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Cancer Genes and Genomics

IKKb Overexpression Leads to Pathologic Lesions in StratifiedEpithelia and Exocrine Glands and to Tumoral Transformationof Oral Epithelia

Angustias Page1, Jos�e L. Cascallana2, M. Llanos Casanova1, Manuel Navarro1, Josefa P. Alameda1,Paloma P�erez3, Ana Bravo2, and Angel Ramírez1

AbstractAlterations in nuclear factor kappaB (NFkB) signaling have been related with several diseases and importantly

also with cancer. Different animal models with increased or diminished NFkB signaling have shown that NFkBsubunits and their regulators are relevant to the pathophysiology of different organs and tissues. In particular, boththe deletion of the regulatory subunit b of the kinase of the inhibitor of NFkB (IKKb) and its overexpression inepidermis lead to the development of skin inflammatory diseases not associated with tumoral lesions. In this work,we have studied the consequences of IKKb overexpression in other organs and tissues. We found that elevatedIKKb levels led to altered development and functionality of exocrine glands (i.e., mammary glands) in transgenicfemale mice. In oral epithelia, increased IKKb expression produced lichenoid inflammation with abundantgranulocytes, macrophages, and B cells, among other inflammatory cells. This inflammatory phenotype wasassociated with high incidence of tumoral lesions in oral epithelia, contrary to what was found in skin. Moreover,IKKb also increased the malignant progression of both spontaneous and experimentally induced oral tumors.These results highlight the importance of IKKb in epithelial and glandular homeostasis as well as in oraltumorigenesis and open the possibility that IKKb activity might be implicated in the development of oral cancer inhumans. Mol Cancer Res; 9(10); 1329–38. �2011 AACR.

Introduction

Nuclear factor kappaB (NFkB) is a conserved family ofubiquitous transcription factors formed by homo- andheterodimers of distinct proteins including RelA/p65, RelB,c-Rel, p50, and p52. It regulates the expression of dozens ofgenes, many of them involved in the control of proliferation,apoptosis, and other essential cellular processes. Impor-tantly, deregulation of this family of transcription factorsis implicated in the origin of several types of cancer andinflammatory diseases (1, 2). NFkB members are usuallymaintained inactive in the cytoplasm by binding to inhi-bitory proteins of the IkB family. When the cells are under

activating conditions, IkB proteins are phosphorylated anddegraded; consequently, NFkB dimers are released, beingable to migrate to the nucleus, and execute their function astranscription factors by binding to specific kB sites in theregulatory region of target genes. Phosphorylation andsubsequent degradation of IkB proteins are controlledmainly by the IKK complex, which comprises the catalyticsubunits IKKa and IKKb and the regulatory subunit IKKg(3, 4). NFkB activity can also be regulated by posttran-scriptional modifications of NFkB proteins, in particularp65 phosphorylation (5).Numerous recent reports have established an important

role for several of the proteins implicated in NFkB activityin epithelial homeostasis (reviewed in refs. 6, 7). Althoughmouse models lacking individual members of the NFkBfamily do not show alterations of skin or other epithelia, thesimultaneous ablation of different combinations of theseproteins produce epithelial phenotypes (8), indicating thattheir activity is needed for the correct function of theepithelia, although there exists some degree of redundancyamong them. The modification of the expression level of thevarious components of the IKK complex in animal modelsleads to different epithelial alterations. IKKa exerts animportant role in skin function and development, and itis also implicated in skin cancer (9, 10). Regarding IKKg , ithas been shown that inactivating mutations of the X-linkedIKKg gene lead to the appearance of the genodermatosis

Authors' Affiliations: 1Department of Epithelial Biomedicine, Centro deInvestigaciones Energ�eticas, Medioambientales y Tecnol�ogicas, Madrid;2Department of Veterinary Clinical Sciences, Laboratory of PathologyPhenotyping of Genetically Engineered Mice, Faculty of Veterinary Med-icine, University of Santiago de Compostela, Lugo; and 3Animal ModelsLaboratory, Instituto de Biomedicina de Valencia, Valencia, Spain

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Angel Ramírez, Department of Epithelial Biome-dicine, Centro de Investigaciones Energ�eticas, Medioambientales y Tec-nol�ogicas (CIEMAT), 28040 Madrid, Spain. Phone: 34-91-346-0882; Fax:34-91-346-6484; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0168

�2011 American Association for Cancer Research.

MolecularCancer

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incontinentia pigmenti in heterozygous females both inhumans and in animal models (11, 12). IKKb activity isalso important for epidermal homeostasis, as its skin-specificablation leads to the development of an inflammatorypsoriasis-like skin disease dependent on TNFa signalingand macrophage activity (13, 14). We have describedpreviously that overexpression of IKKb in mouse skin(K5-IKKb mice) leads, in transgenic (Tg) lines with highexpression levels, to the development of an inflammatoryskin disease with histologic features of interface dermatitis(15). Tg skin keratinocytes, consistent with their increasedIKKb activity, have augmented signaling of the NFkBsurvival pathway and are less susceptible to apoptosis thanwild-type (WT) keratinocytes when treated with TNFa(15). However, less is known about the function of IKKb instratified epithelia different from that in the skin. K5promoter is also active in other stratified epithelia, suchas oral epithelia from the mouth and from the nonglandularstomach (forestomach), as well as in other locations over-expressing IKKb, as myoepithelial cells of exocrine glandsand epithelial appendages (16). On the basis of this knowl-edge, we speculated that transgenic mice overexpressingIKKb under the transcriptional control of keratin K5regulatory elements could suffer pathologic alterations alsoin other stratified epithelia where the K5 promoter is active.In this report, we describe the alterations found in stratifiedepithelia of different IKKb Tg lines. Most of these altera-tions were found even in lines that do not suffer from theserious skin inflammatory phenotype described in the studyof Page and colleagues (15), therefore allowing the study ofother epithelia without the interferences caused by anextended inflammatory skin disease. Our findings indicatethat IKKb plays a fundamental role in the pathophysiologyof stratified epithelia and exocrine glands. In addition,alterations in the activity of IKKb lead to inflammationand tumor development in oral epithelia. Even more, in oralcarcinogenesis experiments, IKKb induces the progressionfrom a benign tumor type toward a malignant one.

Materials and Methods

Mice and treatmentsGeneration of K5-IKKb Tg mice, carrying a VSV-tagged

human IKKb under the control of a 5.2-kbp 50-upstreamfragment of bovine K5 and a rabbit b-globin intron, waspreviously described (15). Tg.AC heterozygous mice wereobtained from Taconic Farm Inc. and mated to hetero-zygous K5-IKKb Tg mice. Tail DNA was used for micegenotyping by PCR. The primers used were 50-TTCAGGGTGTTGTTTAGAAATGG-30 and 50-CAA-TAAGAATATTTCCACGCCA-30 for K5-IKKb and 50-TGGCATTTCTTCTGAGCAA-30 and 50-TTGGACA-AACTACCTACAGAGAT-30for the Tg.AC transgene.4-Nitroquinoline-1-oxide (4-NQO; Sigma) was prepared

and administered following the indications detailed in thework of Tang and colleagues (17). Briefly, a 5 mg/mL stocksolution of the carcinogen 4-NQO was prepared in pro-pylenglycol and stored at 4�C. This solution was further

diluted in drinking water at a final concentration of50 mg/mL. Three-month-old Tg.AC and double K5-IKKb/Tg.AC Tg mice were allowed ad libitum access tothe drinking water containing 4-NQO during the 6-weektreatment period. Bottles were changed twice a week.Animals were maintained on standard rodent chow

(SAFE-A04; SAFE) and kept under a 12-hour light–darkcycle. All experimental procedures were carried out accord-ing to European and Spanish laws and regulations (Eur-opean Convention ETS 123 on the use and protection ofvertebrate mammals used in experimentation and otherscientific purposes; Spanish R.D 1201/2005 of the Ministryof Agricultural, Food and Fisheries on the protection anduse of animals in scientific research) and approved by ourInstitution's Ethics Committee.

Histology and immunohistochemistryUnless other figures are indicated, we conducted histo-

logic analysis of different stratified epithelia of at least 10 Tgand 4 non-Tg littermates adult mice of lines L1 and L2(with lower and higher relative skin expression levels,respectively; ref. 15), as well as 2 additional skin highexpression transgenic founders. Apart from the glandulardysplasia shown in Figure 2C–F and 2I–J, specific of themice with higher expression level, the phenotypes werecommon to all the Tg lines; in fact, the majority of theresults were obtained in the line with lower expression leveldue to practical reasons.Mouse tissues were dissected and immediately frozen in

optimum cutting temperature compound or fixed in 10%buffered formaldehyde or 70% ethanol and embedded inparaffin. Three- to 5-mm thick sections were used forimmunohistochemical preparations or for hematoxylinand eosin (H&E) staining. Cryosections were fixed for10 minutes at �20�C in acetone. The antibodies usedwere from Imgenex (IKKb), Covance (K5), CALTAGlaboratories (F4/80), BD Pharmingen (B220, Gr-1, andCD45), and Dako (CD3). Immunoreactivity was revealedusing an ABC avidin–biotin–peroxidase system and ABCsubstrate (Vector Laboratories), and the sections wereslightly counterstained with hematoxylin. Control experi-ments were carried out omitting the primary antibodies.

Western blot analysisWhole-cell protein extracts frommouse organs and tissues

were prepared as described in the work of Perez and collea-gues (18). The IKKb antibody used inWestern blot analysiswas from Imgenex; b-actin antibody (Santa Cruz Biotech-nology) was used as a loading control. The antibody used forphospho-p65 (Ser536) detection in Supplementary FigureS1 is from Cell Signaling Technology (#3033).

Analysis of mammary glandsWe analyzed mammary glands from K5-IKKb Tg and

WT females (2 of each genotype and condition) fromvirgin females at the ages of 7, 8, and 9 weeks, fromfemales on day 15 of gestation, from lactating females5 days after delivery, and from females with involutive

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Mol Cancer Res; 9(10) October 2011 Molecular Cancer Research1330




mammary glands 2, 5, and 8 days after stopping lactation.For the analysis of involutive mammary glands, femalesfed litters of 5 to 6 mice during 14 days. Then, the pupswere withdrawn and the involutive mammary glandsnumber 4 or 9 were analyzed at the indicated times.For whole mount preparations, inguinal number 4 mam-mary glands were fixed in Carnoy's fixative and stainedin carmine alum, following the protocols described inhttp://mammary.nih.gov/index.html

Results

Expression of IKKb in K5-IKKb miceWe have previously shown that Tg mice expressing

IKKb under the transcriptional control of regulatory

elements derived from the keratin K5 gene express thetransgene in the basal layer of skin and in hair follicles,leading to activation of the NFkB signaling pathwayin keratinocytes and to the development of interfacedermatitis (15).Because keratin K5 is expressed in the basal layer of all

stratified epithelia and in myoepithelial cells of exocrineglands, these cell types are expected to be targeted by thekeratin K5–derived regulatory elements included in thetransgene.We analyzed IKKb expression by Western blotting in

several stratified epithelia and exocrine glands from both L1Tg and WT animals and found consistently higher IKKblevels in the Tg samples (Fig. 1A; the lanes for back skin ofTg mice, included in both Western blot analyses, allow for

Figure 1. Expression of IKKb instratified epithelia and exocrineglands of K5-IKKb mice. A,Western blot analysis of IKKbexpression in different organs andtissues containing stratifiedepithelial or exocrine glands fromline L1 Tg and WT animals. Prep.gland, preputial gland. B,immunohistochemical analysis oftransgene expression in stratifiedepithelia. The genotypes of themice are indicated on the left ofthe figure and the antibodies usedinside the photographs. Theantibody anti-IKKb gave specificsignals in the basal cells of the tailskin, foot sole, palate, andforestomach epithelia of Tg mice(arrows, middle row). Consecutiveparaffin sections were incubatedwith an antibody specific forkeratin K5 (bottom row). Thestomach plica is indicated by asurrounding line. Under thesedetection conditions, almost noIKKb stainingwas seen inWTmice(top row). C,immunohistochemical analysis oftransgene expression in exocrineglands. IKKb and K5 expression inmyoepithelial cells of Meibomianglands are shown. D,immunohistochemical analysis ofK5 and IKKb expression inlactating mammary glands.Myoepithelial cells were positivefor K5 but not for IKKb in WTfemales, but both proteins weredetected in myoepithelial cells ofTg samples (arrows). Bar: 200 mmin B and C and 50 mm in D.

IKKb Leads to Oral Tumorigenesis

www.aacrjournals.org Mol Cancer Res; 9(10) October 2011 1331




the comparison of the expression level between Tg and WTsamples).Immunohistochemical analysis of the expression pattern

of K5-IKKb transgene with antibodies specific for IKKbshowed that it was expressed, as expected, in the basal celllayer of the stratified epithelia studied (tail skin, foot soleskin, and oral epithelia of the palate and forestomach;Fig. 1B, arrows). Of note, IKKb is expressed in the stomachplica, that is, the region where the glandular and thenonglandular parts of the murine stomach join (arrowheadsand arrows, respectively, in the stomach panel of Fig. 1B).IKKb was also expressed in myoepithelial cells of exocrineglands [as mammary, preputial, salivary, and eyelidMeibomian glands; see Fig. 1B for IKKb expression insweat glands (arrowheads in foot sole panel); Fig. 1C forexpression inMeibomian glands; and Fig. 1D for expressionin myoepithelial cells of mammary glands]. The morerestricted basal staining of IKKb relative to that of keratinK5 in the tissues shown in Figure 1B could be because ofdifferences in the stability of these proteins or to differentsensitivity of the antibodies used. IKKb overexpression wasassociated with increased phosphorylation of p65 at serine536 in stratified epithelia and exocrine glands of Tg mice(Supplementary Fig. S1). This posttranscriptional modifi-cation, which enhances p65 transactivation potential, is amarker of NFkB activation (5, 19). It is interesting to notethat IKKb overexpression did not lead to changes in theexpression level of IKKa or IKKg (Supplementary Fig. S2).

Dysplasia associated with inflammation in ectodermalderivatives and exocrine glands in K5-IKKb miceIn those Tg lines with higher expression level (different

from the line L1 shown in Fig. 1), we observed, evenmacroscopically, phenotypic consequences of K5-IKKbtransgene expression in ectodermal derivatives, includingeyelids, nails, and teeth, and in exocrine glands. The processof eyelid fusion that usually takes place in the mouse embryobefore day 18.5 of development frequently failed in Tg mice

and they were born with one or both eyes opened (Fig. 2Aand B, arrows). Some adult animals showed thickening ofeyelids as a consequence of proliferation of dysplasticmyoepithelial and/or reserve cells and substitution of thenormal exocrine cells from Meibomian glands associatedwith discrete inflammation (Fig. 2C–F, arrows). The major-ity of transgenic mice also had nail malformations, some-times accompanied by fragility; they showed abnormallybended and overgrown nails, lacking the usual sharp tip ofthe nails in WT mice (Fig. 2G and H, arrowheads). Thisphenotype was likely the consequence of dysplasia andlichenoid inflammation of the stratified epithelia of thenail bed (not shown). Many Tg animals showed hyperpig-mentation of the foot pads (Fig. 2G and H, arrows) becauseof the presence of macromelanophages as a consequence of apreceding damage in epidermis and hair follicles (see ref.15); this hyperpigmentation was usually more marked inolder mice. In addition to these phenotypic alterations,some Tg males showed atrophic preputial glands (Fig. 1Iand J) as a consequence of proliferation of dysplasticmyoepithelial and/or reserve cells that provoked atrophyof the secretory acini (not shown). Some Tg mice alsodeveloped atrophy of major salivary glands (especially par-otid) and minor salivary glands of the tongue by thedysplastic proliferation of myoepithelial cells (not shown).These alterations were observed mainly in Tg lines withhigh expression level. Nevertheless, we also found epithelialand glandular alterations in Tg lines with lower expressionlevel (i.e., line L1), as well as effects on oral cancer devel-opment (see later).

Mammary gland development and functionality areimpaired in K5-IKKb miceWe analyzed mammary glands from both Tg females

from line L1 and nontransgenic littermates at different ages.In 56-day-old young virgin animals, WT females showed awell-developed tree of ducts, filling the majority of thefat pad of the gland. In contrast, Tg females showed a

A C

B D F H J

G IEWT

Tg

Figure 2.Macroscopic alterations found in K5-IKKb Tg mice. A and B, K5-IKKb Tg mice frequently were born with 1 or the 2 eyes opened. C–F, transformationof Meibomian glands in adult mice from the highest expressing lines. Glandular cells are transformed into dysplastic and basophilic epithelial cellslacking secretory differentiation and associated with periacinar infiltration by mononuclear inflammatory cells (see white arrows in E and F). G and H,K5-IKKb Tg mice also showed altered nails (arrowheads) and hyperpigmentation of the fat pads (arrows). I and J, preputial glands were frequently of smallersize in adult K5-IKKb male mice than in WT mice. Bar: 500 mm in C and D and 50 mm in E and F.

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more rudimentary ductal design, which hardly exceeded thelymph node; in addition, the terminal end buds wereirregularly shaped (arrows in Fig. 3A and B). When westudied lactating females 5 days after delivery, we found thatthe mature alveoli of Tg mammary glands were smaller thanthat of WT females (compare the diameter of alveoli inFig. 3C and D), suggesting deficient functionality of theglands in Tg females. In addition, the luminal secretory cellsin Tg females showed less vacuoles in their apical polesduring lactation, which probably results in low milk pro-duction (Fig. 3E and F, arrows).The remodeling program that takes place at the end of

lactation was also altered in Tg animals. At 2 days ofinvolution, Tg mammary glands showed a macroscopicappearance clearly different from that in WT glands: manyof them were swollen and whitish, apparently still contain-ing milk. Microscopically, 2 days after the cessation oflactation, WT mammary glands showed massive apoptosisof milk-producing alveolar cells. In contrast, Tg glandsshowed a retarded involution process, with scarce apoptotic

cells inside the mammary ducts (Fig. 3G–J, arrows). At thistime, the alveoli showed an increased diameter in Tg mice,also indicative of a delay in the involution process. Thesephenotypes are consistent with the triggering of survivalsignals as a consequence of increased NFkB activation incells of Tg mice. In summary, IKKb overexpression affectsmammary gland at different levels; it leads to reduceddevelopment of mammary glands in female Tg mice, toaltered milk production in lactating females, and to delayedregression of the gland in the involution process.To evaluate the functional effect of these alterations, we

carried out crosses between WT females and Tg males, andvice versa, and compared several reproductive parameters inthe 2 types of crosses. As can be seen in SupplementaryTable S1, the percentage of pups that reached the age of9 days was 100% in WT females and less than 70% whenthe mother was Tg. Furthermore, in this last group, thesurviving pups had reduced weight, reaching only 76.5% ascompared with animals fed by WT females. This impair-ment in newborn survival and fitness is not directly relatedto the presence of the K5-IKKb transgene in the pups, as wedid not find these alterations in the opposite breeding, thatis, WT females crossed with Tg males, see SupplementaryTable S1. In summary, Tg females bring up their litters lessefficiently than WT females, resulting in higher death ratein their pups and less growth of the survivors.

Inflammatory phenotype in oral and forestomachepitheliaMacroscopically, many Tg mice also showed gross altera-

tions of other ectodermal derivatives such as teeth andpalate. Both supernumerary incisors and tooth fragility wererecorded. A consistent finding during the examination ofthe oral cavity of Tg mice was the thickening of the oralmucosa of the palate, even in young mice (Fig. 4A, arrows).Microscopically, the palate epithelium often showed pseu-docarcinomatous hyperplasia in Tg mice, characterized byextreme irregular proliferation of solid epithelial cordsmainly at the base of the rete ridges, bearing a superficialresemblance to invasive squamous cell carcinomas (SCC;see Fig. 4B palate, arrows). This was accompanied by severelichenoid inflammation, basal cell damage, and exocytosis(inset in Fig. 4B). All these features were also observed inoral epithelia of the forestomach at the level of the plica(Fig. 4B stomach, arrow, and Fig. 4C). More than 70% ofthe animals analyzed showed inflammation of the palate andforestomach (in some cases associated with other alterations,see below).In the palate (not shown) and forestomach plica, infil-

trating cells were frequently observed. In these inflamma-tory lesions, eosinophil granulocytes were abundant, asshown by H&E staining of histologic sections (Fig. 4C,white arrows). The immunostaining against CD45 antigen(characteristic of hematopoietic inflammatory cells) showedthat the inflamed oral epithelia were clearly infiltrated by alarge amount of positive cells. Staining with anti-Gr-1antibody, specific for granulocytes, confirmed the presenceof this inflammatory cell type (black arrows in Fig.4C).

A B

C D

E F

G H

I J

Figure 3. Developmental and functional alterations in mammary glands ofK5-IKKb mice. Mammary glands of K5-IKKb females and WT littermateswere analyzed at different ages. A and B, whole mount staining ofmammary glands of 56-day-old virgin females. Arrows indicate terminalend buds. C–F, low- and high-power amplification of day 5 lactatingmammary glands. Arrows indicate vacuoles containing milk. G–J, low- andhigh-power amplification of involutive mammary glands 2 days after thecessation of lactation. Arrows indicate apoptotic cells. Bar: 500 mm in Aand B; 300 mm in C, D, G, and H; and 100 mm in E, F, I, and J.






F4/80- and B220-positive cells (macrophages and B cells,respectively) were also more abundant in the inflammatoryfoci of Tg mice than in WT forestomach (Fig. 4C). CD3staining also showed more T cells in Tg than in WT oralepithelia (not shown). To unravel the contribution oflymphocytes to the overall phenotype found in the oralepithelia of Tg mice, we studied K5-IKKb transgenic micelacking lymphocytes. We generated K5-IKKb Tg mice indifferent immunodeficient genetic backgrounds: K5-IKKb/nude (lacking T lymphocytes) or K5-IKKb/severe com-bined immunodeficient mice (lacking T, B, and naturalkiller cells). The oral phenotype obtained in both immu-nodeficient models was similar to the phenotype obtained inK5-IKKb immunocompetent mice (not shown), indicatingthat T, B, and natural killer cells are not responsible for the

oral phenotype. We also analyzed the implication of TNFsignaling in the oral epithelia phenotype, as TNF signalingis essential for the pathogenesis of skin lesions in transgenicmice lacking IKKb or IKKg in their skin (13, 20). Westudied histologic preparations of palate and forestomachepithelia from K5-IKKb Tg mice treated with etanercept toinhibit signaling through TNFa receptor. The phenotypicalterations observed in these mice were similar to untreatedTg mice (not shown), indicating that these phenotypes areindependent of TNFa signaling.

K5-IKKb mice develop spontaneous tumors in the oralepitheliaA causal relationship between inflammation and cancer

has been known for years, as many human tumors develop

Figure 4. Histologic phenotypeand inflammation in oral epitheliain K5-IKKb mice. A, low-powermagnification of palate. Note thethickening of palate epithelium inTg mice, resulting in less markedwaves of the hard palate thanin WT mice (arrows). B,pseudocarcinomatoushyperplasia in adult palate andforestomach epithelia, showingaberrant proliferation of branchedepithelial cords (arrows),associated with band-likeinfiltration of inflammatory cellsand vacuolar degeneration of thebasal layer (see inset in palatepanel). C, histologic sections fromWT and Tg stomach plica werestained with H&E (white arrows ininset indicate eosinophilgranulocytes) or immunostainedwith antibodies specific forhematopoietic infiltrating cells(CD45), granulocytes (Gr-1),macrophages (F4/80), or B cells(B220), as indicated. In thestomach plica, all types ofinflammatory cells areoverrepresented in K5-IKKbanimals. Bar: 500 mm in A, 300 mmin B (palate), and 200 mm in B(stomach) and C.

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in organs that suffer chronic inflammatory processes (seeref. 21). Because K5-IKKb mice showed persistent inflam-mation in oral epithelia, they could provide a good modelfor studying in vivo the relationship between tumor inci-dence in oral epithelia and the presence of inflammatorycells. We studied the incidence of tumors in oral stratifiedepithelia from the palate and from the forestomach in Tgmice. Histologic analysis of K5-IKKb mice frequentlyrevealed foci of carcinoma in situ in those regions withpseudocarcinomatous hyperplasia associated with inflam-mation; these foci were characterized by high-grade atypia ofkeratinocytes, even presenting epithelial pearls characteristicof well-differentiated SCCs but limited to the oral mucosawith the integrity of the basal membrane (Fig. 5A andTable 1). The spontaneous development of SCCs thatinfiltrated subjacent tissues was also sporadically seen in9.5% of the analyzed animals in the palate and 14.7% in theforestomach (Fig. 5B and Table 1; see malignant epithelialcells infiltrating the deep submucosa of the palate andstomach, arrows). These results indicate that IKKb over-expression, inflammation, and the risk of neoplastic trans-

formation are closely interrelated in oral stratified epithelia,being a high level of IKKb sufficient to trigger the devel-opment of spontaneous tumors in the oral epithelia of Tgmice.

IKKb provokes malignant transformation ofexperimentally induced oral tumorsTo further study the potential of IKKb as a tumor

promoter in oral cancer, we subjected populations of K5-IKKb and control animals to oral carcinogenesis experi-ments. We used K5-IKKb mice that also carried a Tg.ACtransgene, which directs the expression of an active form ofv-Ha-Ras to epithelial cells (22) and thus represent a strainof initiated mice suited for the study of tumor developmentin skin and other stratified epithelia. As a control popula-tion, we used Tg.AC mice that did not carry the K5-IKKbtransgene. Animals of both genotypes were treated with4-NQO in the drinking water, following administrationregimens that result in hyperplasia of oral epithelia and theoccurrence of tumors or preneoplastic lesions (17). Themajority of Tg.AC control animals (6 of 7) developedpapillomas in the epithelia of the forestomach. Thesetumors, although sometimes of great size, were alwaysbenign, showing conjunctive papillary projections coveredby neoplastic proliferation of typical squamous epitheliumwith net limits of the basal membrane (see Fig. 6A and B).All the treated K5-IKKb/Tg.AC transgenic animals (5 of 5)developed tumoral lesions in the forestomach. Strikingly,we found that the majority of them (4 of 5 cases) containednumerous malignant foci of infiltrating carcinoma, wherestrands of squamous malignant cells broke down the basalmembrane and infiltrated the submucosal conjunctive layer(Fig. 6C and D, arrows). These invasive foci were highlydysplastic, with loss of the typical multilayered organizationof the epithelium, and conformed by epithelial cells with ahigh degree of atypia. Similar results were obtained when weanalyzed tumor incidence in the palate epithelia, as malig-nant SCCs were detected in some of the K5-IKKb/Tg.ACmice (2 of 5) whereas Tg.AC mice did not developedany SCC (see Fig. 6E and F). Overall, these results highlightthe importance of IKKb in the development and progres-sion toward malignancy of oral tumors.

Figure 5. Spontaneous tumoral lesions in oral epithelia in K5-IKKb mice.The palate and forestomach showed foci of premalignant lesions(carcinoma in situ, A) as well as infiltrating SCCs (B). Bar: 200 mm in A(stomach) and B and 100 mm in A (palate).

Table 1. Incidence of inflammation and spontaneous premalignant or tumoral lesions in oral epithelia ofK5-IKKb Tg mice

n Percentage of micewithout inflammation

Percentage of mice with inflammation

Nontumoral lesions Carcinoma in situ SCC

Palate 21 19.0 0 71.4 9.5Forestomach 34 26.5 44.1 14.7 14.7

NOTE: Each animal was classified according to the absence or presence of inflammation; animals scoring positive for inflammationwere further classified according to the absence or presence of premalignant or tumoral lesions. The total number of mice analyzed isindicated by n.






Discussion

In this study, we have focused on the involvement ofIKKb in the pathophysiology of stratified epithelia andexocrine glands. Previously published data (13–15, 23)indicate an important role for IKKb in the developmentand functionality of skin. The diverse phenotypes observedin other stratified epithelia and exocrine glands of K5-IKKbTg mice indicate the importance of IKKb also in theseorgans and tissues. Furthermore, increased level of IKKb ledto the appearance of an inflammatory disease in oralepithelia of the palate and the stomach and to a highincidence of tumoral lesions in oral epithelia of transgenicmice.The activity of IKK complex is crucial for the correct

function of mammary epithelial cells. Thus, it is knownthat the kinase activity of IKKa plays an important role incyclin D1 induction and mammary epithelial cells pro-liferation (24). In addition, a proapoptotic function ofIKKb in mammary epithelial cells has been described, asfemale mice with conditional deletion of IKKb directed bythe promoter of the b-lactoglobulin milk protein geneshow delayed apoptosis of epithelial cells in the involutionprocess (25). However, we have also observed a delayed

apoptosis in involutive mammary glands of miceoverexpressing IKKb. This intriguing concordance inthe phenotypes due to IKKb loss of function in milk-producing epithelial cells (25) and IKKb overexpression inmyoepithelial cells (this report) is likely related to thedifferent cell populations targeted in both experiments.Furthermore, it also highlights the complex organizationof the mammary gland and the delicate interrelation andbalance that exist between the different cell types thatbuild up this gland.IKKb, besides acting as an NFkB regulator by phosphor-

ylating IkB proteins, also regulates other cellular responsesthrough phosphorylation of other physiologic targets.Among others, we could point out several proteins relatedto cancer as the forkhead transcription factor FoxO3a, thehistone deacetylase HDAC1, Aurora A, and the tumorsuppressors p16, p53, or Tsc1 (refs. 26–29 and referencestherein). Recently, it has been described that IKKb can exerta novel nuclear function as an adaptor protein for IkBadegradation in a manner independent of its kinase activity(30). Taking into account the variety of phenotypes foundin K5-IKKb mice, including its propensity to oral carcino-genesis, some of them could be partly independent ofNFkB. In this context, it would be of interest to analyzewhether the tendency to develop oral tumors in K5-IKKbTg mice is exerted through NFkB and whether it isdependent on the function of IKKb as part of the IKKcomplex. This knowledge could be clinically relevant, as thepotential therapeutic interventions would be different.Thus, the generation and study of animals expressingmutant forms of IKKb unable to interact with the othermembers of the IKK complex or lacking its kinase activityshould provide interesting information.NFkB is an important mediator of inflammation and

tumorigenesis. It is recognized that persistent inflamma-tion may promote susceptibility to carcinogenesis, partlyby creating a stroma enriched with cytokines and growthfactors (31). Although the response to altered NFkBactivity varies among different cell types, increased NFkBactivity is usually associated with tumor development(1, 32). IKKb also acts as a tumor promoter in differentcell types, as it happens in colitis-associated cancer andmelanoma development in animal models, as well as inhuman breast cancer patients (33–35). In the case of oralepithelia, we have observed inflammation and basal celldamage as a consequence of targeted overexpression ofIKKb. The subsequent epithelial hyperplasia is accompa-nied by cellular atypia and the development of high-gradedysplastic changes of the epithelia. These alterations couldbe ascribed to carcinoma in situ when limited to theepithelium by the basal membrane or to SCC wheninfiltrated the subjacent conjunctive tissue. As IKKboverexpression leads to the development of pretumoralor tumoral lesions in oral epithelia of transgenic mice andpromotes their malignant conversion in a carcinogenesisassay in the presence of activating forms of Ha-ras,deregulation of IKKb activity could underlie some casesof oral cancer in humans. In this regard, it would be of

AA B

C D

E F

A B

E F

Figure 6.Malignant lesions in oral epithelia of K5-IKKb/Tg.ACmice treatedwith 4-NQO. A–D, tumoral lesions in forestomach epithelia of 4-NQO–

treated Tg.AC (A and B) and K5-IKKb/Tg.AC (C and D) mice. B and D,magnifications of the frames indicated in A and C. White lines indicate thebasal membrane, that is discontinuous in D. E and F, H&E-stained sectionsof the palate of Tg.AC (E) and K5-IKKb/Tg.AC (F) mice after 6 weeks oftreatment with 4-NQO. White lines indicate the border between epithelialcells and subjacent conjunctive tissue (E) and islets of malignant epithelialcells that have infiltrated the submucosa (F); other invasive foci areindicated with arrows. Bar: 500 mm in A and C; 200 mm in B; and 100 mmin D–F.

Page et al.





interest to test whether the inhibition of IKKb in oralmalignancies has therapeutic value.Back skin appears to be more resistant than oral epithelia

to carcinogenesis mediated by increased activity of IKKb/NFkB pathway because we have not observed spontaneoustumor development in the skin of K5-IKKb Tg mice (15).This is compatible with the results obtained by otherauthors using other animal models in which the inhibitionof NFkB signaling leads to skin hyperplasia and even tumorformation (36, 37). Our study highlights the complexity ofthe regulatory networks controlled by IKKb and NFkB, astheir manipulation renders completely different outcomesin different cell types. The marked differences in tumordevelopment susceptibility that we have found between skinand oral epithelia when subjected to IKKb overexpression,chronic inflammation, and basal cell damage are surprising.In this regard, K5-IKKb Tg mice represent an interestingmodel for the study of the signaling pathways and regulatorymechanisms underlying tumor formation that could explainthe different tumoral susceptibility in oral epithelia andskin.In summary, the present work indicates that IKKb plays a

central role in epithelial homeostasis and in exocrine glandfunction; IKKb also promotes the formation of tumorallesions in oral epithelia and their malignancy in cooperation

with activating oncoproteins such as viral Ha-ras. Conse-quently, IKKb could be related to the pathogenesis ofhuman tumoral diseases affecting oral epithelia. In thiscontext, it would be interesting to test the potential ther-apeutic value of IKKb inhibition in the treatment of humanoral cancers triggered by inflammation.

Disclosure of Potential Conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank I. de los Santos and F. Sanchez-Sierra for excellent technical assistanceand J. Martinez, E. Almeida and the personnel of the CIEMAT Animal Unit for thecare of the mice used in this work. We also thank Dr. V. Lafarga for advice in theanalysis of mammary glands and Dr. E. Radaelli for critical reading of the manuscript.

Grant Support

This research was supported by grants from the Spanish government(Ministerio de Ciencia e Innovaci�on; SAF2010-22156 to A. Ramírez and PI-10/01480 to M.L. Casanova).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 14, 2011; revised July 29, 2011; accepted August 1, 2011;published OnlineFirst August 5, 2011.

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2011;9:1329-1338. Published OnlineFirst August 5, 2011.Mol Cancer Res Angustias Page, José L. Cascallana, M. Llanos Casanova, et al. Oral EpitheliaEpithelia and Exocrine Glands and to Tumoral Transformation of

Overexpression Leads to Pathologic Lesions in StratifiedβIKK

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