NF-κB functions as a tumour promoter in inflammation-associated cancer

© 2004 Nature Publishing Group

Acknowledgements We thank S. H. Ahn, D. Allis, M. Reyland, U. Siebenlist, the Rockefeller

University Genotyping Resource Center and the MSKCC Genomics Core Laboratory for

providing cells, vectors, reagents and technical assistance. We also thank E. Besmer for help with

manuscript preparation, and A. Patke and members of the Tarakhovsky laboratory for

discussions. This work was supported by The Irene Diamond Fund/Professorship Program

(A.T.), the NIH (A.T.) and The S.L.E. Foundation (I.M.).

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to A.T.

([email protected]).

..............................................................

NF-kB functions as atumour promoter ininflammation-associated cancerEli Pikarsky1*, Rinnat M. Porat1*, Ilan Stein1,2*, Rinat Abramovitch3,Sharon Amit2, Shafika Kasem1, Elena Gutkovich-Pyest2,Simcha Urieli-Shoval4, Eithan Galun3 & Yinon Ben-Neriah2

1Department of Pathology, Hadassah-Hebrew University Medical Center,Jerusalem 91120, Israel2The Lautenberg Center for Immunology, Hebrew University-Hadassah MedicalSchool, Jerusalem 91120, Israel3Goldyne Savad Institute of Gene Therapy, Jerusalem 91120, Israel4Hematology Unit, Hadassah University Hospital, Mount Scopus, Jerusalem91120, Israel

* These authors contributed equally to this work

.............................................................................................................................................................................

The causes of sporadic human cancer are seldom recognized, butit is estimated that carcinogen exposure and chronic inflam-mation are two important underlying conditions for tumourdevelopment, the latter accounting for approximately 20% ofhuman cancer1. Whereas the causal relationship betweencarcinogen exposure and cancer has been intensely investigated2,the molecular and cellular mechanisms linking chronic inflam-mation to tumorigenesis remain largely unresolved1. We pro-posed that activation of the nuclear factor kB (NF-kB), a hallmarkof inflammatory responses3 that is frequently detected intumours4,5, may constitute a missing link between inflammationand cancer. To test this hypothesis, we studied the Mdr2-knock-out mouse strain, which spontaneously develops cholestatichepatitis followed by hepatocellular carcinoma6, a prototype ofinflammation-associated cancer7. We monitored hepatitis andcancer progression in Mdr2-knockout mice, and here we showthat the inflammatory process triggers hepatocyte NF-kBthrough upregulation of tumour-necrosis factor-a (TNFa) inadjacent endothelial and inflammatory cells. Switching off NF-kBin mice from birth to seven months of age, using a hepatocyte-specific inducible IkB-super-repressor transgene, had no effecton the course of hepatitis, nor did it affect early phases ofhepatocyte transformation. By contrast, suppressing NF-kB inhi-bition through anti-TNFa treatment or induction of IkB-super-repressor in later stages of tumour development resulted inapoptosis of transformed hepatocytes and failure to progress tohepatocellular carcinoma. Our studies thus indicate that NF-kB isessential for promoting inflammation-associated cancer, and istherefore a potential target for cancer prevention in chronicinflammatory diseases.

Hepatocellular carcinoma (HCC), the third leading cause ofcancermortality worldwide, commonly develops in the backgroundof chronic hepatitis8. We confirmed and extended previous

results6,9, according to which tumour development in the Mdr2-knockout mouse, similarly to human HCC8, progresses throughdistinct phases: inflammation, dysplasia, dysplastic nodules (ade-noma-like), carcinoma and metastasis (Supplementary Fig. 1a, b).Hepatitis is the earliest sign of disease in the Mdr2-knockout mice,and is characterized by an extensive periductular and periportalmixed inflammatory infiltrate, rich in CD3-positive cells (Sup-plementary Fig. 1c).NF-kB activation is often observed in human HCC, particularly

following hepatitis8. The possibility that NF-kB activation isinvolved in Mdr2-knockout hepatocarcinogenesis was investigatedby RelA (p65) immunostaining (Fig. 1a). Nuclear staining, indica-tive of NF-kB activation, was evident in knockout livers ofmice at allages, but not in normal, age-matched mice; this prompted a searchfor the source of NF-kB activation in the knockout mice. In someneoplasms such as Hodgkin’s disease and certain lymphomas,NF-kB activation is an intrinsic, tumour autonomous feature,possibly related to mutations in its signalling pathway10. If a similarmechanism occurs in Mdr2-knockout HCC, one would observenuclear NF-kB activation in all neoplastic hepatocytes. However,this was not the case; NF-kB activation appeared scattered at theparenchyma, predominantly adjacent to inflamed portal tracts(Fig. 1e, left panel), suggesting an inflammation-induced phenom-enon. To evaluate this possibility, we fed knockout and wild-typemice with the non-steroidal anti-inflammatory drug (NSAID)ibuprofen for 10 days. This treatment resulted in decreased inflam-mation, evident by histological analysis (Fig. 1b), decreased serumalanine aminotransferase (ALT), a marker for hepatocyte damage,(Fig. 1c) and fewer neutrophils (Fig. 1c, d). p65 immunostainingshowed that the degree of hepatocyte NF-kB activation in theNSAID-treated group was significantly lower than in the controls(Fig. 1e). In contrast, ibuprofen had no effect on hepatocyte NF-kBactivation following intraperitoneal TNFa administration (data notshown). Hence, it seems that NF-kB activation in the knockouthepatocytes is secondary to parenchymal infiltration by inflamma-tory cells.One likely mediator of NF-kB activation in the inflamed liver is

TNFa, particularly its membrane form, which is upregulated inknockout livers (Fig. 2a). To study this possibility, we treatedknockout mice with neutralizing anti-TNFa antibodies for threedays and analysed hepatocyte NF-kB activation. Anti-TNFa treat-ment abolished hepatocyte p65 nuclear staining (Fig. 2b), confirm-ing the prediction that NF-kB activation is primarily induced byTNFa. To determine the source of TNFa in the inflamed liver, wefractionated wild-type and knockout livers to hepatocytes (.80%purity by haematoxylin-and-eosin (H&E)) and other cells (,2%hepatocytes). Analysis of the two fractions for TNFa expression byreal-time polymerase chain reaction (PCR) confirmed that thesource of TNFa was the non-hepatocyte fraction, which expressedfivefold more TNFa than the corresponding fraction of wild-typemice (Fig. 2c).To study the relationship between the TNFa-producing cells and

NF-kB activation in the hepatocytes, liver sections were stained forboth TNFa and p65 (Fig. 2d, f). TNFa expression was detectedprimarily at portal spaces, both in endothelial and inflammatorycells (Fig. 2d). Hepatocyte nuclear p65 staining was particularlyabundant close to TNFa-positive portal spaces (Fig. 2f), indicatingthat hepatocyte NF-kB is activated through paracrine TNFa stimu-lation. To confirm the identity of the TNFa producers and dis-tinguish them from TNFa-binding cells, liver sections of wild-type,knockout and ibuprofen-treated mice were analysed by in situmessenger RNA hybridization. Similar to the immunostainingdata, TNFamRNAwas found to be highly expressed by endothelialand inflammatory cells of knockout mice, but far less in wild-typeand ibuprofen-treated knockout mice. TNFa expression was hardlydetected in either knockout or wild-type hepatocytes (Fig. 2e).The frequent activation of NF-kB in knockout hepatocytes

letters to nature

NATURE |VOL 431 | 23 SEPTEMBER 2004 | www.nature.com/nature 461


suggested that inflammation-associated NF-kB activation promotesneoplastic growth4,5. To directly assess the role of NF-kB in hepato-carcinogenesis, we bredMdr2-knockout mice withDN-IkBhepmice,which carry two transgenes: a non-degradableDN-IkB controlled bya tetracycline (tet)-regulated promoter and the tetracycline trans-activator under control of the hepatocyte specific C/EBPb promoter(TALAP1)11. The tet-regulated promoter also directs the expressionof luciferase; consequently, transgene expression levels and its tissuespecificity can be determined using a live-mouse recording ofluciferase activity11. Crossing the bi-transgenic DN-IkBhep micewith Mdr2-knockout mice generates Mdr22/2 DN-IkBhep (doublemutant) mice amenable to NF-kB modulation. Immunohisto-chemical staining of livers from the same animals with anti-luciferase antibodies demonstrated that only hepatocytes, but notthe bile duct epithelium, express the transgene (SupplementaryFig. 2a). As expected, p65 immunostaining detected many positivehepatocyte nuclei in knockout and doxycycline (Dox)-treated(transgene-suppressed) mice, but none in the untreated doublemutant mice (Supplementary Fig. 2b, c). Positively-stained biliaryand Kupffer cells were detectable in all knockout and double mutantmice irrespective of Dox treatment (not shown), attesting to thespecificity and efficiency of the transgene in blocking NF-kBactivation.Because the origin of liver inflammation in theMdr2-knockout is

the biliary system6, it should not be affected by the hepatocyte-

specific DN-IkBhep transgene. Nevertheless, before assessing the roleof NF-kB in tumorigenesis, we wanted to rule out any possible effectof NF-kB inhibition on the inflammatory process. Both the knock-out and the double mutant mice had higher ALT levels than those ofage-matched wild-type mice (Fig. 3b). Histological analysis of liversfrom both groups at all ages revealed comparable non-suppurativecholangitis with portal expansion due to ductular proliferation, amixed portal inflammatory infiltrate and mild to moderate fibrosis(Fig. 3a). Both strains had high numbers of CD3-postive andmyeloperoxidase (MPO)-positive cells in the portal tracts and theliver parenchyma. Thus, it seems that the fundamental inflamma-tory process in Mdr2-knockout mice is maintained in the doublemutant mice and is independent of hepatocyte NF-kB activity.

Mdr2-knockout hepatocytes are distinguishable from wild-typecells by several abnormal features: high proliferation rate, acceler-ated hyperploidy and dysplasia. Whereas the first are also charac-teristics of liver damage and partial hepatectomy12, dysplasia is apremalignant condition13. Hepatocyte proliferation was evaluatedby bromo-deoxy uridine (BrdU) incorporation and Ki-67 antigenstaining. Both markers were clearly enhanced in knockout anddouble mutant mice compared with wild-type mice (Fig. 3b). Inagreement with previous studies14,15, NF-kB deficiency doesn’tcompromise hepatocyte proliferation: there was no significantdifference in either hepatocyte BrdU incorporation or Ki-67 stain-ing between the knockout and the double mutant mice at any age.

Figure 1 NF-kB activation, a prominent component of Mdr2-knockout hepatitis, is

abrogated by NSAID treatment. a, Tissue sections from wild-type and knockout mice were

immunostained with antibodies against p65/RelA. TNFa-treated wild-type liver serves as

a positive control for p65 staining. HCC: a section from a hepatocellular carcinoma that

developed in a knockout mouse. b, H&E-stained sections from livers of 2.5-month-old

wild-type, knockout and ibuprofen-treated knockout mice. Note decreased inflammatory

infiltrate after ibuprofen treatment. c, MPO cell counts and serum ALT levels from

wild-type, knockout and ibuprofen-treated knockout mice (mean ^ s.e.m.).

d–e, Immunohistochemical stains for MPO (d) and p65 (e) were performed on sections

from untreated and ibuprofen-treated knockout mice. Note the NF-kB nuclear staining

in the hepatocytes and bile ducts of knockout mice and the decreased staining in

ibuprofen-treated mice. All scale bars, 50mm. Knockout (KO) ¼ Mdr2 2/2; WT, wild

type. Arrows point to examples of: Kupffer cells (red arrows), NF-kB-positive hepatocytes

(yellow arrows) and bile epithelium (turquoise arrows).

letters to nature

NATURE | VOL 431 | 23 SEPTEMBER 2004 | www.nature.com/nature462


Hepatocyte dysplasia has two major features, architectural dis-organization and cytological atypia. Histological analysis revealedthat anisocytosis was a prominent feature of both knockout anddouble mutant mice; dysplasia was evident at 4 months andincreased in severity with age in both animal strains. We couldnot detect any difference in the extent or degree of dysplasia betweenknockout and double mutant mice at any age group (Fig. 3c). In anattempt to quantify the premalignant changes we analysed hepato-cyte ploidy in both strains in comparison to age-matched wild-typemice. Isolated nuclei from livers were stained with propidium-iodide and subjected to fluorescence-activated cell sorting (FACS)analysis. Both groups had a high degree of hyperploidy, and wenoted no significant difference between the cell-cycle profiles of theknockout and double mutant mice at 4 or 7 months (Fig. 3b). Thefinding that knockout and double mutant mice display comparabledegrees of proliferation, hyperploidy and dysplasia implies thatNF-kB is not required for early neoplastic events.

Whereas NF-kB inhibition had no effect at the tumour initiationphases, magnetic resonance imaging (MRI) and histological analy-sis of older mice clearly discerned the knockout and Dox-treatedmice from the untreated (NF-kB deficient) double mutant mice. At10 months, 60% and 78% of knockout and Dox-treated doublemutant mice, respectively, had liver tumours, compared with only10% of untreated double mutant mice (P , 0.01, Fig. 4). Thus,ablation of NF-kB activity in the hepatocytes led to a dramaticdecrease in tumour progression. A similar tumour suppressioneffect in the NF-kB-deficient animals was noted when comparingthe mean number of tumours per animal for the twomouse groups.

Histological analysis of the livers confirmed the MRI data (data notshown).At which stage of tumour development does NF-kB inhibition

exert its anti-tumorigenic effect? By 7 months no difference in earlytumour development was noticed between knockout and doublemutant mice: neither group had detectable tumours; only dysplasticparenchyma. We therefore switched off the transgene at 7 months,allowingNF-kB activation in the hepatocytes. Tumour developmentwas monitored using MRI for 3 months. Surprisingly, 7 of 9 micefed on Dox (NF-kB proficient) developed MRI-detectable tumourswithin this 3-month period. These were indistinguishable in size(Fig. 4b) and histological appearance (data not shown) fromknockout tumours at a comparable age. Hence, it seems thatwhereas NF-kB is dispensable for the early premalignant phase,(that is, tumour initiation) it is essential for subsequent tumourpromotion.Finally, how does NF-kB support tumour promotion? NF-kB has

a major anti-apoptotic effect in the mouse liver during develop-ment16 and is necessary to protect mature hepatocytes againstimmune attack11 and genotoxic stress17. Compromising the anti-apoptotic function of NF-kB could contribute to the tumour-suppressing effect of the IkB-super-repressor in the double mutantmice. To study this possibility, we assessed apoptosis using immu-nostaining for activated caspase-3. Knockout livers at 7 months (inwhich the majority of hepatocytes are dysplastic; Fig. 3c) showedmore apoptosis than wild-type livers—probably due to the toxiceffects of inflammation (Fig. 5a). However, blocking hepatocyteNF-kB induced a further threefold increase in hepatocyte apoptosis.

Figure 2 NF-kB activation in Mdr2-knockout mice is induced by paracrine TNFa.

a, Western blot analysis of TNFa in knockout versus wild-type liver samples. Anti-TNFa

antibodies detect only the transmembrane TNFa (mTNFa; 26 kD), the expression of

which is enhanced in the knockout samples. n.s., non specific band. b, Anti-p65

immunostaining of knockout livers from 7-month-old mice treated with anti-TNFa

neutralizing antibodies or control IgG as indicated. Quantitative analysis revealed a fivefold

reduction in the number of p65-positive hepatocyte nuclei. c, Real-time PCR analysis

of TNFa in hepatocyte (H) and non-hepatocyte (R) cellular fractions of knockout and

wild-type livers. TNFa-treated (TNF) and non-treated (NT) mouse embryo fibroblasts

(MEF) serve as controls. Values given are TNFa mRNA levels normalized to a ribosomal

standard (mean ^ s.e.m.). d, Immunostaining for TNFa; note the marked endothelial and

mononuclear cell staining in knockout mice. e, In situ mRNA hybridization (ISH) for TNFa;

note the abundant signals in endothelial, mononuclear and Kupffer cells, particularly in

untreated knockout samples. f, Double immunostaining of knockout liver sections for

TNFa (AEC, red precipitate) and p65 (NBT/BCIP, purple-black precipitate). All scale bars,

50 mm. Arrows: red, Kupffer cells; yellow, hepatocytes; turquoise, endothelium; purple,

inflammatory cells.

letters to nature



Figure 4 NF-kB is dispensable for early stages of HCC development, but is critical for

tumour promotion. a, MRIs from the indicated genotypes were taken at 10 months of age;

each image shown is from a different animal. Red dashed lines outline the liver

circumference. The coloured arrowheads indicate the tumours; different colours indicate

individual tumours. b, Liver tumour volume was calculated by analysing all coronal and

axial images of each animal; nodules smaller than 100mm3 were often found to represent

dysplastic nodules rather than HCC in histological analysis and were therefore not scored

as tumours. DM þ Dox ¼ mice fed on Dox between 7–10 months of age. Both knockout

and double mutant þ Dox groups differ significantly (P , 0.01, Mann–Whitney analysis)

from the double mutant group, but not from each other.

Figure 3 Inhibition of hepatocyte NF-kB activity influences neither hepatitis nor

development of the premalignant features in Mdr2-knockout mice. a, H&E-stained

sections of 4-month-old wild-type, knockout and double mutant mice. b, Serum ALT

levels, average number of CD3-positive cells per one high power field (HPF), number of

Ki-67-positive hepatocytes per 20 HPFs and percentage of hyperploid nuclei. All

bar-graphs indicate mean ^ s.e.m. c, H&E-stained sections from livers of 7-month-old

mice of the indicated genotypes, showing comparable dysplasia evident by architectural

disorganization and prominent nuclear pleomorphism in both knockouts and double

mutants, in contrast to the normal nuclei in wild types. All scale bars, 100mm. m.o.,

months old; DM, double mutant.

letters to nature



Notably, a similar effect was achieved by anti-TNFa treatment.Taken together, our results suggest that NF-kB inhibition exerts itstumour-suppressive effect by promoting apoptosis of transformedhepatocytes.

NF-kB regulates multiple anti-apoptotic genes, some of whichcould be relevant to hepatocyte transformation. To identify relevanttarget genes and corroborate the pro-tumorigenic role of theTNFa–NF-kB activation axis, expression of candidate genes wasdetermined by semiquantitative PCR with reverse transcription(RT–PCR) and western blot analyses. Whereas some examined

targets (for example, XIAP) were similarly expressed in NF-kB-proficient and -deficient livers, A1/Bfl1, c-IAP1 andGADD45bweresignificantly enhanced in the knockout livers (Supplementary Fig.3a, b). Concordantly, anti-TNFa treatment prevented the inductionof GADD45b and A1/Bfl1 in knockout mice (Fig. 5b).It is conceivable that the inflammatory process in the liver

contributes to tumorigenesis through two arms that complementone other (Fig. 5d). One arm would be responsible for inducinghepatocyte proliferation in knockout livers. Accelerated DNAreplication is by itself error prone and a ground for further pro-tumorigenic alterations by genotoxic agents18, many of which arespecifically activated in the liver (for example, aflatoxin)19. Inaddition, tumour promotion requires a second arm providingapoptosis protection through NF-kB activation. Our data indicatethat TNFa is a major driver of the anti-apoptotic arm, necessaryfor activation of NF-kB-dependent anti-apoptotic genes, such asA1/Bfl1, c-IAP1 and GADD45b. Although NF-kB is activated earlyin the course of the disease, it is dispensable for a prolonged periodduring which premalignant foci accumulate in the inflamed liver,but is critical at the stage during which these foci turn malignant.Perhaps acquisition of oncogenic mutations during the tumourpromotion phase renders premalignant hepatocytes particularlysensitive to apoptotic factors, which must be neutralized throughNF-kB activity. Putative apoptosis-regulating factors at this vulner-able tumour development phase are p53 and cJun (ref. 20), TNFreceptor family members21 and cJun amino-terminal kinase(JNK)22. JNK activation and augmented cJun expression wereobserved in knockout hepatocytes at 7 months (Fig. 5c andSupplementary Fig. 3a), around the peak period of premalignantchanges (Fig. 3). Similarly to NF-kB, these were dependent oncontinuous TNFa signalling, as anti-TNFa treatment of knockoutmice suppressed liver JNK activation and the expression of hepato-cyte cJun (Fig. 5b, c). JNK activation is subject to NF-kB inhibitionvia the NF-kB target GADD45b (refs 23, 24). NF-kB inhibition inknockout hepatocytes could therefore enhance JNK-mediatedapoptosis through inhibition of GADD45b (Fig. 5b), and promotecaspase-mediated hepatocyte apoptosis through suppression ofmultiple NF-kB-induced anti-apoptotic genes (SupplementaryFig. 3). In parallel, cJun inhibition by anti-TNFa treatment(Fig. 5c) could promote p53-mediated apoptosis20. Thus, by sup-pressing multiple anti-apoptotic pathways (Fig. 5d), anti-TNFatreatment may serve as a potential mode of HCC prophylaxis inchronic hepatitis patients.Our conclusions are supported by recent studies in a colon

inflammation model, in which colonic IkB kinase b (IKKb) in-activation results in epithelial apoptosis during tumour pro-motion25. Whereas inflammation releases many growth factorsand cytokines1,26, our data suggest that TNFa has a central role inactivating NF-kB and protecting transformed hepatocytes againstapoptosis. It has been proposed that anticancer research might bemore effective if aimed at eradicating the cause or the signallingcontext of abnormality rather than just treating the end result27.Whereas eradicating the primary cause, for example chronic hepa-titis, is currently unattainable, disrupting the signalling context ofthe evolving tumour may be a more realistic objective. Our findingthat short-term ablation of epithelial NF-kB activation is sufficientto induce premalignant cell apoptosis and halt tumour progressionsupports this conjecture. Intermittent suppression of a majorsignalling factor, such as NF-kB or TNFa, could thus be a tool toprotract the premalignant phase and inhibit tumour progression inchronic inflammatory diseases with high cancer risk. A

MethodsThe following details can be found in the Supplementary Information: reagents,transgenic mouse construction and genotyping, semiquantitative RT–PCR, real-timePCR, in situ mRNA hybridization, liver cell fractionation, western blot and hepatocyteploidy analyses.

Figure 5 NF-kB inhibition through the IkB-super-repressor or anti-TNFa treatment

results in suppression of anti-apoptotic factors. a, Caspase activity was quantified by

counting the number of hepatocytes stained with antibodies against activated caspase-3

in 10 high-power fields (mean ^ s.e.m.). b, Liver protein extracts from knockout animals

treated for 3 days with anti-TNFa antibodies (anti-TNF), or IgG were subjected to western

blot analysis with the indicated antibodies. c, cJun immunostaining of wild-type and

knockout liver sections from IgG- and anti-TNFa-treated mice. TNFa-treated wild-type

liver is a positive control for cJun staining. All scale bars, 50mm. d, A proposed model for

inflammation-orchestrated signalling at the premalignant phase of HCC development:

TNFa, a major mediator of the inflammatory process, tilts the balance in favour of

hepatocyte proliferation. Carcinogenic metabolites would push tumour progression

further towards malignancy.

letters to nature



Animal studies and tissue preparationAll animal experiments were performed in accordance with the guidelines of theinstitutional committee for the use of animals for research. Mice were killed by a lethaldose of anaesthesia. In vivo luciferase imaging and doxycycline treatment were performedas previously described11. Animals were injected intraperitoneally (i.p.) with 20 ng humanrecombinant TNFa (Roche) and killed after 1 h for liver p65 immunostaining and RNAand protein extraction. For anti-TNFa treatment, mice were injected with 22mg of goatanti-mouse TNFa neutralizing antibodies i.p. (R&D Systems; AF-410-NA) for 3consecutive days, or with control goat IgG. All mice were injected i.p. with BrdU(Amersham; 100 ml per 10 g body weight) 3 and 24 h before they were killed. In someanimals, liver samples were removed and snap-frozen for protein and RNA analyses. Theanimal was then perfused through the left ventricle with 10ml of cold heparinized PBS,followed with 25ml of 4% buffered formalin. Livers were removed, weighed,photographed and fixed in formalin over night. The next day the entire liver wassubmitted for paraffin embedding in three to five cassettes. 5-mM sections were stainedwith H&E and evaluated by a pathologist to whom the genetic makeup or treatment groupwere not known.

MRI analysisMRI was performed on a horizontal 4.7T Biospec spectrometer (Bruker Medical), using abirdcage coil. Mice were anaesthetized (30mg kg21 pentobarbital, i.p.) and placed supinewith the liver located at the centre of the coil. Liver and tumour volumes were determinedfrom multi-slice coronal and axial T1 weighted fast spin echo images covering the entireliver both coronally and axially (repetition time, 400ms; echo time, 17ms; slice thickness,1mm; field of view, 5 cm (coronal) and 3.4 cm (axial) using a 256 £ 256 matrix). Toestimate tumour and liver volumes, the liver and tumour boundaries visualized in eachslice were outlined by using image processing software (NIH image), by an observer towhom the genetic makeup was not known. The number of pixels (for both liver andtumour) were converted to an area by multiplying by the factor ((field ofview)2 £ (matrix)2).

ImmunohistochemistryFor p65 immunostains, 5-mM sections, cut on the same day, were de-waxed and hydratedthrough graded ethanols, cooked in 25mM citrate buffer pH 6.0 in a pressure cooker at115 8C for 3min (decloaking chamber, Biocare Medical), transferred into boilingdeinoized water and left to cool down for 20min. After 5min treatment in 3%H2O2, slideswere incubated with rabbit polyclonal p65 antibodies diluted 1:100 in CAS-Block (Zymed)for 3 h at room temperature, washed three times with Optimax (Biogenex), incubated for30min with anti rabbit Envisionþ (DAKO) and developed with DAB for 15min. Antigenretrieval for TNFa and p65 double immunostaining was performed in 100mM glycinebuffer pH 9.0. After 5min treatment in 3% H2O2, slides were incubated with goatpolyclonal anti-TNFa antibodies diluted 1:50 in CAS-Block for 1 h at room temperature,washed in Optimax, incubated with (1:100) rabbit anti-goat antibodies (JacksonLaboratories) for 30min, washed again with Optimax and incubated with anti-rabbitEnvisionþ (DAKO) and developed with AEC for 15min at 37 8C. Following this, slideswere reboiled for 7min in 100mM glycine buffer in a microwave oven and cooled down toroom temperature. Slides were incubated with rabbit polyclonal anti-p65 antibodiesdiluted 1:100 in CAS-Block for 3 h at room temperature, washed three times withOptimax, post-fixed for 5min in 4% formaldehyde in Tris buffered saline, washed withOptimax, incubated for 30minwith alkaline phosphatase-conjugated polymer anti-rabbitIgG (Zymed) and developed with BCIP/NBT. Slides were counterstained with nuclear fastred. Antigen retrieval for CD3, cJun, BrdU, activated caspase-3, luciferase, MPO and Ki-67were performed in 25mMcitrate buffer pH 6.0. Antigen retrieval for TNFawas performedin 100mM glycine buffer pH 9.0. Detailed protocols for immunostaining with eachantibody are available on request.

Received 14 June; accepted 10 August 2004; doi:10.1038/nature02924.

Published online 25 August 2004.

1. Coussens, L. M. & Werb, Z. Inflammation and cancer. Nature 420, 860–867 (2002).

2. Balmain, A. & Harris, C. C. Carcinogenesis in mouse and human cells: parallels and paradoxes.

Carcinogenesis 21, 371–377 (2000).

3. Li, Q. &Verma, I.M.NF-kB regulation in the immune system.Nature Rev. Immunol. 2, 725–734 (2002).

4. Mayo, M. W. & Baldwin, A. S. The transcription factor NF-kB: control of oncogenesis and cancer

therapy resistance. Biochim. Biophys. Acta 1470, M55–M62 (2000).

5. Lin, A. & Karin, M. NF-kB in cancer: a marked target. Semin. Cancer Biol. 13, 107–114 (2003).

6. Mauad, T. H. et al. Mice with homozygous disruption of the mdr2 P-glycoprotein gene. A novel

animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.Am.

J. Pathol. 145, 1237–1245 (1994).

7. Nakamoto, Y., Guidotti, L. G., Kuhlen, C. V., Fowler, P. & Chisari, F. V. Immune pathogenesis of

hepatocellular carcinoma. J. Exp. Med. 188, 341–350 (1998).

8. Block, T. M., Mehta, A. S., Fimmel, C. J. & Jordan, R. Molecular viral oncology of hepatocellular

carcinoma. Oncogene 22, 5093–5107 (2003).

9. De Vree, J. M. et al. Correction of liver disease by hepatocyte transplantation in a mouse model of

progressive familial intrahepatic cholestasis. Gastroenterology 119, 1720–1730 (2000).

10. Staudt, L. M.Molecular diagnosis of the hematologic cancers.N. Engl. J. Med. 348, 1777–1785 (2003).

11. Lavon, I. et al. High susceptibility to bacterial infection, but no liver dysfunction, in mice

compromised for hepatocyte NF-kB activation. Nature Med. 6, 573–577 (2000).

12. Gupta, S. Hepatic polyploidy and liver growth control. Semin. Cancer Biol. 10, 161–171 (2000).

13. Bruix, J., Boix, L., Sala, M. & Llovet, J. M. Focus on hepatocellular carcinoma. Cancer Cell 5, 215–219

(2004).

14. Chaisson, M. L., Brooling, J. T., Ladiges, W., Tsai, S. & Fausto, N. Hepatocyte-specific inhibition of

NF-kB leads to apoptosis after TNF treatment, but not after partial hepatectomy. J. Clin. Invest. 110,

193–202 (2002).

15. Maeda, S. et al. IKKb is required for prevention of apoptosis mediated by cell-bound but not by

circulating TNFa. Immunity 19, 725–737 (2003).

16. Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S. & Baltimore, D. Embryonic lethality and liver

degeneration in mice lacking the RelA component of NF-kB. Nature 376, 167–170 (1995).

17. Lavon, I. et al. Nuclear factor-kB protects the liver against genotoxic stress and functions

independently of p53. Cancer Res. 63, 25–30 (2003).

18. Friedberg, E. C.,McDaniel, L. D. & Schultz, R. A. The role of endogenous and exogenousDNAdamage

and mutagenesis. Curr. Opin. Genet. Dev. 14, 5–10 (2004).

19. Wang, J. S. & Groopman, J. D. DNA damage by mycotoxins. Mutat. Res. 424, 167–181 (1999).

20. Eferl, R. et al. Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell 112,

181–192 (2003).

21. Ehrhardt, H. et al. TRAIL induced survival and proliferation in cancer cells resistant towards

TRAIL-induced apoptosis mediated by NF-kB. Oncogene 22, 3842–3852 (2003).

22. Kennedy, N. J. & Davis, R. J. Role of JNK in tumor development. Cell Cycle 2, 199–201 (2003).

23. Tang, G. et al. Inhibition of JNK activation through NF-kB target genes.Nature 414, 313–317 (2001).

24. Papa, S. et al. Gadd45 b mediates the NF-k B suppression of JNK signalling by targeting MKK7/

JNKK2. Nature Cell Biol. 6, 146–153 (2004).

25. Greten, F. R. et al. Ikkb links inflammation and tumorigenesis in a mouse model of colitis-associated

cancer. Cell 118, 285–296 (2004).

26. Wilson, J. & Balkwill, F. The role of cytokines in the epithelial cancer microenvironment. Semin.

Cancer Biol. 12, 113–120 (2002).

27. Radisky, D., Hagios, C. & Bissell, M. J. Tumors are unique organs defined by abnormal signaling and

context. Semin. Cancer Biol. 11, 87–95 (2001).

Supplementary Information accompanies the paper on www.nature.com/nature.

Acknowledgements Mdr2-knockout mice were a gift from R. P. Oude Elferink and the TALAP1

mice were received fromH. Bujard.We are grateful to N. Berger, T. Golub andN. Kidess-Bassir for

technical assistance, to A. Hatzubai, O. Mandlboim, N. Lieberman, G. Kojekaro, M. Davis and

H.Harel for helping withMRI, FACS,mRNAand protein analysis.We thankK.Meir andM.Oren

for discussions and a critical reading of the manuscript. This research was supported by grants

from the Israel Science Foundation funded by the Israel Academy for Sciences and Humanities

(Center of Excellence Program), Prostate Cancer Foundation Israel—Center of Excellence,

German-Israeli Foundation for Scientific Research and Development (GIF, in collaboration with

H. Bujard), a grant in memory of H. and F. Brody from H. M. Krueger as trustee of a charitable

trust and the Israel Cancer Research Foundation (ICRF). I.S. is supported by the Lady Davis

Fellowship Trust.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests formaterials should be addressed to E.P. ([email protected]) and

Y.B.-N. ([email protected]).

..............................................................

Mrf4 determines skeletalmuscle identity in Myf5:Myoddouble-mutant miceLina Kassar-Duchossoy1, Barbara Gayraud-Morel1*, Danielle Gomes1,Didier Rocancourt2, Margaret Buckingham2, Vasily Shinin1*& Shahragim Tajbakhsh1

1Stem Cells and Development, 2Molecular Genetics of Development,Department of Developmental Biology, CNRS URA 2578, 25 rue du Dr Roux,75724 Paris Cedex 15, France

* These authors contributed equally to the work

.............................................................................................................................................................................

In vertebrates, skeletal muscle is a model for the acquisition ofcell fate from stem cells1. Two determination factors of the basichelix–loop–helix myogenic regulatory factor (MRF) family, Myf5and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts2–4.In the absence of these factors, progenitor cells remain multi-potent and can change their fate5,6. Gene targeting studies haverevealed hierarchical relationships between these and the otherMRF genes, Mrf4 and myogenin, where the latter are regarded asdifferentiation genes7. Here we show, using an allelic series ofthree Myf5 mutants that differentially affect the expression of thegenetically linkedMrf4 gene, that skeletal muscle is present in the

letters to nature


NF-κB functions as a tumour promoter in inflammation-associated cancer

Documents

Transcript of NF-κB functions as a tumour promoter in inflammation-associated cancer