IN PERSPECTIVE

The High-Risk Benign Tumor: Evidence Fromthe Two-Stage Skin Cancer Model andRelevance For Human Cancer

Adam Glick,1* Andrew Ryscavage,2 Rolando Perez-Lorenzo,1 Henry Hennings,2 Stuart Yuspa,2

and Nadine Darwiche3

1Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, Pennsylvania2Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland3Department of Biology, American University of Beirut, Beirut, Lebanon

Benign tumors that form following chemical initiation and promotion in the mouse skin can be grouped into twoclasses. The majority of papillomas do not progress to squamous cell carcinoma (SCC), and these are designated aslow-risk or terminally benign papillomas. In contrast, a much smaller group forms the true precursor to the SCC, and

these have a significantly higher frequency and rate of malignant conversion than the bulk of low-risk papillomas. Instandard two-stage carcinogenesis studies both tumor types are present, but grossly indistinguishable. Here wedescribe properties and potential origins of high-risk papillomas and discuss the relevance of this model for certain

human cancers with defined premalignant states. � 2007 Wiley-Liss, Inc.

Key words: skin carcinogenesis; SCC; biomarker

EXPERIMENTAL EVIDENCE FOR SUBPOPULATIONS OF

CHEMICALLY INDUCED SKIN TUMORS WITH DIFFERINGRISK FOR MALIGNANT CONVERSION

The multistage mouse skin chemical carcinogen-esis is the prototype model system that has definedthe basic biology of squamous cell carcinoma (SCC)development. In the classical permutation of thismodel, mice are treated once with the carcinogendimethyl benz[a]-anthracene (DMBA) followedby 20 weekly applications of a tumor promoter,such as 12-O-tetradecanoylphorbol-13-acetate (TPA).Benign papillomas representing clonal outgrowthsof keratinocytes with initiating mutations in theHras1 arise within 10–15 wk, and a small percentageprogress to locally invasive SCC. Progression in thismodel is associated with trisomy of chromosomes 6and 7, loss of heterozygosity at the Hras1 locus,followed by amplification of the mutated Hras gene,and increasing aneuploidy and dysplasia [1]. How-ever, there is strong evidence that the benign lesionsproduced by tumor promoters are biologicallyheterogeneous in their potential for malignantconversion. Numerous studies have shown thatsome papillomas require continued TPA to preventregression, while others degenerate even in thepresence of TPA; some never progress to malignancywhile others evolve to malignancy without furtherpromotion [2]. A recent study indicates that thispotential for malignant conversion is largely cellautonomous that is dependent on changes within

the keratinocytes rather than microenvironmentalinfluences [3].Using specific promotion protocols, Yuspa and co-

workers have defined two subclasses of papillomaswith differing rates of premalignant progression [4].When the duration of TPA promotion was variedfrom 5, 10, 20, or 40 wk in DMBA-initiated SENCARmice, thenumberof papillomaspromoted for at least10 wk peaked within 16–20 wk and declined by 35–40% over a subsequent 12 wk period, regardless ofpromotion.While promotionwithTPA for only 5wkreduced the total number of papillomas, howeverthese tumors persisted. Despite the duration ofpromotion the final number of SCC remained thesame. Similarly, the papilloma and SCC incidence inCD-1 mice receiving continuous promotion withTPA was compared to 10 wk TPA treatment followedby acetone. Although the papilloma incidence

MOLECULAR CARCINOGENESIS 46:605–610 (2007)

� 2007 WILEY-LISS, INC.

Abbreviations: SCC, squamous cell carcinomas; DMBA, dimethylbenz[a]-anthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate;K, keratin; TGFb, transforming growth factor beta; AK, actinickeratosis; RAR, retinoic acid receptor; RXR, retinoid X receptor; RA,retinoic acid.

*Correspondence to: Center for Molecular Toxicology andCarcinogenesis, The Pennsylvania State University, University Park,PA 16801.

Received 22 January 2007; Revised 28 March 2007; Accepted 30March 2007

DOI 10.1002/mc.20345

curves were identical at 10 and 16wk, by 28wk therewere twice the number of papillomas inmice treatedcontinuously with TPA compared with 10 wk TPA.Despite this and the decreased latency for the firstcarcinoma in the continuous treatment group, thefinal number of SCC was not significantly differentbetween the two different promotion protocols.These data demonstrate that papillomas that arisewith limited promotion with a strong tumor pro-moter, such as TPA, persist in the absence ofcontinued promotion. These persistent lesions arethe precursor lesions for most of the SCC that formduring the two-stage carcinogenesis protocol, whileTPA-dependent papillomas are very unlikely toprogress to carcinomas. These results also demon-strate the strain independence of this subclassifica-tion of benign tumors. High-risk papillomas are alsoselectively induced by chronic treatment with weakpromoters such as mezerein [4], or chrysarobin [5],and by wounding [6]. In these studies, very fewpapillomas arise because of the relatively weakpromoters that are used, but a high frequency ofthese tumors convert to SCC. Again, these resultshighlight thathigh-risk papillomas require very littlepromotion stimulus. High-risk papillomas also exhi-bit increased sensitivity to malignant conversioninduced by genotoxic agents [7]. Chemicals such as4-nitroquinoline N-oxide (4-NQO) or urethane canincrease the spontaneous rate of malignant conver-sion of papillomas generated by the two-stagecarcinogenesis protocol. However, in tumors thatformed inmice promoted for 5, 10, or 20wk TPA andwere subsequently treated with 4-nitroquinolineN-oxide (4-NQO) or urethane, there was a 2.5- and2-fold increase in conversion frequency in the 5 and10wk promoted tumors but no increase in the 20 wkgroup. Similarly, tumor sensitivity to inducedmalig-

nant conversionwas not lost even after a 15wk delaybefore urethane exposure of 5 wk TPA-promotedpapillomas. Thus, elevated sensitivity to genotoxicagents is an integral property of papillomas that arethe precursor lesions to SCC.

BIOMARKERS THAT DISTINGUISH LOW- ANDHIGH-RISK PAPILLOMAS

Although the histopathology of high-risk papillo-mas classifies it as benign, many of the phenotypicchanges associated with malignant progressionoccur very early in these lesions soon after they aredetectable, within 8–11 wk following the onset ofpromotion. Over the last decade, we have character-ized several biomarkers that distinguish these low-and high-risk benign lesions at early stages (Table 1).In the majority of benign low-risk papillomas, atearly stages of tumor outgrowth, cellular prolifera-tion although elevated is confined to the basal layerof the epithelium which is similar to the normalepidermis. In contrast, high-risk tumors are char-acterized by basal and suprabasal keratinocyte pro-liferation [8]. Because in normal keratinocytes, lossof contactwith the basementmembrane is the signalfor commitment to terminal differentiation andcessation of proliferation, this suggests that initiatedkeratinocytes that form thehigh-risk papillomahavealready become resistant to some of the signals thatregulate the normal segregation of proliferation anddifferentiation in the epidermis. Integrins are impor-tant mediators of cell-cell and cell-extracellularmatrix interactions and, therefore, their distributionhas profound effects on the neoplastic phenotypeand malignant progression. a6b4 integrins connectbasal keratinocytes to the basement membraneproteins in hemidesmosomes, and reorganizationof this integrin probably has important effects

Table 1. Properties of Low-Risk and High-Risk Papillomas

Papilloma type

Property Low-risk High-risk

Histology Benign BenignSensitivity to strong Promoters Low HighPersistence TPA dependent TPA independentSensitivity to 2nd mutagen No increase IncreasedKeratin expression K1þ, K13� K1�, K13þProliferation Increased, basal layer Increased basal and suprabasal layersa6b4 integrin localization a6b4 basement membrane side of

basal cellsa6b4 relocalized around basal cell and

suprabasalTGFb1 protein Basal layer, strong Basal layer, weak or lostRetinoid receptor RARa present RARa absent, RXRa increasedTumor associated CD3þ cells Abundant, multifocal Low, absent

Data summarizes papers cited in text regarding properties of low-risk and high-risk papillomas. For all of the biochemical characterizationof tumors, high-risk papillomas were analyzed shortly after emergence on the dorsal epidermis at 8–11 wk after onset of promotion,either 5 wk TPA, or continuous mezerein. Properties of low-risk papillomas represent initial stages after 12–14 wk continuous TPApromotion.

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on keratinocyte attachment, proliferation, andresponse to detachment from the basement mem-brane [9]. Consistent with this, the normal restrictedexpression of the a6b4 integrin to the basal side ofthe basal keratinocyte compartment in the normalepidermis and low-risk papillomas is lost.Novel a6b4integrin expression around the entire basal kerati-nocyte plasmamembrane aswell as in the suprabasallayers is also observed [9]. The significance of thisearly reorganization of a6b4 expression for malig-nant conversion is emphasized by the finding thatthis integrin is the predominant integrin expressedin SCC [9]. In addition, high-risk papillomas showearly loss of keratin 1 (K1), a suprabasal differentia-tion marker of the normal epidermis, and aberrantsuprabasal expression of K13, a K not present innormal epidermis [8]. This switch in K expression is ahallmark of tumor progression in this chemical skincancer model. However, by 26 wk past promotion,K13 expression is observed in all papillomas indicat-ing that it is not an exclusive marker for high-riskpapillomas, but rather its early expression reflects theaccelerated progression phenotype of these high-riskbenign lesions.The transforminggrowth factor beta (TGFb) family

members are potent cell growth inhibitors of mousekeratinocytes in vitro [10]. In the normal epidermis,TGFb1 proteins are expressed predominantly in thebasal layer, acting as negative regulators of cellproliferation. TGFb2 proteins are expressed in thesuprabasal layers. In low-risk papillomas, TGFb1expression is also largely confined to the basal layersof the tumor. In contrast, TGFb1 protein expressionis reduced or not detected in the basal layers of high-risk papillomas and suprabasal expression of TGFb2is lost [10]. Similar loss of Tgfb1 proteins is observedin high-risk early papillomas induced in p53 nullmice [11] and in chemically induced SCC. Further-more, keratinocytes with a targeted deletion ofTgfb1gene undergo rapid progression tomalignancywhentransduced with the v-Hras1 oncogene [12]. In high-risk tumors, decreased TGFb1 gene expression or lossmay deregulate basal and suprabasal keratinocyteproliferation, contributing to genomic instabilityand, hence, rapid malignant progression in thesehigh-risk tumor cells [13,14].The importance of retinoids in normal epidermal

differentiation and the disruption of their signalingpathways in human and mouse skin tumors is wellestablished. Retinoid receptors are members of thesteroid/thyroid hormone nuclear receptor super-family and comprise the retinoic acid receptors(RAR), and the retinoid X receptors (RXR) genes,designated as a, b, and g [15]. After binding all-transretinoic acid (RA) or 9-cis RA, RARs heterodimerizewith RXRs resulting in transcriptional regulation oftarget genes. The RXR physiological ligand is 9-cisRA, and RXRs act as transcriptional factors throughboth homodimerization and heterodimerization

with other members of the same superfamily,including thyroid hormone and vitamin D3 recep-tors. In the normal epidermis, the two major RARisoforms a1 and g1 are expressed in both the basaland suprabasal layers, while RXRa is abundantlyexpressed in the basal cell layer and concentrated inhair follicles [16]. Both RARa andRARg protein levelsare decreased in mouse papillomas and are almostabsent in carcinomas, consistent with the role ofRARs as growth inhibitors in normal keratinocytes[17]. However, RXRa expression is increased inassociation with the expanded proliferative com-partment [16]. In early papillomas generated bycontinuous TPA promotion (low-risk), RARa proteinlevels were slightly reduced but in continuousmezerein-promoted papillomas (high-risk), theirlevels significantly decreased or were absent. Inter-estingly, high levels of v-Hras1 expression incultured keratinocytes downregulated RAR expres-sionandactivity. Tumorpromoters also reducedRARprotein levels in nontumorigenic epidermis and incultured keratinocytes. Altogether these resultssuggest that epigenetic regulation of these nuclearreceptors by activated Hras1 and tumor promotersmaybe crucial in thehigh-risk phenotype [17].Manystudies have demonstrated that topical retinoids canreduce papilloma incidence in the two-stage carci-nogenesis model. Topical RA reduces papilloma andcarcinoma numbers in both low-risk and high-riskpromotion protocols. However, a subset of RAresistant papillomas persist which express, at earlystages, markers of advanced tumor progression.These lesions have a significantly higher frequencyof malignant conversion [18]. Topical retinoidtreatment may either selectively block the out-growth of all lesions, except those that are the trueprecursors to SCC, or a subset of high-risk papillomashave an altered response, which causes more rapidmalignant progression. Interestingly, despite theincreased frequency of malignant conversion andhigh-risk phenotype, retinoid-treated persistentpapillomas express higher levels of TGFb1 transcriptsand retainedTGFb1expression in thebasal layer [18].This suggests that retinoid-induced overexpressionof TGFb1 may suppress the outgrowth of mostbenign lesions, but may enhance malignant conver-sion in others by selecting for inactivation of theTGFb1 signaling pathway [18].While these studies suggest that low-risk andhigh-

risk papillomas differ significantly in somebiologicalproperties, the underlyingmolecular basis is notwellunderstood. To test if difference in gene expressiondistinguishes these two benign tumor types, wecompared the expression profile between normalmouse skin, low-risk and high-risk papillomas andSCC with oligonucleotide dual fluorescence micro-arrays. Unsupervised hierarchical clustering of 514differentially expressed genes showed that at earlytime points, low-risk and high-risk papillomas differ

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significantly in their pattern of gene expression(Figure 1).High-risk (9/10) papillomas clusteredwiththe SCC, because of several groups of genes withsimilar expression patterns, but not with low-riskpapillomas [19]. Only 1/10 of the high-risk papillo-mas clustered with the low-risk papillomas whichcorrelated with similar expression of the low-risk Kexpression profile. Thus, this microarray analysisshows that subgroups of murine premalignantlesions have reproducible gene expression patternsthat correlate with risk for malignant conversion.These findings will define important signaling

pathways that distinguish these premalignantlesions and could be used as predictive molecular

biomarkers. One set of genes that was identifiedwithprediction analysis for microarray (PAM) to distin-guish low- and high risk-papillomas included manyimmune function genes that were downregulated inthe high-risk papillomas and SCC relative to normalskin and low-risk papillomas. Using immunohisto-chemistry, we found that CD3þ cells were nearlyabsent within the tumor epithelium of the high-riskpapillomas but were abundant within the low-riskpapillomas suggesting that reduced adaptive immu-nity defines papillomas that progress to SCC. Furtherstudies are underway to identify minimal gene setsthat can predict risk for malignant conversion.

ORIGIN OF HIGH-RISK PAPILLOMAS

Although speculative at present, there are severalpossible mechanisms for the early appearance ofthese benign lesions with properties distinct fromthemajority ofpapillomas and their link to SCC.Onepossibility is that the initiated keratinocyte thatforms the high-risk papillomas has distinct oradditional mutations other than the well-character-ized codon 61mutation of the Hras1 oncogenecaused by DMBA. The combination of these muta-tions or the distinct mutation itself could establish apromoter independent and a rapid progressionphenotype. Support for this explanation comes froma study of Hras1 mutations induced by differentchemical carcinogens where the distribution ofspecific mutations differed between papillomas andcarcinomas [20]. Thus, the alkylating agents N-methyl-N0-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea induced exclusively G:A transitions atcodon12, and thismutation is foundpredominantlyin papillomas. However, 3-methylcholanthreneinitiation produced both codon 13 G:T and codon61 A:T transversions in papillomas, but only the G:Tmutation was found in carcinomas. These datasuggest that papillomas that are precursors to SCChave different ras mutations than the majority ofterminally benign papillomas. Another and not amutually exclusive possibility is that the initiatedcell for high-risk papillomas is derived from acompartment distinct from that giving rise to low-risk papillomas. The finding that transgenic miceexpressing oncogenic Hras1 from a suprabasal pro-moter form benign papillomas while basal promo-ters for Hras1 cause rapid SCC development [21]supports the idea that the proliferative or differentia-tion potential of the initiated cell type coulddetermine the fate of the subsequent tumor. Simi-larly, recent studies have shown that the majority ofpapillomas arise from initiated interfollicular kerati-nocytes, while most of the SCC arise from initiatedslow cycling follicular keratinocytes [22]. Thus, ifinitiated ‘‘stem cells’’ are the precursors for high-riskpapillomas and SCC, then the distinct developmen-tal and phenotypic characteristics of these cells mayimpart unique properties to their neoplastic progeny

Figure 1. Hierarchical clustering reveals similar gene expressionpattern between high-risk papillomas and SCC. Microarrays wereperformed with NCI oligo arrays comparing each sample to universalmouse RNA (skin¼ 5, low-risk papilloma¼ 11, high-risk papilloma¼10, SCC¼ 6. 514 genes were selected as significantly differentbetween at least two groups using ANOVA P< .001, and the valuesfor each gene within each group averaged. A heat map wasgenerated with a hierarchical clustering tool on the NCI mAdBwebsite. Representative genes are listed for specific nodes of similarexpression between high-risk papillomas and SCC.

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that produces rapid independence from tumorpromotion and accelerated premalignant progres-sion.

SIGNIFICANCE FOR HUMAN CANCERS

Many human cancers develop from premalignantlesions of variable but unknown risk for malignantconversion. Oral leukoplakia, actinic keratosis (AK)and cervical dysplasia are well-characterized prema-lignant lesions of the oral mucosa, epidermis, andcervix for which risk of malignant progression is animportant clinical issue. Based on numerous studies,there is an estimated risk for malignant progressionover a defined time period of 0.1–18% for oralleukoplakia, and 0.25–20% for AK, suggesting a highdegree of variability between individual lesions[23,24]. In fact, leukoplakias that develop in non-smokers quite probably progress to malignancywhile many leukoplakias that form in smokersdisappear after smoking cessation [25], and leuko-plakias and AK that arise in some anatomic locationshave a higher risk for malignancy [23,26,27]. Theseobservations suggest that not all premalignantlesions have the same potential to form cancers,and as with the mouse skin carcinogenesis modelmay fall biologically into low-risk and high-risksubgroups. Because studies have shown the lack ofstrict correlation between dysplasia, the histopatho-logical standard for diagnosis of risk, and malignantconversion [23], othermore standardizedmarkers ofrisk potential are important to develop. Molecularcharacterization of these cancers has identifiedseveral phenotypic and molecular genetic biomar-kers that are associated with progression/outcome[23], but it is likely that combinations ofmarkers willhave a more beneficial predictive value. If thesepremalignant lesions exist as stable subgroups withdiffering risks for malignant progression, thenprobably that gene expression analysis will segregatethese groups at the molecular level and will identifygroups of genes whose expression pattern predictsthe relative risk for malignancy.

ACKNOWLEDGMENTS

N.D. was supported by the American University ofBeirut in Beirut/Lebanon andby theNational CancerInstitute Scientist Exchange Program Fellowship.R.P-L and A.G. were supported by the Penn StateHuck Institutes of Life Sciences, the Penn StateInstitute of the Environment, and CA117957. Thisresearch was also supported by the IntramuralResearch Program of the NIH, National CancerInstitute, Center for Cancer Research.

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