Genetic Predisposition and Parameters of Malignant Progression in

American Journal of Pathology, Vol. 149, No. 6, December 1996

Copyright C) Amenican Societyfor Investigative Pathology

Genetic Predisposition and Parameters ofMalignant Progression in K14-HPV16 TransgenicMice

Lisa M. Coussens,*t Douglas Hanahan,*tand Jeffrey M. ArbeitttFrom the Department ofBiochemistry and Biophysics,*Hormone Research Institute,t and Department of Surgery,University of California, San Francisco, SanFrancisco, California

Reproducible multi-stage progression to inva-sive squamous carcinoma of the epidermis hasbeen achieved in transgenic mice expressing theHPV16 early-region genes, including the E6/E7oncogenes, under the control of the human ker-atin-14 promoter/enhancer. Although 100% ofK14-HPV16 transgenic animals develop hyper-plastic and/or dysplastic lesions in several in-bred backgrounds, including C57BL/6, BALB/c,and SSIN/SENCAR, only mice backcrossed intothe FVB/n background progress to malignantsquamous cell carcinomas of two pathologicalgrades, weU differentiated and moderate/poorlydfferentiated (WDSC or MPDSC, respectively),each displaying characteristicpatterns ofmalig-nant behavior. WDSCs typically arise within theepidermis of the ear and invade deeply into theunderlying dermis but fail to metastasize,whereas MPDSCs develop on the chest and trun-cal skin and invariably metastasize to regionallymph nodes. The transition to the malignantstate, in 21% ofFVB/n transgenic mice, is char-acterized by alteration of the repertoire of ker-atin intermediate filament proteins expressedwithin neoplastic epidermis, such that WDSCsmaintain expression of keratins common to ter-minaly differentiating stratified keratinocytes(K10), whereas MPDSCs are distinguishedfromWDSCs by activation of embryonic and mucosalkeratins (K13, K8, and K19). Precursor hyper-plastic and dysplastic lesions are characterizedby aprogressively increasedproliferative index,striking morphological alterations in keratino-cyte cell-cell and cell-matrix interactions, and ex-

tensive remodeling of the underlying dermalstroma. Remarkably, this extensive stromal re-modeling, which may facilitate both angiogene-sis and eventual tumor ceUl invasion, developsearly at the dysplastic stage in aU animals weUbefore malignant conversion. (Am J Pathol1996, 149:1899-1917)

In humans, the most common cancers are epithelialin origin, accounting for approximately 80% of allmalignancies.1 It is increasingly evident that the pro-gression to an invasive carcinoma from precursorlesions represents a rate-limiting step in multi-stagecarcinogenesis.2 Human papillomaviruses (HPVs)have been implicated in the genesis of several hu-man epithelial malignancies, particularly squamouscell carcinomas of the anogenital region.3-5 In par-ticular, HPV types 16 and 18 are found in up to 90%of invasive carcinomas of the cervix.6 However, alarger percentage of women, also HPV-positive,never develop invasive disease.347 Therefore, al-though expression of HPV oncoproteins may be suf-ficient to elicit hyperproliferative disorders in vivo,they are insufficient to induce invasive carcinomasalone. In addition to genetic predisposition, otherevents may underlie malignant conversion. Thus, un-derstanding the molecular and biochemicalchanges, in addition to HPV infection, that mandatemalignant conversion are of paramount importanceto understanding and eventually inhibiting anogeni-tal carcinogenesis.

In models of murine epidermal carcinogenesis,either two-stage carcinogenesis or targeted expres-

Supported by a National Institutes of Health grant (CA 09043), aNational Cancer Institute grant (RO1CA47632-07A1), an AmericanCancer Society grant (PRTF 12A), and a grant from the MarkeyCharitable Trust.

Accepted for publication August 12, 1996.

Address reprint requests to Dr. Douglas Hanahan, Hormone Re-search Institute, University of California, San Francisco HSW 1090,513 Parnassus Avenue, San Francisco, CA 94143-0534.

1899

1900 Coussens et alAJP December 1996, Vol. 149, No. 6

sion of oncogenes or growth factors to the epider-mis, the incidence of malignant conversion is alsotypically low.8-10 Moreover, strain-specific suscepti-bility appears to influence malignant conversion toinvasive carcinoma in several epithelial carcinogen-esis models.11-13 We have developed a transgenicmouse model of epidermal neoplasia wherein theoncogenes of HPV16 are expressed in basal kerati-nocytes of squamous epithelium as directed by ahuman keratin-14 (K14) promoter regulatory re-gion.14 We previously reported that the epidermis ofK14-HPV16 transgenic mice, maintained in theC57BL/6 or BALB/c inbred backgrounds, developedhyperplasia and high-grade dysplasia, respectively,with no progression to malignancy. We now reportthat squamous cell carcinomas develop in 21% ofK14-HPV16 transgenic mice by 8 to 12 months ofage when backcrossed one or more generations intothe FVB/n genetic background. The cancers ariseout of premalignant lesions and possess distinctgrades of differentiation corresponding with charac-teristic degrees of malignant aggressiveness. Nota-bly, molecular and biochemical changes first ob-served in premalignant precursor lesions appear toset the stage for malignant conversion long beforeinvasion actually occurs.

Materials and Methods

Transgenic MiceCreation of transgenic mice has been reported else-where.14 All tissues used in this study were obtainedfrom animals of one lineage (K14-wt-1) derived byinjection of plasmid pK14-1203, containing wild-typeHPV16 early-region DNA, which had been back-crossed several generations (minimum of five) intothe FVB/n inbred strain, and maintained in the het-erozygous state.

HistologyAll animals sacrificed in this study were first anes-thetized with Avertin (0.018 mg/g body weight), fol-lowed by perfusion through the left ventricle with3.75% paraformaldehyde in calcium- and magne-sium-free phosphate-buffered saline (PBS). Tissueswere then postfixed overnight at 40C in 3.75% para-formaldehyde in PBS, followed by processingthrough graded alcohols and three changes of xy-lene and embedding in paraffin. Five-micron sec-tions were then stained with hematoxylin and eosin(H&E) for histological analysis. All histology shown is

representative for a particular stage of malignantprogression as described in the text.

ImmunohistochemisttyDeparaffinized 5-p.m sections were stained with thefollowing specific antisera or affinity-purified antibod-ies: rabbit anti-human K5, 1:2000 (obtained from E.Fuchs, Chicago, IL); affinity purified rabbit anti-mouse K10, 1:2000 (obtained from S. Yuspa, Be-thesda, MD); rabbit anti-mouse filaggrin, 1:2000 (ob-tained from B. Dale, Seattle, WA); rat monoclonalantibodies to K8 (Troma 1) and K19 (Troma 3), 1:50(obtained from R. Kemler, Freiburg, Germany); affin-ity-purified rabbit anti-mouse K6 and K13, 1:2000(both obtained from D. Roop, Houston, TX), andmurine monoclonal antibody to proliferating cell nu-clear antigen (PCNA), 1:200 (Biogenex, San Ramon,CA). For specific staining to K5, K10, K6, K13, andfilaggrin, deparaffinized sections were passedthrough graded alcohols, rehydrated in PBS, andthen incubated in 5% H202, methanol (20 minutes) toblock endogenous peroxidase activity. After briefPBS washes, 10% normal serum or 3% bovine serumalbumin (Sigma Chemical Co., St. Louis, MO) wasapplied as a blocking agent (30 minutes). Retrievalof the antigens for K8, K19, and PCNA was achievedby first blocking endogenous peroxidase activity andthen microwaving the tissue sections twice for 5minutes each in antigen retrieval solution (Citra, Bio-genex). Tissue sections were then incubated withprimary antibodies, at the dilutions given above, inPBS and 3% bovine serum albumin followed by over-night incubation at 40C. After three washes in PBS, asecondary antibody (biotinylated goat anti-rabbitIgG for K5, K10, K6, K13, and filaggrin; biotinylatedrabbit anti-rat IgG for K8 and K19; and biotinylatedgoat anti-mouse IgM for PCNA) was added at adilution of 1:200 in PBS, 3% goat serum or bovineserum albumin (30 minutes at room temperature).Again, after three washes in PBS, Vectastain EliteABC reagent (Vector Laboratories, Burlingame, CA)was applied (30 minutes). The sections were thenwashed in 0.1 mol/L Tris (pH 7.4) three times andvisualized by treatment with 3,3'-diaminobenzidine(Sigma) and H202. After brief washes in PBS anddehydration through graded alcohols, the sectionswere mounted in Entellan new mounting medium(EM Science, Gibbstown, NJ). PCNA immunohisto-chemistry was essentially as described previously,15except the antibody was diluted 1:200 in 3% bovineserum albumin, 0.5% Triton X-100 in PBS. Beforemounting, PCNA-stained sections were counter-stained briefly with hematoxylin. The immunohisto-

Squamous Carcinogenesis in HPV16 Transgenic Mice 1901AJP December 1996, Vol. 149, No. 6

chemical analyses presented are representative foreach particular stage of malignant progression andencompassed biopsy specimens removed from 25animals representing 2 nontransgenic negative littermates, 13 transgenic mice with premalignant le-sions, 5 well differentiated squamous cell carcino-mas (WDSCs) and 7 moderate/poorly differentiatedsquamous cell carcinomas (MPDSCs; two animalspossessed multi-focal tumors where one WDSC andone MPDSC were present). All immunohistochemicalexperiments included negative controls for determi-nation of background staining, which was negligible.A Nikon microphoto FM microscope fitted with No-marsky optics was used for photomicroscopy.

In Situ HybridizationsParaffin-embedded 5-,tm sections were processedfor in situ hybridizations as described previously.15 Ariboprobe for HPV1 6-E6 and -E7 was constructed bypolymerase chain reaction from a plasmid templatecontaining the entire HPV16-E6 and -E7 open read-ing frames. The resultant polymerase chain reactionproduct contained nucleotides 104 to 853. This frag-ment was subcloned into pBluescript 11 (Stratagene,La Jolla, CA) enabling synthesis of sense and anti-sense RNA from the linearized vector using T3 andT7 polymerase (Promega Corp., Madison, WI), re-spectively.

PCNA Labeling IndexImmunohistochemical analysis of PCNA labelingwas determined as previously described.16 Briefly,the proportion of tumor cells staining positively forPCNA was determined from 2000 cell counts byselecting four to six high-power fields (x40) showingthe greatest concentration of stained nuclei.

Electron MicroscopyTissues for electron microscopy were obtained fromanimals that had undergone perfusion as above with2% paraformaldehyde, 0.2% glutaraldehyde in 0.15mol/L cacodylate buffer at pH 7.4, followed by im-mersion fixation in 4% paraformaldehyde, 0.15 mol/Lcacodylate buffer at pH 7.4, overnight at 4°C. Post-fixation was performed using 1 % osmium tetroxide incacodylate buffer (overnight at 4°C) followed by de-hydration through graded alcohols and embeddingin epoxy resin (Epon 812). Semi-thin sections (1 ,um)were stained with toluidine blue and ultra-thin sec-tions (60 nm) were picked up on uncoated grids andexamined with a Philips 300 electron microscope

after staining with uranyl acetate and lead citrate at60 kV.

Results

Genetic Background Restricts Progressionto Carcinoma

We have previously reported the development ofeight independent K14-HPV16 transgenic lines ofmice, each derived from a different B6D2 F2 trans-genic founder.14 The transgenic founders containedone of three types of HPV16 early-region DNA: wildtype or two mutant variants in which function of eitherthe El or the E2 gene was abrogated by insertion ofa translation termination linker (ttl) mutation.14 Theseeight transgenic lines varied in their epidermal phe-notype, which was limited to development of neo-plastic lesions without malignant conversion. No cor-relation was found between type of HPV DNA(mutant versus wild type) and the extent or severity ofepidermal pathology.14

Based on the results of chemical carcinogenesisstudies on murine skin that suggest that susceptibil-ity to epidermal carcinogenesis varies according togenetic background, 13,15,17 we selected two inbredmurine strains (SSIN/SENCAR and FVB/n) for cross-breeding to investigate the possibility that geneticbackground may influence susceptibility to malig-nant conversion of neoplastic lesions in K14-HPV16transgenic mice in comparison with the C57BL/6 andBALB/c backgrounds analyzed previously.14 Inter-crossing K14-HPV16 mice for five generations intoSSIN/SENCAR18 and nine generations in the BALB/cinbred strains resulted in more advanced dysplasticphenotypes as compared with C57BL/6, all of whichfailed to undergo malignant conversion (Table 1). Incontrast, mice in four transgenic lines (K14-wt-1 and-3 and K14-E2ttrl and -9) developed invasive epi-dermal squamous carcinomas when backcrossedinto the FVB/n strain (Table 1 and Figure 1). One ofthese transgenic lines (K14-wt-1) was selected foradditional backcrossing into the FVB/n strain. Squa-mous cancers of the epidermis developed by 8 to 12months of age in 21% (38/169) of the K14-wt-1 micebackcrossed into FVB/n (Table 1). Malignant conver-sion was observed in the first hybrid (Fl, C57BL/6 xFVB/n) intercross into this strain and has persistedupon continuous intercrossing into FVB/n. In addi-tion, a recent backcross into C57BL/6 after nine gen-erations of breeding in FVB/n also resulted in elabo-ration of squamous carcinomas on transgenicanimals, further suggesting that susceptibility to ma-


Table 1. Degree qf Neoplastic Progression in K14-HPV16 Tranisgenic Mice as a Function of Genetic Strain

Transgenic mice*

355453

227

Mice at riskt

294139169

Precursor pathologyt

HyperplasiaHyperplasiaDysplasiaDysplasia

*Indicates the total number of transgenic mice, from all litters, within each inbred strain.tlndicates the total number of transgenic animals alive at 8 to 12 months.tlndicates the most advanced stage of neoplastic progression exclusive of malignancy.51ndicates the total number of invasive cancers verified by histopathological analysis.

lignant conversion is a dominant trait of FVB/n (datanot shown). As progression to invasive carcinomawas a reproducible feature of K14-wt-1 transgenicmice in the FVB/n genetic background, we used thistransgenic lineage for a more detailed analysis of themolecular and biochemical changes accompanyingmulti-stage epithelial carcinogenesis.

K14-HPV16 Transgenic Mice Develop BothWell Differentiated and Moderate/PoorlyDifferentiated Squamous Cell Carcinomas

We assessed the progressive histopathologicalchanges that occur in K14-HPV16 transgenic miceby analyzing biopsy specimens representing distinc-tive morphological lesions. Overall, the temporal ap-

pearance and characteristics of hyperplasia anddysplasia were similar in K14-HPV16 transgenicmice crossed into the FVB/n background (Figure 1, Band C) to that observed in the C57BL/6, BALB/c, or

SSIN/SENCAR backgrounds (not shown). However,two distinct types of squamous cell carcinoma de-velop in K14-HPV16 x FVB/n transgenic mice (Fig-ure 1, D and F). The tumors arise at characteristicanatomic sites, differ markedly in their visible ap-

pearance, show distinguishing grades of differentia-tion, and display disparate malignant behavior. Al-though the two types of carcinoma are distinct, they

both develop from fields of high-grade dysplasia(Table 1 and Figure 1C).The well differentiated form of invasive squamous

carcinoma typically develops on the ventral surfaceof the ear as a protuberant lesion capped by a largekeratin horn. These lesions resemble keratoacantho-mas, as seen both in humans19 and in other trans-genic models of epidermal carcinogenesis.2"Whereas these lesions in the HPV1 6 transgenic miceare persistent, similar lesions (keratoacanthomas) inhumans spontaneously regress.19 Histologically, theear cancers invade deep into the dermis and occa-

sionally destroy the underlying cartilage (Figure 1, Dand E). Malignant keratinocytes within invading le-sions maintain the overall pattern of keratinocyte ter-minal differentiation, such that invading clusters ofmalignant cells within the dermis are arranged inkeratin pearls, concentrically organized structuresthat differentiate internally, accumulating keratinwithin their centers (Figure 1, D and E). These fea-tures are consistent with these lesions being classi-fied as Broders grade WDSCs.21 Despite extensiveinvasion, WDSCs do not metastasize to regionallymph nodes.The second type of invasive cancer, which arises

on chest and truncal skin, is typically a low-profilelesion with heaped margins and a central ulceration.Histologically, these lesions are markedly heteroge-

Figure 1. Histopathology of malignant progression in K14-HPV16 transgenic mice, shoun itn 5-pn. paraffin-embedded sections of epidenmisfromnontransgenic anid transgenic animals stained with H&E. A: H&E stainintg ofear skinfrom a 1-month-old nontransgenic animal demonstratinig thenonnally thin, fouir-layered mnuirine epidernmis composed of basal ( b), spinionis (s), granular(g), and corneum (c) cellsjuxtaposed to the underlyingdermis (d). B: Hyperplastic ear epidermis from a 1-month-old transgenic anlimal. 7here is an overall twofold thickening ofeach epidermal cell layerwith overlying hyperkeratosis (arrow). C: Dysplastic cear epidermisfrom a 3-month-old transgentic animal. Arceas stuch as this conttain hyperchromaticnuclei thronighout the epidennis (arrowheads) extendingfrom the epidermal-dermaljuntctioni to the renmnants ofthe stratu ni conzeun. Feau granularand corneum cells are observled int these region.s. D: Grade I well differentiated squamous carcinonia fromn the ear of a 9-mnonth-old trauisgenicanimal. The arrow represents the dircection of tumor growth doun intto the dermal stroma (s). The asterisk indicates the presence of keratin pearlsarising from keratinocytes that have undergone positionally inappropriate terminal differentiationz. E: Higher-pou'er magnification of keratin pearland inv)adinig keratinocytes wuithitn the WDSCshoun in D. F: Trunical skit from an 11-montth-old trautsgenic animal conttaining a metastatic MPDSC.H&F staining of this primar tuimor rei.eals anastomosing clusters of keratinocyte-s colontizintg the dermis below the highly dysplastic, overlyingepidermis. 7he arrow indicates the site at which initial inivasion likeVly occurred. G: High-pou'er magnification ofa malignant cell ncestgrou'ing unithina grade II MD)SC surrounded by dermal stroma (s). Some degree of keratinocyte differenitiation is present in this tunior as evidenced by thekeratobyalin granules (arrowheads) in the centter of the cell nest, uhich also contain numerous mitotic figures (arrows). H: Maligntant cell nests ina grade III MPDSC. The arrows indicate mitotic figures, nzote the reductiont in tuimor stroma surrounding the cell nests and blurring qf the tuniorstroma interface. I: H&E staining of a regional lymph node removed from the same animal shown in H revealing metastatic keratinocytes (K)aggressively growing int and around lyniphatic tissue (L). Bar, 5.4 gin (A to C), 21.6 p.m (D), 10.8 p.n (E), 21.6 ,um (F), 5.4 Aiu) (G atnd H), and21.6 p.ni (I).

Strain

SSIN/SENCARC57BL/6BALB/cFVB/n

Cancers§

000

35

Squamous Carcinogenesis in HPV16 Transgenic Mice 1903A/P December 1996, Vol. 149, No. 6

neous, containing regions with gradations of differ-entiation (Figure 1, F-H). Some areas are character-ized by invading nests of concentrically arrangedmalignant keratinocytes, which evidence partial epi-dermal differentiation, in the form of granular-likecells containing keratohyalin granules surrounded

by less prominent accumulations of more amor-phous keratin (Figure 1G). Such regions are consis-tent with the criteria of a Broders grade 11 cancer.21Other areas within these cancers are considerablyless differentiated, being composed of sheets orclusters of anaplastic squamous cells with frequent

w ~ ~* *;S #E t

r. *.~ - -~~ ~~A

'-*.'.' #d

--w- AV.W,40:'It., r, 0.,Lir. '0!'.. O..4OW0 *A -"'


Figure 2. Incremental increase in transgene expression at the malignant conversion step, as shown by dark-field microscopy showing hybrdizationof HPV16 E6/E7 mRNA to 35S-radiolabeled antisense E6/E7 riboprobes. A: Normal, nontransgenic ear epidermis. B: One-month-old hyperplastictransgenic ear epidermis. C: Three-month-old dysplastic transgenic ear epidermis. D: Apical tip of a WDSC. E: Cell nests from a primary MPDSCtumor. F: Regional lymph node removedfrom the anzimal shown in E. Note that 1ymphocytes (zupper regionz ofpaniel) do niot express £61E7 mRNAs.Bar, 10.8 j.tm (A to C), 21 ,lm (D and E), and 10.8 ,um (F).

mitotic figures and pronounced nuclear atypia voidof keratohyalin-granule-containing cells (Figure 1, Fand H). The histopathology of these areas possess

features of a Broders grade Ill squamous carcino-

ma."1 Overall, we have categorized these lesions as

MPDSCs, reserving the category of a strict poorlydifferentiated squamous carcinoma (grade IV malig-nancies) for spindle cell lesions, which have beenobserved in only six K14-HPV16 transgenic mice of169 mice observed over the last 2 years (Table 1).Notably, the MPDSCs invariably metastasize to re-

gional lymph nodes (Figure 11), such as the axillaryor cervical nodes, after attaining a 0.8- to 1.0-cmdiameter. No basal cell carcinomas have been ob-served among the eight independent K14-HPV16transgenic lines, in any of the genetic backgroundstested; this result is indicative of the inability ofHPV16 expression in basal keratinocytes to com-

pletely block their intrinsic propensity to enter a dif-ferentiation pathway.15

Incremental Increases in HPV1 6-E6/E7Oncogene Expression duringCarcinogenesisPathological evaluation of neoplastic progressionin K14-HPV16 transgenic mice reveals increasednumbers of basaloid cells appearing throughoutskin epidermis and diminished numbers of termi-nally differentiated cells (Figure 1). As transgeneexpression in K14-HPV16 mice is regulated by aK14 promoter/enhancer, expression of the trans-genes should be restricted to basaloid cells andnot present in suprabasal terminally differentiatingcells. Previously, we reported that the HPV16-E7

11

A

i

i

k

Squamous Carcinogenesis in HPV16 Transgenic Mice 1905A/P December 1996, Vol. 149, No. 6

Table 2. Summary of Pbenotypic and Biochemical Changes during Malignant Progression

Premalignant

Normal Hyperplasia Dysplasia WDSC

MalignantMPDSC Mets

E6/E7 mRNAPCNA indexK5/K1 4K10K6K13K8K19DesmosomesHemidesmosomesBasal laminaCollagen fibrilsAngiogenesis*

32%BSB

51%B/SBSBSB

++78%B/SBSBSB

80%B/SBSB

++ 4++ +

+++X~~~~~~~-

++

88%Uniform

3B SB- Heterogeneous

UniformHeterogeneous

p + +

p + _+l- +l-

85%Uniform

HeterogeneousHeterogeneous

UniformHeterogeneous

NDNDNDNDND

PCNA labeling index was quantitatively determined as discussed in the text. Other phenotypic and biochemical changes werequalitatively assessed by scoring parameter indicated for both presence and/or level of detection: -, parameter was not detected; +,parameter was detected at lowest level; ++++, maximal detection level of parameter. B, basaloid cells; SB, suprabasal cells; ND, notdetermined.

*Onset of angiogenesis has been demonstrated by Smith-McCune et al, manuscript submitted, and Arbeit et al.43

oncoprotein could be detected in the skin of2-day-old neonatal transgenic mice by a com-bined immunoprecipitation/Western blotting pro-cedure.14 Although this analysis verified E7 ex-pression, it did not reveal the specificity of thatexpression. We have now used in situ mRNA hy-bridization to determine the intensity and distribu-tion of epidermal transgene expression within neo-plastic keratinocytes at each stage of neoplasticprogression (Figure 2). We also determined thepattern and intensity of expression of K14 mRNA toinvestigate whether HPV oncogene expressioncontrolled by 2 kb of the human K14 enhancer/promoter was concordant with expression regu-lated by the endogenous murine K14 regulatoryelements (data not shown). Briefly, hyperplasiashad a twofold expansion of the number of kerati-nocyte layers containing K14 mRNA and dyspla-sias possessed striking changes in the distributionof K14 mRNA, such that almost the entire neoplas-tic epidermis from basement membrane to surfaceepithelium contained silver grains; K14 mRNA wasalso diffusely present in WDSCs, MPDSCs, andlymph node metastases. There was no evidence,however, for any change in the levels of K14 mRNAexpressed per cell.

In contrast to the increased distribution, but notincreased expression per cell, of endogenous K14mRNA, the expression of the HPV16 transgene wasincrementally increased in basaloid cells during neo-plastic progression (Figure 2 and Table 2). In earlyhyperplastic lesions, transgene expression was atthe threshold of detection (Figure 2B). Advanced

hyperplastic lesions of older transgenic mice (datanot shown) and dysplastic lesions showed definiteup-regulation of transgene expression in basaloidcells throughout transgenic epidermis (Figure 2C). Inthe cancers, an incremental increase in the level ofE6/E7 mRNA was detected in both differentiationgrades of carcinoma as well as in the metastases ofthe MPDSCs (Figure 2, D-F). Thus, although trans-gene expression mirrors the distribution of endoge-nous K14 expression in keratinocytes, the level oftransgene expression throughout neoplastic pro-gression is discordant with the expression of K14.

Progression to Invasive Cancer Is NotDefined by Increased Cell ProliferationTo address the issue of whether cellular proliferationincreases during the transition to invasive malig-nancy, we examined the proliferative status of kera-tinocytes in preneoplastic lesions and invasive tu-mors by immunostaining to detect the expression ofPCNA (Figure 3 and Table 2). PCNA is an auxiliaryprotein to DNA polymerase-6 and is expressed dur-ing the cell division cycle in late Gl, throughout S,and in early G2. 16,22PCNA expression in nontransgenic FVB/n control

animals is detectable in interfollicular and outer rootsheath basal cells (Figure 3A). In hyperplastic trans-genic epidermis, PCNA-positive keratinocytes are ob-served in basal and suprabasal cell layers, accountingfor 51% of the epidermal keratinocytes (Figure 3B) and78% in dysplastic epidermis (Figure 3C). These prolif-

c.


.z-.

.$.

.Figure 3. Progressive increase in epidernmal PCNA during maligntant progression. Five-micron sections adjacent to those shown in Figures 1, 2, and4 were utsedfor immunohistochemistry with an antibody recognizing PCNA and counterstained uith hematoxvlint. A: In 1-month-old nontratnsgenicear epidermis, only basal cells express PCNA. B: In areas of hyperplasia, cells occuipying allfour epidermal la yer express PCNA. C: Developedcdysplastic epidermisfrom a 3-month-old transgenic animal shous homogeneous PCNA staining throughout the epidermis in 78% ofthe keratinocytesas compared uwith 51% in B and 32% in A. D to F: Homogeneouis PCNA staininig is also observed in a WDSC ( 80%; D), MPDSC (88%; E), anzdkeratinocytes residitng in a lymph nodce metastasis (85%; F). Note the increased size of the nuclei itn B antd C compared uith A and the increaseddegree of nuclearpollymorphism u'ithin the neoplastic cells (D to F). Ini F, L refers to lymphocytes within the lym)nph nzode. Bat; 5.4 p.m (A to C) anid10.8 um (D to F).

erative indices, derived from PCNA expression, are

numerically twice that obtained from our previous studyusing bromodeoxyuridine incorporation.14 This discor-dance reflects expression of PCNA during parts of theGl and G2 phases of the cell cycle in addition toS-phase (see above). In WDSCs (Figure 3D), MPDSCs(Figure 3E), and lymph node metastases (Figure 3F),the PCNA index plateaued at 80 to 88%, similar to thatof advanced dysplasias, indicating that the incidenceof cells in cycle, and inferentially their characteristic cellcycle times, are unlikely determinants of progression tofull malignancy.

Aberrant Cytokeratin ExpressionAccompanies Malignant Progression

The organization of multilayered epithelium is in partfacilitated by keratinocytes undergoing self-renewal

concomitant with terminal differentiation. This processis accompanied by changes in the expression of epi-thelial intermediate filament proteins, the keratins,which are, under normal conditions, temporally regu-lated in an epithelial-cell-type (simple or stratified), dif-ferentiation-program-type (cornifying or noncornifying),and proliferation-rate-dependent manner.23 Hence, astrong correlation can be established between the his-tological category of an epithelium and the differentia-tion status of keratinocytes based on the repertoire ofexpressed keratins.24 To examine the alterations inkeratinocyte terminal differentiation during neoplasticprogression at a biochemical level, we have deter-mined the expression patterns of the major keratinproteins (Figure 4 and Table 2), specifically, the basalkeratins K5 and K14, the suprabasal cornifying keratinK10, the suprabasal noncornifying keratin K13, the hy-perproliferative-associated keratin K6, the simple epi-

II

I

I4

PI


K6

Figure 4. Keratin andfilaggrin immunohistochemistry during malignant progression. Five-micron paraffin-embedded sections (adjacent to thoseused in Figures 1, 2, and 3) ofnontransgenic earskin (A to D), hyperplastic transgenic earskin (E to H), dysplastic earskin (I to L), WDSC ear tumor(M to P), and chest skin with MPDSC (Q to T) were stained with antibodies recognizing specific epithelial proteins: keratin 5 (A, E, I, M, and Q),keratin 10(B, F, J, N, and R); filaggrin (C, G, K, 0, and S), and keratin 6(D, H, L, P, and T). Arrows, keratin 5 staining cells in suprabasal layers;--- the epidermal-dennal border or basal lamina; sg, sebaceous gland surrounding the keratin 6 staining cells of the hairfollicle. Bar, 5.4 Jim (Ato L), 8.6 pm (M to P), and 10.8 p.m (Q toT).

thelial keratins K8 and K19, and filaggrin, which isexpressed only in terminally differentiated corneum

cells.Normal murine epidermis is characterized by

expression of K5 and K14 (data not shown) in

basal cells, K10 in suprabasal spinous and gran-ular cells, and filaggrin in the outer corneum layer(Figure 4, A-C, respectively). In transgenic hyper-plastic and dysplastic epidermis, expression of K5and K14 (data not shown) is expanded to supra-

K5 K10

ffV.}.' -.: ~ '-- .¢iiWlvil; -10 %

f.i

7.:

I.'i

:A%,! ..-Aj -, _., . f'.. ,. ., :!--.-'.vl,-,- ..---,


K13

Figure 5. Aberrant expression of keratins 13, 8, and 19 in metastatic moderate/poor/y differentiated squamotis carcinoma, as shouw hy imnnti.io-stainingfor keratin 13 (A), keratin 8(B), and keratin 19(C) in a grade IIIMPDSC. Note that keratin 13 and keratinz 19 arefocally expressed withinthe tumor whereas keratin 8 expression is ubiquitous in all malignant keratinocytes within the cell nests. Bar, 10.8 ,um.

basal basaloid cells and K10 expression remainsrestricted to suprabasal spinous and granularcells, whereas filaggrin expression diminishes co-

ordinately with reduction in keratinocyte terminaldifferentiation (Figure 4, E-L, respectively). Thispattern of keratin expression is similar to thatwhich has been previously reported for K14-HPV16 transgenic mice bred in the C57BL/6 andBALB/c genetic backgrounds.14 In normal epider-mis, K6 expression is restricted to the inner rootsheath cells of the hair follicle (Figure 4D), kerati-nocytes homologous to spinous cells that are com-

mitted to terminal differentiation.2325 K6 expres-

sion is diffusely up-regulated in interfollicularsuprabasal keratinocytes in hyperplastic and dys-plastic lesions in K14-HPV16/FVB/n mice (Figure4, H and L) similar to previous reports in othermurine models of epithelial carcinogenesis.2026There was no detectable expression of the muco-

sa-specific K13 in high-grade dysplastic lesions(data not shown). This result is notable in thatsimilar lesions arising in other murine models ofepidermal neoplasia have been reported to aber-rantly express this keratin (see Discussion).202728

Analysis of invasive squamous cancers in K14-HPV16/FVB/n transgenic mice revealed that theprofile of keratin expression in WDSCs is similar tothat observed in the premalignant lesions, whereexpression correlates with differentiation and strat-ification. Hence, K5 is found in basaloid cells (Fig-ure 4M), and K10, filaggrin, and K6 (Figure 4, N-P,respectively) are prominently expressed in supra-

basal spinous, granular, and corneum cells, re-

flecting the well differentiated nature of this tumor.This repertoire of keratin expression also persistswithin keratinocytes forming keratin pearls deepwithin the dermis (data not shown). Similar to thepremalignant dysplastic lesions, K13 and the sim-ple epithelial keratins K8 and K19 are not detect-able in WDSCs (data not shown). This result is alsonotable in that low-grade WDSCs arising in othermurine epithelial models of neoplastic progressionoften express K13 and less often K8, in addition tothe normal repertoire of keratins.13

In contrast to the retention of differentiationmarkers seen in the premalignant stages and WD-SCs, MPDSCs display striking alterations in theirrepertoire of keratin expression. K5 is uniformlyexpressed within these lesions rather than re-

stricted to basal-type cells (Figure 4Q). K14 ex-

pression mirrored K5 expression in both the WD-SCs and the MPDSCs (data not shown). Both K10(Figure 4R) and filaggrin (Figure 4S) are undetect-able in MPDSCs, whereas K6 expression is prom-

inent in all suprabasal keratinocytes within the in-vading clusters of malignant cells (Figure 4T). Inaddition, MPDSCs, and their lymph node metasta-ses (data not shown) display de novo expression ofK13 (Figure 5A) as well as the simple epithelialkeratins K8 and K19 (Figure 5, B and C, respec-

tively). K8 is uniformly expressed within these le-sions (Figure 5B), whereas both K13 (Figure 5A)and K19 (Figure 5C) are focally detectable in in-dividual keratinocytes, or small clusters of malig-nant keratinocytes, scattered throughout MPDSCs,presumably marking the presence of transdifferen-

K8 K19


FFigure 6. Electron microscopy of niormal mnurine skin and dysplastic tranisgenic epidermis. Electron iniicrographs of norimial (A to C) anid tr-anisgeniicclysplastic (D to F) ear skitin are shown. A: Normial murine ear skin is shoun comnposed of allbour epidermal (e) cell layers (B, basal, S, spinous, G,

granilar; C, con))meoIm1) overlVing the dermi.s (d). The epidermnal-dermnal border (arrow) and de.smosomes (circles) are inidicated. B: Higherinagnijication of dcesmnosomnes (arrows) attachinbig two ba.sal layer keratinocytes. C: The dermal-epidermal bordcer is shown uhere hemidesmosomees(arrowheads) tether basal keratinocytes to the basal lanmina overIving the denise papillary dermis (P). wbicb is packed uwitb collagent fibers (c). D:Dysplastic ear epidermis is shown at the samne mtiagnification as the epidermis in A. Basal layer keratinocytes (B), desmosomces (circled), antdepidermal-dermal border (arrow) are inidicated. Note the increased extracelliilar space anid forrmation of microvilli by plasma membranes ofdysplastic cells as nell as the loosened papillary dermis where a nmast cell ( m) antd a dermalfibroblast (f) are located. E: Higber mnagnlificationi ojextracelldlar space (asterisks) uhere desmosomes (arrows), adjacent to nmicrovilli. attach two dysplastic basal laryer keratinocvtes. F: Hemidesmo-somes (arrowheads) atnchor-inig the alysplastic basal keratinocytes to the basal lamina overlying a loosenedpapillary dernmis. K, keratinfilaments; LL,lamina Itlcida; LI), lamina densa; n, nuicleu-is. Bat- 640 nin (A anid D) anid 185 nm (B, C, E, anid F).

tiated cells uncommitted to terminal squamous dif-ferentiation. Collectively, the analysis of keratinexpression during epidermal carcinogenesis inK14-HPV16 transgenic mice demonstrates that,whereas WDSCs retain a similar repertoire andstratification of keratin expression as the anteced-ent neoplastic lesions, MPDSCs are characterizedby a distinctive program of aberrant differentiation.

Cell Junctions and Extracellular MatrixRemodeling during Malignant Progression

Alterations in the architecture of neoplastic skin andsquamous cancers seen by histopathological analy-sis, concomitant with de novo expression of non-

stratified keratin intermediate filaments in the more

aggressive carcinomas, led us to further analyze thestages of carcinogenesis in K14-HPV16 transgenic

mice by electron microscopy (Figures 6 to 8 andTable 2). First, as a baseline, we evaluated normalskin, using nontransgenic mice to avoid any subtlealterations produced by transgene expression in ap-

parently normal skin. Normal epidermis is character-ized by tightly packed adjacent basal cells (Figure6A) that are mutually adherent via broad-based des-mosomes into which intermediate filaments conver-

gently insert into desmosomal inner plates (Figure6B). Normal basal cells are also notable for theirclose apposition to the underlying basal lamina viamultiple hemidesmosomes, also associated with a

convergent insertion of intermediate filaments (Fig-ure 6C). Another important feature of normal skin inrelation to neoplastic progression and malignantconversion is the architecture of the dermis immedi-ately subjacent to the overlying keratinocytes. Thecollagen fibers in this region are arranged in a

I -pI T.pC

\ o ,

t-l

,. .1

W. i

IVB .4


Figure 7. Invasive keratinocytes in a well differentiated squamous carcinoma. Shown are electron micrographs of basal layer and malignantkeratinocytes with surrounding neoplasia removedfrom a 10-month-old transgenic animal containing a WDSC, fixed and embedded in Epon. A:An invading keratinocyte (asterisk) is shown successfully traversing the basal lamina (arrow). The cell nucleus (n) is located on the dermal side ofthe basal lamina. Desmosomes (circled) are shown attaching the invading keratinocyte to basal keratinocytes (bc) located in the epidermis as wellas to keratinocytes located within the dermis (d). The arrowheads point to prominent hemidesmosomes attaching epidermally located basalkeratinocytes to the basal lamina. The Inset a: A higher magnification ofdesomosomes (arrows), keratin filaments (k), and the extracellular space(asterisk) between two basal layer keratinocytes in this tumor. Inset b: A higher magnification of hemidesmosomes (arrowheads) facilitatingheterophyllic attachment of basal keratinocytes to the basal lamina. B: A malignant cell nest is shown located approximately 13 ,um below theepidermal-dermal border (thick arrow). Malignant keratinocytes (asterisks) are discerned from other stromal cells by the electron-dense keratinfilaments within the cytoplasms as well as by desmosomal junctions (circled), which attach adjacent cells. C: Higher magnification of the areaenclosed by the square in B, showing a desmosomal junction (arrow) attaching two adjacent keratinocytes and a hemidesmosome (arrowhead)tethering one of the two malignant keratinocytes to a discontinuous basal lamina (--- -). n, nucleus; mc, mast cell; LL, lamina lucida; LD, laminadensa; C, collagen fibrils; k, keratin filaments. Bar, 540 nm (A), 230 nm (insets), 2.14 ,um (B), and 118 nm (C).

Squamous Carcinogenesis in HPV1 6 Transgenic Mice 1911AJP December 1996, Vol. 149, No. 6

.. .

Figure 8. Finte structuire analysis ofa moderately differentiated squamous carcinoma. All data shown arefrom a grade H MDSC removedfrom thetrnik of a 10-monith-old transgenic animal. A: Toluidine-blue-stained semi-tbin section of tumor showing darklv stained neoplastic keratinocytesanzd lighterstaining dernmal stroma (S). B: Flectroii micrograph oftwo leading-edge keratinocytes revealing homophyllic attachmentfacilitated by adesmosomal junction (arrow) that spans the cextracellular space (asterisk) between uwell developed microvilli. Keratin filaments (k) attaching to thedesmosomes are indicated. C: The epidermal-dermal junction shown in this panel reveals a loosely formed basal lamina ( - ) where ahemidesmosome (arrowhead) isfound at the plasma membrane ofa leading-edge keratinocyte within the tumor. Keratin filaments are indicated.D: This micrograpb represents a more typical ultrastructure of a malignanzt keratinocyte or tumor cell (tc) observed within the MDSCs wheredesmosomes between cells (circled) are dimnittished in numbers and hemidesmosomes and basal lamina are, in general, absent at the dermal-epidermal borders (arrows). Although keratin filaments are present in the ribosome- and lysosome-rich cytoplasm, they do not abut the plasmamembranes. The stromal (s) component of this tumor is composed ofpoorly organized collagen fibers. n, nucleuis. Bar, 10.8 ,um (A), 118 nm (B),146 nim (C), anid 350 nm (D).

densely ordered array oriented both longitudinallyand transversely (Figure 6, A and C).

Examination of both hyperplastic (data notshown) and dysplastic lesions (Figure 6, D-F) re-vealed alterations in the architecture of both epi-dermis and dermis. The keratinocytes are charac-terized by cellular hypertrophy, and increases inthe nuclear/cytoplasmic ratio. There is a strikingappearance of villous projections circumferentiallyaround the lateral and superior surfaces of thebasal and suprabasal keratinocytes (Figure 6D).Although desmosomal architecture is apparentlyunchanged in the hyperplastic and dysplastic le-sions, the hemidesmosomal junctions are remark-

able for an overall diminution in the presence ofintermediate filaments inserted into their innerplates (Figure 6, D and F). In addition, the contactsurface area of the hemidesmosomes with the un-derlying basal lamina appears diminished in theneoplastic compared with normal basal cells. Alsonotable in neoplastic keratinocytes is the ruffling ofthe plasma membrane surfaces abutting adjacentkeratinocytes (Figure 6, D and E) and of the inferioraspect in contact with the basement membrane(Figure 6F). There was also a suggestion that thebasement membrane itself was focally thinned inneoplastic compared with nontransgenic skin(compare Figure 6, C-F). Another feature is the

1t..b.

41,

1912 Coussens et alA/P December 1996, Vol. 149, No. 6

dissolution of the underlying dermal collagen net-work of the dermis in dysplastic lesions. Theseareas are typified by large scalloped areas withresidual collagen fibers (Figure 6, D and F), withan almost complete disruption of the ordered fibril-lar array evidenced in normal skin (compare Fig-ure 6, C-F). The dissolution of the dermal collagenfibrillar array and the thinning of the basementmembrane suggest an activation of matrix pro-teases concomitant with dysplasia, before malig-nant conversion (see Discussion).

Ultrastructural analysis of the low-grade squa-mous cancers confirmed the hallmark of invasivecancer, namely, focal or discrete disruptions of thebasement membrane through which malignant kera-tinocytes invade the dermis (as exemplified in Figure7A). It is noteworthy that keratinocytes found activelytraversing basal lamina apparently lose hemidesmo-somal structures while retaining desmosomal attach-ment to adjacent cells within the epidermis and der-mis (Figure 7A). In turn, malignant keratinocytesresiding within the dermis form keratin pearls andregain, or perhaps reorganize, hemidesmosomalstructures (Figure 7, B and C). Beyond that, theWDSCs demonstrate similar, if somewhat exagger-ated, aberrant features to those seen in the prema-lignant lesions (Figures 6 and 7), namely, dissolutionof extracellular matrix between adjacent keratino-cytes, between the keratinocytes and the basementmembrane, and within the underlying dermis (Figure7, A-C).

Ultrastructural analysis of the MPDSCs revealedinvading clusters of malignant squamous cells withextensive interdigitating microvilli (Figure 8, B andD). Desmosomes, although not as prevalent asthose seen in normal or antecedent lesions, remainfocally dispersed over these microvilli (Figure 8B).Within the cytoplasm, intermediate filament bundlearchitecture is aberrant, in that the bundles appearthinned, elongated, and randomly oriented withinthe cytoplasm and are typically not abuttingplasma membranes at dermo-epidermal borders(Figure 8D). Hemidesmosomal junctions are sel-dom observed within these cancers; however,when present, these structures are typically adja-cent to a poorly organized, thinned basal lamina(Figure 8, C and D). The tumor-stroma interfacesurrounding malignant cell nests within MPDSCsare more typically devoid of basal lamina. More-over, there is further disruption of the dermal col-lagen array surrounding these malignant cell nests(Figure 8, C and D).

DiscussionThis study describes and characterizes a new trans-genic mouse model of epidermal carcinogenesissharing many features with multi-stage progressionin the squamous epithelium of the human cervix inassociation with expression of high risk HPV onco-genes. The functions of the HPV16 oncogenes haverecently been reviewed.10'29 The inbred FVB/nmouse line appears to contain a dominant suscepti-bility locus, which, in the context of targeted expres-sion of the HPV16 oncogenes to the basal keratino-cytes of the epidermis, allows progression to bothWDSC and MPDSC, the latter of which metastasizeto regional lymph nodes. Transgene expression inK14-HPV16 transgenic mice is appropriately re-stricted to epithelial keratinocytes and parallels ex-pression of endogenous K14; however, an incremen-tal increase in E6/E7 mRNAs is observed at thetransition to invasive carcinoma correlating with pro-gression to malignancy. This increase does not cor-relate with proliferation indices, as both histologicaltypes of cancer are similarly high, and comparableto that of advanced dysplasias, implicating other asyet undefined changes in the progression to malig-nancy. Characterization of alterations in cell differen-tiation during neoplastic progression, revealed byexpression patterns of keratin, demonstrated 1) theactivation of K6 beginning at the hyperplastic stageand persisting thereafter, 2) loss of expression ofK10 and filaggrin in the high-grade cancers, 3) lateactivation of K13, only in higher-grade cancers, and4) the specific activation of the simple epithelial kera-tins, exemplified by K8, exclusively in the MPDSCs.Ultrastructural analyses revealed profound changesin keratinocyte shape and cell-cell and cell-matrixinteractions accompanying the early stages of car-cinogenesis. Paralleling the cell-cell and cell-matrixalterations was an extensive remodeling of the der-mal architecture, first evident at the dysplastic stage,as seen in the disruption of orderly fibrillar arrays ofcollagen fibers underlying neoplastic epidermis. Thisloosening of the dermis occurs reproducibly in dys-plastic lesions, well before the basement membraneis breached and invasion begins. Table 2 summa-rizes the parameters of this squamous carcinogen-esis pathway.

Genetic Background and Susceptibility toMalignant ConversionInfection of genital epithelia by one of the high riskHPVs is a common situation in many human popu-lations; -60% of American women are infected.5 3f


Yet only a small fraction (-3%) develop neoplasticdisease (cervical intraepithelial neoplasia to 111),and of those, approximately 20% progress to inva-sive cancer accounting for -90% of squamous car-cinomas of the cervix.5 These facts implicate addi-tional factors in cervical carcinogenesis, with geneticpredisposition being one of them.31 Targeted ex-pression of the HPV1 6 oncogenes to the epidermis inK14-HPV16 mice produces neoplasia in 100% of themice, and much as in the human condition, only-20% of affected individuals progress to malig-nancy, and only when they are at least heterozygousfor the FVB/n genotype. Thus, FVB/n appears tocontain a dominant susceptibility locus that contrib-utes to malignant conversion. The repertoire of in-bred mouse lines present a fixed set of genetic poly-morphisms that are proving to show divergentinfluences upon the penetrance both of environmen-tal agents and of genes the expression of which isinappropriately up-regulated or selectively abro-gated in transgenic and gene knockout mice, re-spectively. Among the classical examples of geneticsusceptibility is the two-step model of epidermal car-cinogenesis, in which marked inbred strain differ-ences exist; C57BL/6 are resistant, whereas BALB/cand SSIN/SENCAR are permissive for neoplastic de-velopment and papilloma formation yet resistant tomalignant conversion. More recently, FVB/n hasbeen reported to be very susceptible to chemicalcarcinogenesis protocols; approximately 10 to 15%of treated FVB/n mice progress to carcinoma.9'10These susceptibility patterns mirror the phenotypesin the K14-HPV16 mice in different genetic back-grounds; C57BL/6 mice are the most resistant, de-veloping only hyperplasia, whereas BALB/c andSSIN/SENCAR develop dysplasia and papillomas,and only FVB/n is permissive for malignant conver-sion. Taking into account the two other transgeniclines that develop squamous cell cancer in FVB/n,11 12 the data are consistent in implicating a sus-ceptibility locus for malignant conversion of the epi-dermis in this genetic background. As we haveshown remarkable histological similarities betweenthe stages in carcinogenesis in the epidermis ofK14-HPV16 mice and in the human cervix (Smith-McCune K, Zhu Y-H, Hanahan D, Arbeit J, submit-ted), there is reason to believe that the dominantsusceptibility locus in FVB/n may have a humancounterpart that influences malignant progression inHPV-infected individuals. This possibility clearly mo-tivates genetic mapping studies to localize the mu-rine locus and identify its region of synteny in thehuman.

Patterns of Keratin Expression and AberrantDifferentiation in HPV16-InducedCarcinomasTargeted expression of the HPV16 early-regiongenes to basal cells of epidermis does not result inbasal cell tumors but rather in two grades of squa-mous cell carcinoma, distinguished by varying de-grees of differentiation. This fact indicates that theHPV16 oncogenes do not block differentiation butrather elicit hyperproliferation in cells with varied ter-minal differentiation capacity. This variance is mostevident by comparing the keratin pearls character-istic of the well differentiated cancers with the quasi-differentiated cells scattered throughout the moder-ate/poorly differentiated cancers. The propensity forvaried differentiation is evident not only histologicallybut also with molecular markers. Thus, in a previousanalysis, we demonstrated that the c-myc oncogene,which is expressed in proliferating cells, and its an-tagonistic analogue mad-1, which marks differentiat-ing cells, define proliferative and differentiated re-gions in both grades of cancer.15 Now we show thatkeratin profiles reveal partial, aberrant differentiationas well and, furthermore, distinguish the two gradesof cancer. All of the neoplastic and malignant lesionsexpress K6, which is normally expressed in a subsetof basal keratinocytes and in keratinocytes tran-siently proliferating during wound healing. K6 is sim-ilarly expressed both in human cervical neoplasia32and in the two-stage chemical carcinogenesis mod-el,9 suggesting it is a general marker for continuallycycling keratinocytes. In contradistinction to neo-plastic progression induced by the two-stage chem-ical protocols or by directed expression of otheroncogenes or growth factors to the epidermis (eg,Ha-ras, v-fos, and transforming growth factor-a), thesuprabasal mucosal keratin K13 is not activatedearly in the HPV16 pathway. K13 expression, undernormal conditions, is restricted to suprabasal differ-entiating cell layers of internal stratified squamousepithelia that line the oral cavity and the upper di-gestive tract.33 Mutational activation of Ha-ras (viachemical mutagens) has been associated with in-duction of K13 in papillomas resulting from two-stage protocols.9 Instead, we see K13 as a latemarker, heterogeneously expressed in the moder-ate/poorly differentiated cancers, suggesting K13 isnot obligatory for development of squamous cellcancer; as such, the two models appear to followdistinct pathways to squamous cancer. Inferentially,one might suspect that ras mutation will prove infre-quent in the HPV16-induced cancers, and indeed,our preliminary surveys of the three murine ras alleles


support this inference (Coussens and Cronin, un-published observations).

Finally, the simple epithelial keratin K8 is uniformlyexpressed in the MPDSCs and their metastases butis not expressed in WDSCs or progenitor lesions,thus providing a marker for the more aggressivecancer. Expression of K8 and K19 in the adult istypically restricted to simple epithelial tissues suchas tubular epithelium of the kidney and intestine, aswell as complex epithelia of the lung, trachea, mam-mary gland, bladder urothelium, oviduct, uterine ep-ithelium, and mesothelium.3435 Interestingly, duringembryogenesis, these keratins are expressed in thebilayered embryonic epithelia and are localized tothe flattened upper layer of periderm cells.36 Thecells of this layer possess desmosomes but are nei-ther stratified nor terminally differentiating and, like-wise, are not associated with a basement mem-brane, reminiscent of the keratinocytes that reside inthe MPDSCs. Thus, activation of simple embryonickeratins in more aggressive grades of squamouscancers is indicative of the less differentiated char-acter of the MPDSCs, perhaps indicative of a paralleldifferentiation program, eg, retention of aberrant dif-ferentiation and partial reactivation of an embryonalprogram. Activation of K8 has also been reported ina variety of late-stage human carcinomas.32'37 41 Ofwhat consequence to the cell is the de novo expres-sion of these keratins? Casanova et a135 have dem-onstrated that transgenic expression of K8 in adultmurine skin is without apparent consequence to theepithelium. Thus, it would seem that, in the context ofa normal terminally differentiating cell, de novo ex-pression of simple epithelial keratins imposes nodominant phenotype on the cell.A more plausible explanation for K8 and/or K19

expression in malignant keratinocytes takes into ac-count changes in cell polarity as a result of alteredinteractions between the cytoskeleton and theplasma membrane or between the plasma mem-brane and the extracellular matrix. It has been re-ported that disruption of integrin binding to the ex-tracellular matrix can result in the dissociation ofcytoskeletal proteins from the plasma membrane42and that these disruptions profoundly affect cellshape and cell polarity, thus affecting differentiationand possibly differentiation programming.43 Giventhat the tumor-stroma interfaces in the MPDSCs dis-play loss of organized basal lamina, loss of hemides-mosomes, relocation of intermediate filament bun-dles away from plasma membranes, and variedexpression of intermediate filament proteins in tumorcells, it is possible that expression of the simpleepithelial keratins, in this malignant context, is more

an indicator of altered cell polarity and/or differenti-ation programming gone awry.

Dermal Remodeling Begins at the DysplasticStageInvasion into underlying stroma is a hallmark ofsquamous carcinomas, and degradation of thestromal architecture is a necessary component ofthat process. The surprising result from the ultra-structural analysis is that dissolution of the array ofcollagen fibrils in the dermis begins at the dysplas-tic stage and then persists in the carcinomas. Earlyactivation of dermal remodeling has several impli-cations. First, this capability is apparently not suf-ficient for progression to invasive cancer. How-ever, a qualification is that dysplastic samplesanalyzed above were from relatively old mice (6 to8 months), which now provides motivation for atemporal analysis to follow the appearance andprogression of dermal remodeling from the dys-plastic switch at 2 to 3 months to malignant con-version in 20% of affected mice at 7 to 12 months.Second, disruption of the stromal architecture mayserve a more immediate purpose in the dysplasticstage; one possibility is to release matrix-boundheparin-binding growth factors and consequentlyactivating angiogenesis, which is characteristic ofthe dysplastic stage44 (Smith-McCune K, Zhu Y-H,Hanahan D, Arbeit J, submitted), wherein abun-dant new capillaries grow in tight apposition to thebasement membrane underlying the dysplastic le-sion. Thus, disruption in the dermal architecture,along with the development of a new capillarynetwork, may together lay the groundwork for sub-sequent invasion by the tumor cells after progres-sion to carcinoma and breaching of the basementmembrane. The third implication of the dermal re-modeling is mechanistic, in that dissolution of col-lagen fibrils is a known consequence of activatedmatrix metalloproteinases and is associated withinvasive capabilities of both endothelial and tumorcells.45 Interestingly, D'Armiento et al46 have re-cently targeted the expression of a matrix metallo-proteinase to the suprabasal layer of murine skinepidermis. Histological examination of skin inthese mice reveal acanthosis, hyperkeratosis, andepidermal hyperplasia. Treatment of these micewith traditional two-stage carcinogenesis proto-cols resulted in a marked increase in tumor inci-dence among transgenic mice compared withcontrol littermates.46 A similar result has been re-ported by Sympson et al,47 who found that expres-


sion of stromelysin-1 in mammary epithelium alsoelicits a multi-stage pathway of malignant progres-sion resulting in mammary adenocarcinomas. Theimplications of these results are that aberrant ex-pression of proteolytic enzymes can elicit a sensi-tivity of the epithelium toward malignant conver-sion. In preliminary studies, we have detectedenhanced gelatinase A and gelatinase B mRNAand proteinase activity in biopsies of both earlyand late dysplastic skin (Coussens and Werb, un-published observations), providing motivation foradditional investigation into matrix-degrading pro-teinases and their regulation in both dysplasticand carcinoma stages.

ConclusionTaken together, the present study (summarized inTable 2) and additional analysis from this laborato-ry141544 (Smith-McCune K, Zhu Y-H, Hanahan D,Arbeit J, submitted) validate K14-HPV16 transgenicmice as a model of squamous cell carcinogenesis.The results raise an important question for futureresearch. What defines the switch from dysplasia toinvasive carcinoma? The dysplasias possess manyof the characteristics of the carcinomas: a high pro-liferation index, changes in cell shape and reduc-tions in cell contacts, dermal remodeling, and angio-genesis. Of the parameters investigated in thepresent study, transgene and keratin expressionwere altered between premalignant and malignantlesions, as well as hemidesmosomal junctions beingapparently lost in the higher-grade carcinomas. Thetime lapse between the appearance of dysplasiasand the progression to carcinomas implicates othergenetic alterations in addition to the genetic predis-position conferred by the dominant susceptibility ofthe FVB/n background, a possibility that is the sub-ject of an ongoing investigation. Given the similaritybetween this model and human cervical cancer, itcan be anticipated that the genetic and cell biolog-ical knowledge obtained through the continuinganalysis of carcinogenesis in this manipulatablemouse model will provide insight into critical featuresof HPV-associated cervical carcinogenesis and per-haps suggest novel therapies.

AcknowledgmentsWe are grateful to E. Fuchs, S. Yuspa, B. Dale, R.Kemler, and D. Roop for their generosity in providingreagents. We also thank P. Goldsmith and E.L. Gar-rett for technical advice with electron microscopy; E.

Bergsland for assistance with immunohistochemis-try; Z. Werb, E. Fuchs, and C. Damsky for discus-sion; and Z. Werb and 1. Moll for critical readings ofthe manuscript.

References

1. Ruddon R: The epidemiology of human cancer. CancerBiology. Oxford, Oxford University Press, 1987, pp25-67

2. Knudson A: Genetic events in human carcinogenesis.Origins of Human Cancer. Edited by J Brugge, T Cur-ran, E Harlow, and F McCormick. Cold Spring Harbor,NY, Cold Spring Harbor Laboratory Press, 1991, pp17-26

3. Zur Hausen H: Human papillomavirus in the pathogen-esis of anogenital cancer. Virology 1991, 184:9-13

4. Zur Hausen H: Molecular pathogenesis of cancer of thecervix and its causation by specific human papilloma-virus types. Curr Top Microbiol Immunol 1994, 186:131-156

5. Lowy D, Kirnbauer R, Schiller J: Genital human papil-lomavirus infection. Proc Natl Acad Sci USA 1994, 92:2436-2440

6. Bosch F, Munoz N, deSanjose S, Izarzugaza I, Gili M,Tormo M, Moreo P, Ascunce N, Gonzalez L, Tafur L,Kaldor J, Guerrero E, Aristizabal N, Santamaria M,Alonso deRuiz P: Risk factors for cervical cancer inColumbia and Spain. Int J Cancer 1992, 52:750-758

7. Koutsky L, Holmes K, Critchlow C, Stevens C,Paavonene J, Beckmann A, DeRouen T, Galloway D,Vernon D, Kiviat N: A cohort study of the risk of cervicalintraepithelial neoplasia grade 2 or 3 in relation to pap-illomavirus infection. N Engl J Med 1992, 327:1272-1278

8. Yuspa S: The pathogenesis of squamous cell cancer:lessons learned from studies of skin carcinogenesis.Cancer Res 1994, 54:1178-1189

9. Brown K, Balmain A: Transgenic mice and squamousmultistage skin carcinogenesis. Cancer Metastasis Rev1995, 14:113-124

10. Arbeit J: Transgenic models of epidermal neoplasiaand multi-stage carcinogenesis. Cancer Surv 1996,26:7-34

11. Leder A, Kuo A, Cardiff D, Sinn E, Leder P: v-Ha-rastransgene abrogates the initiation step in mouse skintumorigenesis: effect of phorbol esters and retinoicacid. Proc Natl Acad Sci USA 1990, 87:9178-9182

12. Lambert P, Pan H, Pitot H, Liem A, Jackson M, Griep A:Epidermal cancer associated with expression of hu-man papillomavirus type E6 and E7 oncogenes in theskin of transgenic mice. Proc Nati Acad Sci USA 1993,90:5583-5587

13. Hennings H, Glick A, Lowry D, Krsmanovic L, Sly L,Yuspa S: FVB/n mice: an inbred strain sensitive to thechemical induction of squamous cell carcinomas in theskin. Carcinogenesis 1993, 14:2353-2358


14. Arbeit J, MOnger K, Howley P, Hanahan D: Progressivesquamous epithelial neoplasia in K14-human papillo-mavirus type 16 transgenic mice. J Virol 1994, 68:358-4368

15. Hurlin P, Foley K, Ayer D, Eisenman R, Hanahan D,Arbeit J: Regulation of Myc and Mad during epidermaldifferentiation and HPV associated tumorigenesis. On-cogene 1995, 11:2487-2501

16. Garcia R, Colters M, Gown A: Analysis of proliferativegrade using anti-PCNA/cyclin monoclonal antibodiesin fixed, embedded tissues. Am J Pathol 1989, 134:733-739

17. Slaga T, Fischer S: Strain differences and solvent ef-fects in mouse skin carcinogenesis experiments usingcarcinogens, tumor initiators, and promoters. Prog ExpTumor Res 1983, 26:85-109

18. Gimenez-Conti I, Bianchi A, Fischer S, Reiners J, ContiC, Slaga T: Dissociation of sensitivities to tumor pro-

motion and progression in outbred and inbred SEN-CAR mice. Cancer Res 1992, 52:3432-2358

19. Miracco C, DeSanti M, Lio R, Biagioli M, Tosi P, Luzi P:Quantitative evaluation and ultrastructural findings can

add to the differential diagnosis between keratoacan-thoma and well differentiated squamous cell carci-noma. J Submicrosc Cytol Pathol 1992, 24:315-321

20. Greenlaugh D, Rothnagel J, Quintanilla M, Orengo C,Gagne T, Bundman D, Longley M, Roop D: Induction ofepidermal hyperplasia, hyperkeratosis, and papillomasin transgenic mice by a targeted v-Ha-ras oncogene.

Mol Carcinogen 1993, 7:99-1 1021. Broders A: Practical points on the microscopic grading

of carcinoma. NY State J Med 1932, 32:667-67122. Demeter L, Stoler M, Broker T, Chow L: Induction of

proliferating cell nuclear antigen in differentiated kera-tinocytes of human papillomavirus-infected lesions.Hum Pathol 1994, 25:343-348

23. Eckert R: Structure, function, and differentiation of thekeratinocyte. Physiol Rev 1989, 69:1316-1346

24. Lane E, Alexander C: Use of keratin antibodies in tumordiagnosis. Semin Cancer Biol 1990, 1:165-179

25. Schweizer J, Mitsuru K, Furstenberger G, Winter H:Sequential expression of mRNA-encoded keratin setsin neonatal mouse epidermis: basal cells with proper-

ties of terminally differentiating cells. Cell 1984, 37:150-170

26. Vassar R, Hutton M, Fuchs E: Transgenic overexpres-

sion of transforming growth factor-a bypasses theneed for c-Ha-ras mutations in mouse skin tumorigen-esis. Mol Cell Biol 1992, 12:4643-4653

27. Sutter C, Strickland J, Welty D, Yuspa S, Winter H,Schweizer J: v-Ha-ras-induced mouse skin papillomasexhibit aberrant expression of keratin K13 as do their7,12-dimethylbenx[a]anthracene/1 2-0-tetradecanoyl-phorbol-13-acetate-induced analogues. Mol Carcino-gen 1991, 4:467-476

28. Tennenbaum T, Yuspa S, Grover A, Castronovo V,Sobel M, Yamada Y, De Luca L: Extracellular matrixreceptors and mouse skin carcinogenesis: altered

expression linked to appearance of early markers oftumor progresssion. Cancer Res 1992, 52:2966-2976

29. Scheffner M, Romanczuk H, Munger K, Huibregtse J,Mietz J, Howley P: Functions of human papillomavirusproteins. Curr Top Microbiol Immunol 1994, 186:83-99

30. Schiffman M: Epidemiology of cervical human papillo-maviruses. Curr Top Microbiol Immunol 1994, 186:55-81

31. Apple R, Erlich H, Klitz W, Manos M, Becker T, WheelerC: HLA DR-DQ disease associations with cervical car-cinoma show papillomavirus-type specificity. NatureGenet 1994, 6:157-162

32. Smedts F, Rameaekers F, Troyanovsky S, PrusczynskiM, Link M, Lane B, Leigh I, Schijf C, Vooijs P: Keratinexpression in cervical cancer. Am J Pathol 1992, 141:497-511

33. Kuruc, N, Leube, R, Moll I, Bader B, Franke W:Synthesis of cytokeratin 13, a component of charac-teristic of internal stratified epithelia, is not inducedin human epidermal tumors. Differentiation 1989, 42:111-1 23

34. Leube R, Bosch F, Romano V, Zimbelmann R, Hofler H,Franke W: Cytokeratin expression in simple epithelia.Differentiation 1986, 33:69-85

35. Casanova L, Bravo A, Were F, Ramirez A, Jorcano J,Vidal M: Tissue-specific and efficient expression of thehuman simple epithelial keratin 8 gene in transgenicmice. J Cell Sci 1995, 108:811-820

36. Byrne C, Tainsky M, Fuchs E: Programming gene ex-pression in developing epidermis. Development 1994,120:2369-2383

37. Moll R, Franke W, Schiller D, Geeger B, Krepler R: Thecatalog of human cytokeratins: patterns of expressionin normal epithelia, tumors, and cultured cells. Cell1982, 31:11-24

38. Quinlan R, Schiller D, Atzfeld S, Schistatter T, Moll R,Jorcano J, Mogin T, Franke W: Patterns of expressionand organization of cytokeratin intermediate filaments.Ann NY Acad Sci 1985, 455:282-306

39. Markey A, Lane E, Churchill L, MacDonald D, Leigh I:Expression of simple epithelial keratins 8 and 18 in epi-dermal neoplasia. Invest Dermatol 1991, 97:763-770

40. Suo Z, Holm R, Nesland J: Squamous cell carcinomas:an immunohistochemical and ultrastructural study. An-ticancer Res 1992, 12:2025-2032

41. Schaafsma H, VanDer Velden L, Manni J, Link M, RuitterD, Ramaeers F: Increased expression of cytokeratins 18and vimentin in the invasive front of mucosal squamouscell carcinoma. Am J Pathol 1993, 170:77-86

42. Luna E, Hitt A: Cytoskeleton-plasma membrane inter-actions. Science 1992, 258:955-964

43. Eaton S, Simons K: Apical, basal, and lateral cues forepithelial polarization. Cell 1995, 82:5-8

44. Arbeit J, Olson D, Hanahan D: Upregulation of fibro-blast growth factors and their receptors during multi-stage epidermal carcinogenesis in K14-HPV16 trans-genic mice. Oncogene (in press)


45. Liotta L, Stetler-Stevenson W, Steeg P: Cancer invasionand metastasis: positive and negative regulatory ele-ments. Cancer Invest 1991, 9:543-551

46. D'Armiento J, DiColandrea T, Dalal S, Okada Y, HuangM-T, Conney A, Chada K: Collagenase expression intransgenic mouse skin causes hyperkeratosis and ac-

anthosis and increases susceptibility to tumorigenesis.Mol Cell Biol 1995, 15:5732-5739

47. Sympson C, Talhouk R, Bissell M, Werb Z: The role ofmetalloproteinases and their inhibitors in regulatingmammary epithelial morphology and function in vivo.Perspect Drug Discovery Design 1994, 2:401-411

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