracts were sub-jected to electrophoretic mobility shift assay (EMSA) using asprobe the upstream NF-�B element (URE-�B) from the c-mycgene.49 The nuclear extracts from 3 normal murine livers dis-played low levels of a single binding complex (band 1) (lanes1-3, Fig. 1A). In contrast, 2 bands were seen in the dysplasticlivers (lanes 4-6) and in the HCCs (lanes 7-16). Higher levelsof binding of complex 1 and an additional slower migratingcomplex (band 2) were observed. In addition the intensity ofband 2 appeared higher in the HCCs compared with the dys-plastic livers (Fig. 1A). Equal loading of the samples was con-firmed by using a radiolabeled probe specific for the oc-tamer-1 (Oct-1) (Fig. 1A, bottom panel). To characterize thenature of the subunit components of bands 1 and 2, supershiftEMSA was performed with extracts from each of the normal(10W1), dysplastic (MA11W1), or HCC (267F1T1) livers(Fig. 1B). These were compared with supershift EMSA withnuclear extracts from Hs578T human breast cancer cells,which express constitutive NF-�B (Fig. 1B).63 Addition of anantibody that preferentially recognizes NF-�B1 in a ho-modimer complex supershifted band 1 in all of the 4 samples(lanes 6, 10, 14, and 18), whereas an antibody against the RelAsubunit supershifted only band 2 (lanes 5, 9, 13, and 17).Addition of I�B-�-GST fusion protein, which interacts prefer-entially with the RelA subunit in classical NF-�B comparedwith NF-�B1 in a homodimer form, selectively reduced for-mation of band 2 (lanes 7, 11, 15, 19); whereas, addition ofGST alone had no effect on binding (data not shown). Fur-thermore, addition of an antibody against c-Rel had no effecton binding (lanes 8, 12, 16, and 20). Taken together, theseresults indicate that band 1 contains NF-�B1 homodimers,and band 2 is a heterodimer of NF-�B1/RelA or classicalNF-�B.

To determine whether the increased NF-�B binding seen inthe dysplastic livers and in the HCCs was a result of highercellular levels of RelA, WCEs were subjected to immunoblotanalysis. The resulting Western blot showed comparable lev-els of RelA protein expression in the normal versus neoplastictissues (Fig. 1C), suggesting that the accumulation of RelA inthe nucleus in the neoplastic tissues is a result of differentialregulation of its translocation in these cells. No significantbinding to the PU.1 or TCF oligonucleotides was detected innuclear extracts from the normal, dysplastic, or tumor livertissues (Fig 2A and B, respectively), indicating that the NF-�Bbinding activity seen in the dysplastic and tumor samples isnot a result of B or T lymphocyte infiltration. Thus, TGF-�/c-myc HCCs display increased NF-�B binding activity relative

to tissues from normal liver. Intriguingly, NF-�B transloca-tion is seen as an early event during TGF-�/c-myc–inducedneoplastic development, because it could be detected in dys-plastic livers, which precedes the onset of tumor growth.

FIG. 1. NF-�B is activated in dysplastic livers and HCCs of TGF-�/c-myctransgenic mice. (A) NF-�B binding activity. EMSA was performed using theupstream (URE) NF-�B element from the c-myc gene, as a probe.49 Nuclearextracts were isolated from normal (lanes 1-3), dysplastic (lanes 4-6), andneoplastic (lanes 7-16) livers (upper panel). EMSA was also performed withan Oct-1 probe, confirming equal loading of samples (bottom panel). (B)Dysplastic livers and HCCs express classical NF-�B. For supershift analysis,following a 30-minute incubation of nuclear extracts from the breast cancercell line 578T, the normal liver 10W1, the dysplastic liver MA11W1, and theHCC 267F1T1 with the URE NF-�B probe, 1 �L of antibody against either theNF-�B1 (sc-114), RelA (sc-372), or c-Rel (sc-070) protein was added asindicated. The reaction was incubated for an additional hour and subjected toEMSA. Alternatively, 100 ng of I�B-�-GST protein was added to the reaction.(C) RelA expression is comparable in normal, dysplastic, and transformedliver. For immunoblotting, 40 �g of WCEs were separated according to mo-lecular weight by SDS-PAGE and subjected to immunoblot analysis using thesc-372 antibody preparation raised against the RelA product. WCEs fromF22-Ras RLE cells, which express high levels of classical NF-�B,38 were usedas positive control.

34 FACTOR ET AL. HEPATOLOGY July 2001

Next, NF-�B binding activity in murine HCCs from singlec-myc and bitransgenic mice was compared. Nuclear extractsfrom peritumorous tissue (PT) and tumors (T) of 3 c-myctransgenic mice, and from the 267F1T1 tumor were subjectedto EMSA for NF-�B binding. On a light exposure, only lowlevels of band 2 were seen with the nuclear extracts from thec-myc transgenic mice (data not shown). On prolonged expo-sure, low levels of band 2 were detectable in all 3 HCCs (T) ofc-myc single transgenic mice (Fig. 3, lanes 3, 5, 7, and 8, toppanel). EMSA of the nuclear extract from the double TGF-�/c-myc 267F1T1 HCC displayed substantially higher levels ofNF-�B binding activity (lane 1). Interestingly, the low levelsof NF-�B binding in the tumors from the single transgenicmice were similar to those found in peritumorous tissues (PT)from the same animals (lanes 2, 4, and 6). Thus, higher levelsof NF-�B binding are seen in tumors from TGF-�/c-myc trans-genic mice compared with single c-myc transgenic mice.Overall, these data suggest that c-myc alone is not able toinduce high levels of NF-�B binding, suggesting that neoplas-tic development of the liver by this oncogene is less reliant onNF-�B.

IKK Complex Mediates Constitutive NF-�B Activation in HCCs ofTGF-�/c-myc Transgenic Mice. Recently, we and others haveshown that oncogenic Ras- or Raf-mediated translocation ofNF-�B in epithelial cells functions through activation of theIKK complex activation,38,50 and functions to promote onco-genic transformation. Thus, we explored the potential roles ofIKK-1 and IKK-2 in the increased NF-�B activity seen in thedysplastic livers and HCCs. Kinase assays were performed asdescribed previously10 using GST fusion proteins of eitherwild type I�B-� or of Ser32/36 double mutated I�B-�, whichcannot be phosphorylated, as control for specificity of thereaction. WCEs, prepared from multiple c-myc/TGF-�-in-duced dysplastic livers and HCCs and from normal liver tis-

FIG 2. Specificity of NF-�B acti-vation in TGF-�/c-myc dysplasticlivers and HCCs. EMSA was per-formed with nuclear extracts de-scribed in the legend to Fig. 1A witheither the TCF (A) or PU.1 (B) oligo-nucleotide, as probe. Extracts fromthe Jurkat T or the WEHI 231 B cellline were used as positive controlsfor TCF-1 or PU.1 binding, respec-tively.

FIG. 3. HCCs of single c-myc transgenic mice display low levels of NF-�B.The levels of NF-�B binding activity to the URE-�B NF-�B probe were mon-itored by EMSA analysis, as in Fig. 1A, using nuclear extracts from peritu-morous tissue (PT) or HCCs (T) from mice overexpressing c-myc (upperpanel). EMSA was also performed with an Oct-1 probe, confirming equalloading of samples (bottom panel).

HEPATOLOGY Vol. 34, No. 1, 2001 FACTOR ET AL. 35

sues, were immunoprecipitated with antibodies against IKK-1or IKK-2 and subjected to kinase assays. We were unable todetect IKK kinase activity in 3 samples of normal liver tissue(lanes 2-4, Fig. 4A). In contrast, both immunoprecipitatedIKK-2 and IKK-1 from either the dysplastic (lanes 5-7) orfrom c-myc/TGF-�–derived HCC-cell extracts were able tophosphorylate wild type (lanes 5-17, Fig. 4A, top and middlepanels, respectively) but not the Ser32/36 mutant I�B-�-GSTprotein (Fig. 4B). The presence of approximately equalamounts of IKK-1 or IKK-2 protein in the immunoprecipitateswas verified by immunoblotting (Fig. 4A and 4B, respectively).Thus, IKK complex is active and likely mediates the inductionof NF-�B through phosphorylation and degradation of I�B-�on serine 32 and 36 in TGF-�/c-myc–derived dysplastic andneoplastic liver tissues.

Previously, casein kinase II has been implicated in phos-phorylation of I�B-� at its C-terminal PEST sequence.48,51-53

To assess the potential role of this kinase in HCCs, we ana-lyzed the levels of expression of the CKII� and � subunits byimmunoblot analysis. Total cell extracts of normal, TGF-�/c-myc dysplastic livers, and HCCs revealed equal levels of ex-

pression of the CKII� catalytic subunit (Fig. 5A, top panel). Incontrast, the level of expression of CKII� was found to beupregulated in the HCCs but not in the dysplastic or normallivers (Fig. 5A, bottom panel). This prompted us to assesswhether CKII� upregulation had any effect on CKII-mediatedphosphorylation of I�B-�. A kinase assay was performed us-ing GST fusion proteins of either wild type I�B-� or a PEST-deleted I�B-� (I�B-� �2-GST), which lacks the 5 C-terminalsites phosphorylated by CKII.48 Because CKII preferentiallyuses guanosine triphosphate (GTP) as a phosphate donor,kinase assay was carried out in the presence of [�-32P] GTP,which allowed us to distinguish CKII-mediated phosphoryla-tion from that of the IKKs, which preferentially use ATP asphosphate donor. Total extracts from normal, dysplastic, andtumor tissue resulted in basal phosphorylation of wild typeI�B-�-GST, whereas phosphorylation of the I�B-� �2-GSTmutant was of much lower intensity consistent with measure-ment primarily of CKII activity (Fig. 5B). Furthermore, thelevels of I�B-� phosphorylation in the extracts from the nor-mal liver were comparable with those from the dysplastic andtumor tissues, with only the exception of the HCC 267C2T2

FIG. 4. IKK complex mediates constitutive NF-�B activation in dysplastic livers and HCCs of double TGF-�/c-myc transgenic mice. (A) Kinase assays.Following immunoprecipitation of extracts (80 �g), with antibodies against IKK-2 (�IKK-2, top panel) or IKK-1 (�IKK-1, middle panel), one half of thematerial was subjected to a kinase assay using wt I�B-�-GST. An equal aliquot of each immunoprecipitate was subjected to immunoblotting for IKK-1 protein.WCEs (80 �g) from F22-Ras RLE cells were used as positive control. (B) Alternatively, WCEs (80 �g) were immunoprecipitated with anti-IKK-2 and subjectedto kinase assay with either the wt or the Ser32/36 I�B-�-GST version, which cannot be phosphorylated by IKKs (top panel). To confirm equal amounts of IKK-2protein in the immunoprecipitates, immunoblotting was carried out as described above (bottom panel).

36 FACTOR ET AL. HEPATOLOGY July 2001

sample. These results suggest that the enhanced NF-�B levelsin the HCCs compared with normal and dysplastic tissue seenearlier in Fig. 1 is predominantly a result of activation of theIKK kinases.

IKK Kinase Activation Is Mediated by Akt/PKB. Recently, aphosphatidylinositol 3-kinase (PI(3)K) to Akt/PKB pathwayhas been shown to activate IKK via phosphorylation of IKK-1on threonine 23.8 To assess the possible involvement of Akt insignaling to the IKK complex, we carried out a kinase assayusing GST fusion proteins of either wild type IKK-1 or anIKK-1 mutant (T23A), harboring a threonine to alanine mu-tation at position 23. Total cell extracts were prepared from 4TGF-�/c-myc-induced HCCs, one dysplastic, one normal livertissue, and from 293T cells, as positive control. Followingimmunoprecipitation with an antibody against Akt, sampleswere subjected to kinase assays. We were unable to detect IKKkinase activity in either the normal or dysplastic liver tissue(lanes 2-3, Fig. 6). In contrast, all of the 4 tumors displayedvariable amounts of IKK-1-GST phosphorylation, with thetumor 267F1T1 expressing a level comparable with that of the293T cells. Intriguingly, the same extracts were able to phos-phorylate the T23A mutant IKK-1 to a similar extent as thewild type IKK-1. This result suggests that Akt can phosphor-ylate IKK-1 at multiple, presumably alternative sites; althoughthe possibility of a coprecipitating kinase responsible for theactivity cannot be excluded. Overall, these results suggest thatAkt may, in part, participate in the activation of the IKK com-plex in HCCs of TGF-�/c-myc transgenic mice.

Constitutive NF-�B Binding Activity in Human HCC Cell Lines.To determine whether aberrant activation of NF-�B occurs inhuman HCCs, we selected several human HCC cell lines,whose altered genotype has been shown to resemble that ofprimary tumors.43-46 SK-Hep-1, HLE, Huh-7, SNU387, andHep3B HCC nuclear extracts displayed 2 specific bindingcomplexes, labeled 1 and 2, and one nonspecific band (Fig.7A, and data not shown). In contrast, the HepG2 cell linedisplayed only low levels of band 1. To characterize the NF-�B

subunits present in complex 1 and 2, nuclear extracts of SK-Hep-1 cells were subjected to supershift analysis (Fig. 7B).Addition of an antibody that recognizes the NF-�B1 (p50)subunit ablated formation of both bands, leaving the nonspe-cific band (ns) unaffected. Addition of the cognate peptideblocked the supershift of band 1, but not of band 2, suggestingthat the antibody anti-p50 interacts with the protein con-tained in complex 1 through a different epitope. Incubationwith an antibody against the RelA (p65) subunit supershiftedonly band 2, and this effect was in part reduced by peptidecompetition. Addition of I�B-�-GST fusion protein elimi-nated band 1 and reduced formation of band 2. Furthermore,addition of an antibody against c-Rel had no effect on binding.Taken together, these results indicate that band 2 is a het-erodimer of p50/p65 or classical NF-�B and band 1 containsp50 homodimers. Similar results were obtained with extracts

FIG 5. CKII activity does not cor-relate with hepatic neoplastic devel-opment of mice overexpressingTGF-� and c-myc. (A) WCEs (40�g) were subjected to immunoblotanalysis using antibody preparationsagainst the CKII-� (top panel) or theCKII-� subunit (bottom panel). (B)CKII kinase assay. WCEs (40 �g)were subjected to kinase assays us-ing wt I�B-�-GST (Wt) or I�B-� D2-GST (D2) mutant, which cannot bephosphorylated by CKII.48

FIG. 6. Akt/PKB phosphorylates the IKK complex in HCCs of TGF-�/c-myc transgenic mice. Kinase assay. Following immunoprecipitation with an-tibody against Akt/PKB �Akt, WCEs (40 �g) were subjected to kinase assaysusing either wt IKK-1-GST (upper panel) or IKK-1-GST-T23A mutant (mid-dle panel), which is not phosphorylated by Akt in response to TNF-� stimu-lation.8 Alternatively, an equal aliquot of each immunoprecipitate was sub-jected to immunoblotting for Akt protein (bottom panel). WCEs (80 �g)from 293T cells were used as positive control.

HEPATOLOGY Vol. 34, No. 1, 2001 FACTOR ET AL. 37

from the other HCC cell lines (data not shown). Thus, humanHCC cell lines express constitutive binding of classical NF-�B.

DISCUSSION

In this study we show constitutive activation of NF-�B inhuman HCCs and that induction of NF-�B activity in thelivers of mice overexpressing TGF-� and c-myc expressionparallels neoplastic development. In mice, this induction cor-related with activation of the 2 critical kinases in the I�Bkinase complex, IKK-1 and IKK-2. Based on our knowledge ofcontrol of I�B turnover, we conclude that the induction ofNF-�B was achieved through phosphorylation of I�B-� by theactivated IKK complex. Previously, HCCs have been shown tooccur in TGF-�/c-myc double transgenic mice with a muchhigher frequency index compared with single c-myc trans-genic lines. This effect was due both to the stimulation ofhepatocyte cell proliferation by TGF-� through disruption ofthe pRb/E2F pathway40 and the reduction of the apoptoticindex, indicating that survival factors are also involved indevelopment of HCC. Here we have identified NF-�B as one ofthese anti-apoptosis factors.

We could detect NF-�B binding activity as early as dyspla-sia of the liver, suggesting that NF-�B induction precedes theonset of tumor growth and is likely to contribute to the posi-tive selection of preneoplastic hepatocyte population. Earlierresults showing appearance of hepatocellular carcinoma intransplantation experiments with dysplastic liver cells fromthe TGF-�/c-myc mice further support this notion.54

Previously, we and others have shown NF-�B/Rel factorsplay a key role in regulating hepatocyte cell survival in vitroand in vivo. Inhibition of NF-�B activity led directly to theinduction of apoptosis of murine hepatocytes and ectopic ex-pression of the c-Rel subunit rescued these cells from TGF-�1–mediated cell death.38,55 Similarly, oncogenic Ras- andRaf-mediated transformation of RLEs enhanced basal levels ofNF-�B promoting cell survival. Inhibition of NF-�B upon ec-topic expression of I�B-� restored the sensitivity of Ras andRaf-transformed RLEs to apoptosis upon TGF-�1 treatment,whereas, RelA or c-Rel subunits rescued normal RLEs from

TGF-�1 cell killing. It has been shown recently that IKK-2 orIKK-� knockout mice died early in utero because of massiveliver degeneration by apoptosis.56,57 This phenotype is similarto that of mice lacking the RelA subunit, which also die due toliver cell death.58 Both IKK-2 and RelA deficient mice could berescued by null mutation of the TNFR1 gene,56,59 indicating aprotective role of NF-�B against TNF-mediated cell death ofhepatocytes. Similarly, NF-�B induction after partial hepatec-tomy promoted cell survival and cell-cycle progression ofhepatocytes.60 Overall, these finding indicate NF-�B as a keyregulator of cell death of hepatocytes. Thus, it is likely thatsurvival promoted by NF-�B contributes to preneoplastic andneoplastic lesions of the liver.

Consistent with our hypothesis, NF-�B has been implicatedin both carcinogen- and viral-induced neoplastic develop-ment of mammary and liver epithelial cells. Recently, we haveshown that over 85% of DMBA-induced mammary tumors inthe Sprague-Dawley rat display elevated levels of NF-�B bind-ing.24 This NF-�B activation was evident after only 3 weeks ofDMBA exposure when tumors have not yet developed, againsuggesting that NF-�B translocates early to the nucleus duringepithelial tumor formation.30 Consistently, nuclear NF-�B ac-tivity was detected in specimens from primary human breastcancer, which could be attributed either to activation of IKK-1or IKK-2, or elevated levels of CKII.24,61 In addition, NF-�Bhas also been implicated in viral-induced hepatocarcinogen-esis. Recently, hepatitis C virus (HCV)-infected livers showedenhanced NF-�B binding activity.62 Similarly, HCV core pro-tein-transfected MCF-7, HeLa, and HepG2 cells displayed in-creased NF-�B binding, which rendered them more resistantto TNF-� cell killing.62-64 HCV infection is thought to lead tothe development of chronic and acute hepatitis whose out-come is often cirrhosis and ultimately HCCs,65,66 suggestingthat NF-�B activity may mediate some of these processesthrough inhibition of apoptosis. In addition, the upregulationof NF-�B has been detected in hepatitis B–associated livercancer, possibly because of X-protein activation.67 Impor-tantly, in this study we provide evidence that aberrant NF-�Bactivation also occurs in human HCCs. As cell models, we

FIG 7. Human HCC cell lines display constitutive NF-�B activity. (A) Nuclear extracts were prepared from the indicated HCC cell lines and EMSAperformed as in Fig. 1 (upper panel). As control for equal loading, the nuclear extracts were similarly subjected to EMSA with an Oct-1 probe (bottom panel).ns: nonspecific. (B) Human HCC cell lines express classical NF-�B. For supershift analysis, 1 �g of antibody against either the p50 (SC114-G), p65 (sc-372-G),or c-Rel (SC-070) protein was added for 10 minutes to nuclear extracts from the SK-Hep-1 cell line with the URE NF-�B probe, as indicated. The reaction wasincubated for an additional 30 minutes and subjected to EMSA. Alternatively, 100 ng of either I�B-�-GST or cognate peptide (pep) was added to the reaction.Similar results were obtained using nuclear extracts from the other HCC cell lines (data not shown).

38 FACTOR ET AL. HEPATOLOGY July 2001

used a panel of HCC lines43-46 whose biological and cytoge-netic characteristics closely mimic the genetic alterations seenin primary tumors.45 It is, therefore, likely that dissection ofthe mechanism leading to aberrant NF-�B activation in theseHCC cell lines will provide an important resource for definingthe functional genomic pathways relevant for pathogenesis ofliver cancer.

Overall, our results indicate that NF-�B may play a role inTGF-�–induced hepatocarcinogenesis by acting as a survivalfactor hence accelerating the neoplastic development of c-myc-transformed cells. Accordingly, AML-12 hepatocytes,which derive from a dysplastic liver of a TGF-� transgenicmouse, express constitutive NF-�B activity.30 Moreover, EGFdeprivation of the EGF-dependent normal murine hepatocytecells (NMH) inhibits NF-�B activity rendering these cellsmore sensitive to TGF-�1 cell killing. Conversely, stimulationof starved NMH cells with EGF leads to NF-�B activationdelaying TGF-�1-induced apoptosis (Arsura M., and Sonen-shein G.E., unpublished data, 2000).

Epidermal growth factor receptor (EGFR) signalling canbe stimulated by different EGF family members, includingTGF-�.68 One of the downstream effectors of the EGFR cas-cade has been shown to activate the PI(3)K-Akt pathway.69,70

Recently, we showed that in Ras transformed RLEs NF-�Btranslocation is a result of IKK complex activation by the ERKand PI(3)K. Here we show that the PI(3)K-Akt pathway isinvolved in NF-�B activation through phosphorylation ofIKK-1. From our data we cannot conclude that Akt is requiredfor the early activation of NF-�B during HCCs developmentbecause we did not detect Akt kinase activity on the substrateIKK-1 using extracts from dysplastic liver. Although the pos-sibility that our kinase assay was not sensitive enough to de-tect IKK-1 phosphorylation in the dysplastic livers, whichdisplay only low levels of NF-�B activation, cannot be ex-cluded. Our data are consistent, however, with a role for Aktin NF-�B activation later and hence in development of HCCtumors.

Recently, IKKs has been shown to mediate many of thesignaling cascades leading to NF-�B activation,71 althoughsome exceptions have been reported.72,73 Our findings indi-cate that overexpression of TGF-� can activate NF-�Bthrough IKKs conferring a survival advantage to liver epithe-lial cells. Thus, IKK complex, which represents a ubiquitousmediator of NF-�B activation, might represent a candidatetarget for anticancer therapy. Previously, a dramatic increaseof reactive oxygen species (ROS) in TGF-�/c-myc transgenichepatocytes has been reported.42 Because oxidative stress hasbeen reported to be a potent activator of NF-�B,2 it is temptingto speculate that ROS might mediate NF-�B induction in theHCCs. In support of this hypothesis, it has been recentlyshown that vitamin E, a potent free radical scavenger, reducesliver dysplasia, increases hepatocyte cell survival, and pre-vents malignant conversion in TGF-�/c-myc mice.74 Work isin progress to test the possibility that the inhibition of hepaticneoplastic development by antioxidants is a result of NF-�Binhibition.

Acknowledgment: The authors gratefully acknowledgeJohn Hiscott, Carlos Paya, David Donner, and C. Harris forkindly providing cloned DNAs and cell lines. We are thankfulto Ms. Mariya Nazarova for excellent technical assistance. Theauthors thank D. Sloneker for assistance in preparation of thisarticle.

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