The Pathogenesis of Squamous Cell Cancer: Lessons Learned ... · derstanding molecular aspects of...

(CANCER RESEARCH 54, 1178-1189, March 1. 1W4]

Special Lecture

The Pathogenesis of Squamous Cell Cancer: Lessons Learned from Studies of SkinCarcinogenesisâ€”Thirty-third G. H. A. Clowes Memorial Award Lecture1

Stuart H. Yuspa

Laboratory of Cellular Carcinogenesi.s and Tumor Promotion, National Cancer Institute, Bethesdu, Maryland 20892

Abstract

The multistage nature of cancer pathogenesis was first defined over 50years ago by the sequential topical application of chemical agents to mouseskin. Since then, the skin model has provided remarkable insights into thebiology, biochemistry, pharmacology, and genetics of carcinogenesis. Discoveries from studies of mouse skin have proved to be landmarks in cancerresearch including: the binding of carcinogens to DNA; the monoclonalorigin of benign and malignant tumors; the powerful tumor-promoting

action of phorbol esters; the antipromoting potency of retinoids and steroids; the modifying role of age, caloric intake, and specific dietary constituents on cancer induction; the variable risk for benign tumors toprogress to cancer; and the requirement for multiple genetic changes inmalignant conversion. Many of these concepts are now widely applied tothe interpretation of specific molecular discoveries both in simple experimental systems and in human cancers, but the power of this quantitative,multistage skin carcinogenesis model has made these assessments possible.It has also provided the separation of mechanistically distinct stages incancer pathogenesis: initiation; promotion; premalignant progression;and malignant conversion. This paper will review our current understanding of the genetic, biological, and biochemical alterations that contributeto the evolution of each of these stages in skin carcinogenesis. These newinsights provide an opportunity to replace the traditional operational-

based schemata defining the process of carcinogenesis with a workingmodel designed around functional alterations in neoplastic cells.

Delivering the Clowes Lecture in 1993 was an extraordinary experience for me, and I would like to thank the selection committee, mymentors, and colleagues for making this possible. When I was asecond year medical student, HarÃanFirminger, the chairman of thePathology Department, introduced me to the excitement and intellectual challenge of cancer research and made it possible for me toexperience the thrill of scientific discovery. Richard Bates gave me anopportunity to join the National Cancer Institute, introduced me to thepower and versatility of the skin carcinogenesis model, trained me inscientific methods, and provided me with a chance to prove myindependence. Umberto Saffiotti fostered a national carcinogenesiscollaborative program in the 1970s that was extremely important inthe scientific development of the field, and provided a forum andsupport for a cadre of young investigators who became major contributors to carcinogenesis research. Isaac Berenblum, Philippe Shu-

bik, and Roswell Boutwell were pioneers in skin carcinogenesis research, and their extraordinary contributions have inspired me andconceptualized the process of carcinogenesis for all of us.

My work and my life experience have been enriched by long-term

associations with a number of senior colleagues. Henry Hennings andI have worked together throughout our careers, and he shares thecredit for many of our observations on multistage carcinogenesis.Miriam Poirier and I, in collaboration with I. Bernard Weinstein,initiated the first studies on detection of carcinogen-DNA adducts by

Received 1/5/94; accepted 1/5/94.1 Presented at the 84th Annual Meeting of the Americal Association for Cancer Re

search, May 20, 1993, Orlando, PL.

immunological methods (1). These experiments contributed to theemergence of biochemical epidemiology as a new field in cancerresearch and remain an important component of our research programunder the leadership of Dr. Poirier. Ulrike Lichti and Jim Stricklandhave made significant contributions to our biochemical and biologicalstudies and have been dedicated and diligent colleagues for manyyears. Peter Blumberg and Luigi De Luca are Section Heads in theLaboratory of Cellular Carcinogenesis and Tumor Promotion. Individually, they have made major contributions to cancer research. I ampersonally grateful that they have shared their insights and expertisewith me and members of my group and have helped to create an openand collÃ©gialatmosphere in the laboratory that has enhanced ourprogress tremendously. A number of outstanding students, postdoctoral fellows, visiting scientists, and collaborators have made valuablecontributions to our research over the years, and I would like to thankthem as well. My colleagues and I have been privileged to work at theNational Cancer Institute, entrusted with support to pursue our visionsin a stable research environment. I acknowledge the unique opportunity this has been for us, and I hope the recognition provided by thisaward is some justification for this trust.

Advances in understanding cancer pathogenesis have come at anastounding pace in the last decade. Much of this progress is due to theintroduction of molecular biology and new methods for cell culture astools for cancer research. Experimentally derived molecular information is integrated with the process of carcinogenesis by tests in animalmodel systems that recapitulate the pathogenesis of cancer in humantissues. This is exemplified by the induction of squamous cell cancerson mouse skin by the sequential application of chemical agents. Theconceptual framework established for the biology of carcinogenesis inepithelial tissues evolved from over 50 years of research on this targetsite. These studies indicated that progressive stages were predictableduring the clonal evolution of a normal epidermal cell through thebenign squamous papilloma stage into a squamous cell carcinoma,and reproducible genetic and epigenetic events contributed to thesechanges (2). The development of other tissue-targeted experimentalcarcinogenesis models revealed that cancers of internal lining epithe-

lia, particularly the aerodigestive and genitourinary systems, followedthe predictable pattern of skin carcinogenesis (3). Thus, experimentalskin carcinogenesis serves as a prototype for understanding the pathogenesis of squamous cell cancer.

In parallel with cancer research in general, recent progress in understanding molecular aspects of skin carcinogenesis has been remarkably rapid. This progress was facilitated by major advances inunderstanding both the regulation of growth and differentiation of theskin keratinocyte and the pathogenesis of diseases of the epidermis(4, 5). The development of methods for the cultivation of functionallyintact epidermal cells from mice and humans, the application of techniques to graft experimentally altered keratinocytes in vivo, and thecloning and characterization of keratinocyte-specific genes have fu

eled these advances. In this paper I will review certain aspects ofepidermal physiology that are relevant for understanding the pathogenesis of squamous cancer and provide an overview of current con-

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PATHOGENESIS OF SQUAMOUS CELL CANCER

Marker CompartmentGrowth Factorsand Receptors Gradients

Fig. 1. Schematic presentation of the stratifiedepidermis depicting the compartments of maturation of keratinocytes, markers expressed in eachcompartment, and biochemical pathways whichregulate this phenotype. The scheme is compiledfrom a number of studies conducted on mouse andhuman skin in vivo or from cultured keratinocytes.FGFR2, fibroblast growth factor receptor 2; EGFR,epidermal growth factor receptor; PI, phosphati-dylinosito!; /V, nucleus.

LoncnnFilaggrinTransglutaminase

Keratin 1Keratin 10

Keratin 5Keratin 14

BasementMembrane

cepts of skin carcinogenesis derived from our studies and those ofothers in this field. With this foundation, I will briefly summarizeresults of four experimental approaches we are currently taking tofurther elucidate the biochemical basis for a particular stage of skincarcinogenesis. I firmly believe that understanding the biochemistryof carcinogenesis will drive the design of novel strategies for earlydiagnosis, prevention, and treatment of premalignant and malignantsquamous cell tumors.

The Regulation of Epidermal Growth and Differentiation

The stratified epidermis displays a highly coordinated program ofsequential changes in gene expression that are coincident with thephenotypic evolution from a proliferating basal cell to the mature,nonviable squame (Fig. 1) (6). Basal cells contact an epidermis-

specific basement membrane and express characteristic markers including cytokeratins 5 and 14. The migration of basal cells into themore superficial spinous layer is associated with the loss of prolifera-

tive capability, suppression of cytokeratin 5 and cytokeratin 14 geneexpression, and the up-regulation of transcripts for Kl2 and K10,

markers for an early stage of keratinocyte differentiation. Upon furthermaturation and migration into the granular cell compartment, Kl andK10 are suppressed and loricrin, filaggrin, and keratinocyte transglu-taminase are up-regulated. These unique proteins are essential com

ponents for the terminal phase of maturation. The activation of keratinocyte transglutaminase in granular cells cross-links loricrin and

other substrates to form the permeable, rigid cornified envelope thatreplaces the plasma membrane in cells of the cornified layer. Cornified envelope formation is associated with the loss of intracellularorganelles and programmed cell death of mature squames (7). Whilebasal cells and putative keratinocyte stem cells (8, 9) may be mostrelevant to carcinogenesis, the mature keratinocyte responsible for theskin barrier function is the nonviable squame consisting of cytokeratinbundles in a filaggrin matrix contained within the cornified envelope.It is now recognized that important immunological and endocrinefunctions are performed by skin keratinocytes during maturation (10),and disturbances in these functions may contribute to the developmentof cutaneous and systemic diseases.

Compartmentalization and differential expression of signaling molecules in skin and cultured keratinocytes have provided clues to the

2The abbreviations used are: Kl, cytokeratin 1; KIO, cytokeratin 10; TGF, transform

ing growth factor; EOF, epidermal growth factor; FGF, fibroblastic growth factor; PLC,phospholipase C; PKC, protein kinase C.

regulatory pathways which are involved in epidermal differentiation(Fig. 1). The discovery that mouse and human basal keratinocyteswith a high growth fraction can be cultured in medium with a Ca2+concentration of 0.05 mM, whereas medium with Ca2+ greater than

0.1 mMinduces terminal differentiation (11-13), has greatly facilitated

the analysis of the regulation of keratinocyte growth and differentiation. In culture, Ca2+-induced maturing keratinocytes cease prolifera

tion, stratify, express the suprabasal markers of differentiation, activate keratinocyte transglutaminase, cornify, and desquamate as a sheetof mature squames (11, 14). As is the case in vitro, a principalregulator for keratinocyte maturation in vivo is a gradient of extracellular and intracellular calcium across the epidermis that is low in thebasal cell compartment and high in the granular cell layer (15, 16). Anopposing vitamin A gradient may also contribute to the regulation ofkeratinocyte gene expression (17). Restraint on basal cell proliferationis contributed by autocrine expression and response of basal cells tothe growth inhibitor TGFÃŸl, while restraint on growth of suprabasalcells is contributed by autocrine expression and response of suprabasalcells to the growth inhibitor TGFÃŸ2(18). Positive growth control ismediated by local expression of TGFa and several other EOF receptorligands, but the proliferation response is confined to the basal cellcompartment by the down-regulation of the EOF receptor in su

prabasal cells (19, 20). The epidermis contains abundant FGF receptors of the FGFR2 class, a splice variant that has a high affinity forkeratinocyte growth factor synthesized by cells of the dermis, and thiscomplex may contribute to epidermal morphogenesis and to thewound healing response (21, 22). The interaction of basal cells withthe basement membrane is mediated by the expression of specificintegrins, particularly the a6ÃŸ4integrin complex that is polarized onthe basal surface of basal cells (23-26). Other integrins mediate cell-cell and cell-matrix interactions to maintain the stratified phenotype(24, 25). Transduction of the Ca2+ signal for maturation is associated

with increased activity of PLC (27, 28). In basal cells PLCyl, -y2,and -ÃŸ3are detected; in addition, differentiating keratinocytes expressPLC-S1 (28). The associated rise in PLC-generated diacyglycerol may

contribute to the activation of PKC that is essential to the terminalphase of keratinocyte maturation (27, 29-31). Four isoforms of PKC

(a, o, e, Â£)are present in keratinocytes throughout the mouse epidermis, and PKCÃ¯)is also expressed in the granular cell compartment (32,33). PKC activity is essential to down-regulate Kl and KIO andup-regulate loricrin, filaggrin, and transglutaminase during the spi

nous to granular cell transition (34). The importance of PKC, both inregulating keratinocyte differentiation and in skin tumor promotion

1179




(35-37), indicates that alterations in this enzyme family are likely to

contribute to epidermal carcinogcnesis.

Operational Stages in the Development of Squarnous CellCarcinoma of Mouse Skin

Operational definitions based on tumor induction protocols in micehave been useful in dissecting the complex process of skin cancerpathogenesis (Fig. 2), and these definitions have been adopted toanalyze stages in carcinogenesis for most epithelial neoplasms ofhumans and experimental animals (3, 38, 39). Initiation is carcinogeninduced and mutational and produces a subtle change in keratinocytephenotype, unrecognizable in the context of the intact epidermis.

Initiated keratinocytes in vitro display an altered response to signalsfor terminal differentiation, a characteristic which provides a selectivegrowth advantage under culture conditions favoring differentiation(40-42). Exploitation of this difference has been particularly helpful

in isolating initiated keratinocytes of mouse and human origin in vitro(43-46).

Important insights into the genetic alterations associated with theinitiated phenotype have emerged from the identification of mutationsin the c-rasf1" gene associated with papilloma formation (47-49).These genetic analyses indicated that; (a) c-rasf" gene mutations

were frequently heterozygous in papillomas and could be detected ininitiated skin prior to the emergence of tumors (50); (b) the initiatingagent determined the existence, nature, and site of the c-ras"" muta

tion (51, 52); and (c) many human benign and malignant skin tumors,presumably induced by UV light, also contain ras gene mutations(53-58).

Application of tumor promoters to initiated epidermis causes theselective clonal outgrowth of initiated cells to produce multiple benignsquamous cell papillomas, each representing an expanded clone ofinitiated cells (59-61). Thus exogenous exposure to initiators and

promoters is required for the earliest premalignant component of skincarcinogenesis, as is the case for the early stages of tumorigenesis inmost internal tissues as well. In internal tissues, promoting exposuresmay be endogenous to the organism but exogenous to the target tissue,such as tumor promotion mediated by hormones or growth factors. Inskin carcinogenesis, selective clonal expansion of the initiated population may result from differential sensitivity of initiated and normalkeratinocytes to either cytotoxic (62) or growth-stimulatory (63) ef

fects of exogenous tumor promoters. The most potent exogenous skintumor promoters are the phorbol esters which activate PKC and accelerate terminal differentiation of normal keratinocytes (34, 64, 65).Initiated keratinocytes are resistant to terminal differentiation inducedby activators of PKC (45, 66, 67), and the differential response ofnormal and initiated cells favors the growth of the neoplastic sub-

population, enhancing clonal outgrowth and producing papillomas.Therefore, the process of tumor promotion by phorbol esters in vivorecapitulates the clonal selection of initiated cells by differentiation-inducing agents in keratinocyte culture (67-69).

In skin, squamous papillomas are characterized by a high rate ofproliferation and by delayed expression of differentiation markers,properties which are analogous to the phenotype of individual initiatedcells in vitro (18, 70). The mechanism of exogenous promotion islikely to be epigenetic in most cases since (a) papillomas are diploidwhen they first emerge (71, 72), (b) a single genetic change in normalkeratinocytes is sufficient to produce a papilloma phenotype (73-75),

and (c) most promoting agents are not mutagens (2). In the absence ofexposure to exogenous tumor promoters, initiated skin rarely developstumors. Thus, exogenous promotion in general is a rate-limiting early

event in carcinogenesis, but the actual tumor yield is determined byadditional hereditary factors influencing both initiation and promotion(76, 77). The genes which determine hereditary susceptibility to initiators and promoters in mouse skin remain largely to be defined, andsusceptibility appears to be multigenic (78, 79).

Premalignant progression of a papilloma to a carcinoma in mouseskin is generally a spontaneous process that is not enhanced by mostexogenous tumor promoters (80, 81). Genetic studies indicate thatnonrandom, sequential chromosomal aberrations are associated withpremalignant progression of mouse skin papillomas; particularlyprominent are trisomies of chromosomes 6 and 7 (72, 82-85). Pre

malignant progression and malignant conversion can be enhanced andaccelerated by exposing animals bearing papillomas to a mutagen (80,81, 86), further supporting a genetic basis for prcmalignant progression. This stage of cancer pathogenesis constitutes the major time-

dependent component of carcinogenesis and must involve repeatedepisodes of cell selection since modal dominance of a specific chromosomal aberration indicates clonal outgrowth. Thus, at least onefunction of the relevant genetic events occurring during premalignantprogression must result in a growth advantage for the affected cell.The genetic changes which contribute to premalignant progression donot accumulate with sufficient frequency in the few single initiatedcells in nonpromoted skin to produce a significant risk for cancerdevelopment. Furthermore, genetic changes occurring spontaneouslyin noninitiated keratinocytes will be inconsequential unless the terminal differentiation program of the cell is altered. However, persistentexogenous exposures of initiated skin to carcinogens (complete carcinogenesis) or clonal expansion of the initiated population inducedby tumor promoters can increase the probability for the occurrence ofrelevant additional genetic changes required in the initiated populationfor premalignant progression. Subpopulations of papillomas with ahigh risk for spontaneous premalignant progression have been identified (87-90). High risk papillomas are also more sensitive to muta-gen-induced progression (88). Thus, the factors which determine risk

for premalignant progression and malignant conversion must increasesusceptibility to spontaneous or induced genetic changes. As for earlier events in carcinogenesis, hereditary background influences therisk for prcmalignant progression, and the genes involved are distinctfrom those responsible for risks to initiators and promoters (91, 92).

Fig. 2. Operational stages in experimental skincarcinogenesis. Each stage is defined by the biological consequences of a specific experimentalprotocol. This scheme serves as a framework formolecular analysis of multistage carcinogenesis.See text and Ref. 2 for details.

Exogenous Exposures Spontaneous

Initiation(Mutation)

Premalignant ProgressionPromotion (Chromosomal Changes,

(Epigenetic) Clonal Outgrowth)Malignant Conversion(Genetic, Epigenetic)

InitiatedCell1MILClonal

ExpansionPromoter

DependentEarly

PapillomabnPromoter

IndependentProgressing

PapillomaCarcinoma1180




Malignant conversion of benign tumors is a relatively rare occurrence; less than 5% of squamous papillomas spontaneously convertto clinically obvious cancers (80, 81). Squamous papilloma celllines in vitro can be converted to squamous carcinoma cells by introducing specific oncogenes (Table 1), supporting a genetic basis forconversion and distinguishing this as a discrete step in cancer patho-genesis. Changes in two cellular genes, c-rasfl" and p53, have been

closely identified with malignant conversion of skin tumors. Themutated ras"" oncogene, which is heterozygous in papillomas, is

frequently homozygous in carcinomas (84, 93), and an oncogenicras"" gene can cause malignant conversion of papilloma cells witha heterozygous c-rasfj" gene mutation (52, 94). This suggests thatras"" gene dosage is important in determining the neoplastic pheno-

type. Mutations in the p53 tumor suppressor gene are rarely foundin chemically induced papillomas but are frequently detected insquamous carcinomas, particularly those induced by benzo(a)pyrene(95-98). In addition, topical administration of initiators and promoters

to mice lacking an intact p53 gene results in the rapid progressionof papillomas to the carcinoma stage (99). Mutations in the p53gene are frequently detected in human squamous and basal cellcarcinomas of the skin (100-103) and in mouse skin carcinomas

induced by UV light (97, 104). The most frequent mutagenic changeis a Câ€”>Ttransition commonly found at C-C dinucleotides (100, 101,

103, 105) suggesting that UV light causes these mutations directly.The high frequency of specific mutations indicates that the mutationalchange in p53 produces a growth advantage for the incipient neoplastic cells in UV-induced skin cancer. While these genetic changes are

associated with malignant conversion, many progressing papillomasexpress markers of the malignant phenotype in a multifocal pattern(23, 82), a finding inconsistent with a mutational basis for conversion.Furthermore, permanent malignant conversion can be induced byagents that are not mutagenic in epidermal-derived mouse JB-6 clones

(106). It therefore seems probable that multiple mechanisms mayinfluence this late stage of carcinogenesis.

My laboratory has used a combined in vivo (mouse skin) andin vitro (cultured keratinocytes) approach to identify metabolic pathways involved in these predictable stages of skin carcinogenesis. Ingeneral, in vivo studies have defined new biological properties associated with a particular stage of skin carcinogenesis, and wherepossible we have attempted to confirm an in vitro discovery by testsin animals. Using cultured keratinocytes, we have been able to identify signal transduction pathways, growth factors, and transcriptionregulators that appear to be significantly and causally involved inproducing a particular neoplastic phenotype in epidermis. Examplesof these approaches and results are briefly described in the next foursections.

Table 1 Oncogenes tested for converting action in benign neoplastic keratinocytes

Cultured papilloma cells were transfected with expression vectors encoding theparticular oncogene and a selectable marker, and selected transfcctants were testedin vivo.

Table 2 Comparison of biochemical changes induced in cultured mouse keratinocvtesby treatment with 12-0-letradecanoylphorbol-13-acelate (TPA) iir by transduction of

the v-ras11" oncogene in a replication-defective retroviral vector

OncogeneHu-c-raj"Â°-(mul)Â°\f-rasiiav-fosHVV

(fos)c-mycEIATGFaÃŸ-Actin

(mut)p53

(mut)neuv

-junRef.5294,

17213813813813863a172172aCell

linePA-PE308,

pli308.SP-308,SP-308.SP-308,SP-308.SP-308,SP-pll7pll7308,

SP-!

ResultCarcinoma7

CarcinomaCarcinomaCarcinomaPapillomaPapillomaPapillomaPapillomaPapillomaPapillomaPapilloma

Phenotype0.05

mMCa2+Keratins

5 and 14I25I-EGF binding

TGFaTransglutaminase

Ornithine decarboxylaseIntercellularcommunication>0.1

mMCa-*Keratins

1 and10Loricrinand filaggrin

TransglutaminaseTerminal differentiationv

-/W"Unchanged

DecreaseIncreaseIncrease

IncreaseDecreaseDecreaseIncrease

DecreaseDecreaseTPAUnchanged

DecreaseIncreaseIncrease

IncreaseDecreaseDecreaseIncrease

IncreaseIncrease

"Unpublished results.

The Biochemical Basis for the Phenotype of InitiatedKeratinocytes: Mutations of the c-ras"" Gene and

Alterations in Protein Kinase C

The analysis of the biological and biochemical basis for initiation inskin carcinogenesis was enhanced by the development of methods toselect keratinocytes with a carcinogen-altered phenotype (11, 40-42).A subpopulation of keratinocytes isolated from carcinogen-initiated

skin or normal basal keratinocytes exposed to carcinogens in vitroresists the Ca2+ signal for terminal differentiation and evolves as fociwhich continue to grow in medium with >0.1 mM Ca2 +. These focidisplay properties consistent with the initiated phenotype. "Ca2+-resistant" foci (a) develop from keratinocytes initiated in vivo or in

vitro (40, 41, 107), (b) are more frequent with exposure to stronginitiators and higher initiator doses (42), (c) develop even if a delay of10 weeks is interposed between initiation in vivo and Ca2+ selection

in vitro (107), and (d) develop into cell lines which produce papillomas or carcinomas when grafted to nude mice (44, 46). These celllines have provided a crucial component for the analysis of the biochemistry of initiation.

Relating the oncogenic activation of the c-rasfi" gene to the initia

tion of skin carcinogenesis has facilitated an in vitro analysis of themetabolic basis for the initiated phenotype. When the v-ras"" onco

gene is introduced into cultured normal mouse keratinocytes, recipientcells form papillomas when grafted to nude mice, indicating that thissingle genetic change is sufficient to produce the initiated phenotype(73). When an oncogenic ras gene is introduced into normal humankeratinocytes, recipient cells become growth factor independent inculture and aberrantly express keratin 19 in vivo but do not producetumors (108). In contrast, when an oncogenic ras gene is introducedinto an immortalized human keratinocyte cell line, recipient cellsproduce either benign or malignant squamous tumors in vivo suggesting that other changes, in addition to ras gene mutations, are requiredto transform human keratinocytes (109). In vitro, mouse keratinocytesexpressing v-ras"" have a high proliferation rate and fail to terminallydifferentiate in response to medium with >0.1 mM Ca2+ (110-113),although Ca2+ metabolism is not altered in these cells (114). Thehyperproliferation induced by v-ras"" transduction in mouse kera

tinocytes is caused by overexpression and autocrine response toTGFa, a growth factor known to be elevated in papillomas (115-117).

When cultured as basal cells in 0.05 mM Ca2+ medium, v-ras""

keratinocytes express a phenotype with striking similarity to normalkeratinocytes exposed to activators of protein kinase C, such as phor-bol esters or diacylglycerol (34, 66, 113, 118) (Table 2), and diacyl-glycerol levels are constitutively elevated in v-ras"" keratinocytes(115). When v-ras"" keratinocytes are exposed to medium with >0.1mM Ca2+, they also share features with phorbol ester-treated epider

mal cells (Table 2). For example, cytokeratins Kl and K10 are notinduced while loricrin and filaggrin are overexpressed (34). However,

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PATHOOENESIS OF SOUAMOUS CELL CANCER

PKCÃ”Immunoprecipitates

mi(O

E ^*â€” (0o VZ >

Antip-tyr -PKCÃ”

AntiPKC5

Fig. 3. Protein immunoblot of PKC8 immunoprecipitated from control and \-ras""

kcratinocytes. Treatment, lysis, and immunoprecipitation conditions; gel electrophoresis;and antibodies are described in Ref. 120. Top, immunoblot using antiphosphotyrosineantibody: boitim. filter reblotted with a PKCS antibody to show that the isoform is presentand equal in both lanes.

transglutaminase mRNA decreases in Ca2+-shifted v-ras"" keratino-cytes, and terminal differentiation is blocked. In fact, v-ras"" kera-tinocytes maintained in >0.1 ITIMCa2 + medium resume a basal cellphenotype when returned to 0.05 rtiMCa2+ medium whereas control

keratinocytes are irreversibly committed to terminal differentiationonce exposed to >0.1 HIMCa2+ medium (111). An essential role forPKC activation in the v-ras"" keratinocyte phenotype is supported by

studies with the functional PKC inhibitor, bryostatin (119). In thepresence of bryostatin, Kl and K10 are expressed and filaggrin, loric-rin, and transglutaminase are suppressed in either v-ras"" keratino

cytes or phorbol ester-treated normal keratinocytes. Furthermore,Ca2+-dependent PKC activity is elevated in lysates from v-ras""keratinocytes, but Ca2 +-independent PKC activity is reduced whencompared to normal keratinocyte lysates.3 This suggests that qualita

tive or quantitative changes in keratinocyte PKC isoforms might be aconsequence of v-ras"" oncogene transduction.

Cultured normal and neoplastic mouse keratinocytes express 5 isoforms of PKC (a, o, e, TJ,Ã‡)(32). While the amount of each isoformis similar in normal and v-ras"" keratinocytes (120), higher activity ofPKCa must account for the greater Ca2 +-dependent activity in v-ras"" keratinocytes, since this is the only Ca2+-dependent isoform

detected in these cells. This isoform may be responsible for the alteration in cytokeratin profile in v-ras ""keratinocytes. The reduction ofCa2+-independent PKC activity in v-ras"" keratinocyte lysates may

be linked to a specific tyrosine phosphorylation of PKCÃ´ (Fig. 3)(120). An identical modification is found in neoplastic keratinocytesinitiated by carcinogens which induce mutations in the c-ras"" gene

(120). Modified PKC8 is not stimulated by phorbol esters and has lowconstitutive activity when assayed in immunoprecipitates (120). Thisstructural and functional change in PKCÃ´may be associated with theblock in Ca2+-induced terminal differentiation characteristic of initiation of keratinocytes by a ras"" oncogene. Support for this concept

comes from studies of staurosporine, an inhibitor of tyrosine andserine kinases, which prevents the tyrosine phosphorylation of PKCS

(or causes phosphate removal) in v-ras"" and carcinogen-initiated

keratinocytes and induces terminal differentiation at the same concentrations (120, 121).

From these studies we conclude that alterations in PKC are essentialfor mediating the initiated phenotype in squamous tumors of skintransformed via activation of the ras"" gene. Differential modification

of isoforms, particularly activation of PKCa and inhibition of PKCS,produce keratinocytes with enhanced proliferative capacity and reduced sensitivity to signals for terminal differentiation. These studiesimplicate PKCS as an essential mediator of the terminal steps inepidermal maturation, particularly the induction and activation oftransglutaminase and the formation of cornified envelopes, functionswhich are blocked in initiated keratinocytes. Since tyrosine phosphorylation of PKCÃ´ is associated with the reduction in its kinaseactivity, identification of the responsible tyrosine kinase may providea novel target for the design of reagents to reverse the earliest stagesof squamous neoplasia. In vivo studies, in which staurosporine causedirreversible papilloma regression, suggest this approach has somemerit (122).

The TGFÃŸFamily of Growth Inhibitors Is a Suppressor ofPremalignant Progression

The hyperproliferative phenotype of squamous papillomas couldcontribute to the spontaneous accumulation of chromosomal changes,including trisomies, recombinations, and deletions, which have beenassociated with premalignant progression in skin carcinogenesis (83-

85). Papillomas at high risk for premalignant progression can beselectively induced after initiation by limited exposure to strong promoters or prolonged exposure to weak promoters (90). Thus, theanalysis of this class of benign squamous tumors should yield information regarding factors that influence premalignant progression.High risk papillomas erupt early, grow large and do not regress whenpromotion is stopped, suggesting that tumor cell growth may beespecially enhanced in this group. Phenotypic markers distinguishhigh and low risk papillomas at the time when they are first clinicallydetected (8-11 weeks of tumor promotion) (18, 123) (Table 3; Fig. 4).

In contrast to low risk papillomas, high risk tumors do not expresscytokeratin Kl but instead synthesize keratin K13. The a,,ÃŸ4integrinis detected in suprabasal and basal cells in high risk papillomas whileexpression is polarized to the basal surface of basal cells in normalepidermis and low risk papillomas (23, 123, 124). These early changesin high risk papillomas are characteristic of most papillomas in muchlater stages of premalignant progression (23, 125). High risk papillomas also lack immunodetectable TGFÃŸl and TGFÃŸ2,whereas TGFÃŸlin low risk papillomas and normal epidermis is localized to the basalcell compartment and TGFÃŸ2is detected in the suprabasal compartment (Table 3) (18, 126). Neither papilloma subtype expresses markers associated with the malignant phenotype such as keratin 8 or7-glutamyltranspeptidase (72, 127, 128). These distinct phenotypic

Table 3 Markers distinguish high risk from low risk papillomas at ihc first clinicalsign of tumor formation

Papilloma risk type

Marker"High

(8wk)Low

(11 wk)

Keratin IKeratin 13Suprabasal a,,ÃŸ.jTGFÃŸlTGFÃŸ2TGFaKeratin 8â€¢¿�y-Glutamyltranspeptidase

3 A. Dlugosz, C. Chen, E. K. Williams, and S. H. Yuspa, manuscript in preparation." Tumors were induced and markers were detected by immunohistochemistry or his-

tochemistry (y-glutamyl transpeptidase) as described in Ref. 123.

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a6ÃŸ4 KERATIN 1 KERATIN 13 KERATIN 8

LOW RISKPAPILLOMA

HIGH RISKPAPILLOMA

CARCINOMA

IFig. 4. Immunohistochemical markers distinguish high risk and low risk papillomas. Papillomas and carcinomas were generated on the skin of SENCAR mice by initiation with 10

fig of 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate (2 fig, once weekly) to produce papillomas at low risk for malignant progression ormezerein (4 fig. twice weekly) to produce papillomas at high risk for malignant progression (90). High risk papillomas were excised at 8 weeks when Ihey were first detected clinicallyand only about 1-2 mm in diameter. Low risk papillomas were excised at II weeks, also at first detection. Serial frozen sections were processed for immunochemislry using specificantibodies for the aâ€žintegrin subunit, keratin 1, keratin 13. and keratin 8. Reagents and procedures for frozen sections and immunochemistry have been described previously (23). Eachpanel represents a 5-/im frozen section cut through the entire tumor.

differences in papillomas detected at the earliest sign of benign tumorformation suggest that each tumor class is derived from a distinctpopulation of initiated cells, representing either different target cells inthe epidermis or initiated keratinocytes with distinct genetic or epi-genetic alterations (129). While differences in mutations at the c-ras1'"

alÃeledo not distinguish these papilloma subtypes (89), the dose ofinitiator may influence the frequency of each tumor type (130).

The TGFÃŸsare potent growth inhibitors for cultured keratinocytes,and TGFÃŸlregulates basal cell proliferation in vivo since the absenceof TGFÃŸl in normal epidermis of TGFÃŸl "knockout" mice causes

basal cell hyperproliferation (18).TGFÃŸl and TGFÃŸ2mRNA is detected in high risk papillomas by

in situ hybridization, indicating that posttranscriptional changes eitherin the translation of the mRNA. stability of the peptides, or extracellular secretion, processing, or binding of the peptides account for theabsence of the growth inhibitors (18, 126). The absence of detectableTGFÃŸl and ÃŸ2peptides in most high risk papillomas could accountfor their more rapid emergence, larger size, or lack of regression andcould enhance the accumulation of genetic or chromosomal changes ifthese tumors were particularly hyperproliferative. Studies using bro-modeoxyuridine labeling prior to sacrifice of tumor-bearing animalsindicate that both low risk papillomas and phorbol ester-treated epi

dermis have labeling indices about 4 times greater than that of normalskin, but labeled cells are confined to the basal cell compartment(Table 4). High risk papillomas, devoid of TGFÃŸland TGFÃŸ2,havea substantially higher (3-fold) labeling index than low risk papillomas,and 40% of labeled cells are in the suprabasal compartment. A sub-

population of high risk tumors loses TGFÃŸlbut retains TGFÃŸ2.Thisgroup also has a substantially higher labeling index than low riskpapillomas (2-fold) but fewer labeled suprabasal cells, suggesting that

both the expression and activity of TGFÃŸsare compartmentalized inthe epidermis and in squamous papillomas (18). About 90% of mouseskin squamous cell carcinomas are devoid of TGFÃŸland TGFÃŸ2,andthe TGFÃŸsare also lost in many low risk papillomas when examinedat 36 weeks in a tumor induction protocol suggesting that TGFÃŸlossand hyperproliferation are common in premalignant progression, buttumor cells acquire this trait at vastly different rates (18). The char-

Table 4 Relationship of TGFÃŸpeptide expression to tumor induction protocol andDNA synthesis invivo"TissueNormal

skinPapillomaPapillomaPapillomaTreatment

protocolPromoter

treatedHigh

riskLowriskHigh

riskLowriskHigh

riskLow risk%

TGFÃŸphenotypcÃŸi*

0'*23

850r0?*14

063"

15%

BrdUrd h

positivenuclei18.5

153175%

suprabasalBrdUrd-positive

nuclei0

0.85.232

"Protocols and results laken from Ref. 18.''5-Bromodeoxyuridine (BrdUrd) injected l h prior to sacrifice.

acterization of some high risk tumors as TGFÃŸlnegative and TGFÃŸ2positive suggests that TGFÃŸl loss may occur first and that basal cellhyperproliferation or some other change regulated by TGFÃŸlthat mayinfluence genomic stability may be sufficient to increase the risk forprogression of squamous neoplasia.

Direct evidence linking TGFÃŸl loss and accelerated premalignanttumor progression comes from studies using keratinocytes culturedfrom transgenic mice constructed with a targeted disruption in theTGFÃŸlgene (131). These mice do not express TGFÃŸlin the basal cellcompartment of the epidermis, but suprabasal TGFÃŸ2is expressednormally. Introduction of the v-ras"" oncogene into cultured kera

tinocytes of TGFÃŸl null mice or wild-type and heterozygous litter-

mates results in papillomas when the cells are grafted to nude mice.However, multifocal carcinomas develop in the TGFÃŸlnull homozy-gous papillomas (but not in those of the other genotypes) within 18-25days of grafting.4 These results clearly demonstrate that the TGFÃŸ

family of growth inhibitors can serve as suppressors of tumor cellproliferation and premalignant progression, and the absence of TGFÃŸ

J A. Click, M. Lee, A. Kulkarni. S. Karlsson, and S. H. Yuspa, manuscript in prepa

ration.

1183



PATHOGENESIS OF SOUAMOUS CELL CANCER

is an important marker of prognostic significance for squamous cancerdevelopment in the mouse skin model.

The p53 Tumor Suppressor Protein May Inhibit PremalignantProgression through the TGFÃŸPathway

Approximately 10% of carcinomas and 20% of high risk papillomasretain TGFÃŸ.These tumors may contain cells that are resistant to thegrowth-inhibitory effects of TGFÃŸ,a phenotype described for somekeratinocyte-derived tumor cells in vitro (132-135). Several labora

tories have reported that the introduction of a mutant p53 gene intoneoplastic epithelial cells with endogenous wild-type p53 impartsresistance to the growth-inhibitory effects of TGFÃŸ(105, 136), sug

gesting that a link could exist between mutations in p53, TGFÃŸ,andpremalignant progression. When skin keratinocytes from p53 nullmice and their wild-type and heterozygous littermates (137) werecultured and transduced with the \-ras"" oncogene, both wild-typeand heterozygous v-ras"" keratinocytes produced papillomas whengrafted onto nude mouse hosts, while homozygous p53 "knock-out"

v-ras11" keratinocytes produced carcinomas. Over 3-5 weeks, multi-

focal carcinomas also developed in about 50% of papilloma graftsdeveloping from v-ras"" keratinocytes of p53 heterozygous mice.5 Invitro, v-ras"" keratinocytes from the homozygous p53 null genotype

were less sensitive to growth inhibition by TGFÃŸl or TGFÃŸ2thanwild-type v-ras"" keratinocytes. The p53 heterozygous genotype was

intermediate in responsiveness. Resistance to TGFÃŸgrowth inhibitionwas greater in thepJJ null genotype after v-ras"" transduction than in

the p53 null controls without the oncogene. Together, these resultssuggest that alterations in the p53 gene may cooperate with the rasoncogene to produce cells less responsive to negative growth regulators such as TGFÃŸ.Such cells would have a growth advantage in abenign tumor, particularly those tumors retaining TGFÃŸ,and may beat greater risk for tumor progression. Cells with a mutant p53 genecould contribute to the subpopulation of squamous carcinomas thatretain expression of TGFÃŸafter initiation and promotion in the mouseskin model. The high frequency of p53 mutations in human skincancers and in UV light-induced mouse skin carcinomas may reflect

a more vigorous environment for growth restraint in tumors inducedby chronic exposure to UV light. Such an environment requires adaptation by tumor cells for clonal evolution.

Integrin Changes and Malignant Conversion: An EpigeneticContribution to the Cancer Phenotype

The potent activity of the v-fos oncogene in converting papillomacell lines to the carcinoma phenotype and the ability of v-fos to causemalignant conversion in concert with v-rasfl" in normal keratinocytes

(138, 139) suggest that changes in gene expression, without structuralgenetic alterations, could be sufficient to convert benign cells tomalignancy. As far as we know, the oncogenic activity of v-fos de

pends on its influence on gene transcription as a component of theAP-1 family of transcription factors (140). The squamous cancersproduced by v-fos oncogene-mediated conversion are highly invasive

and vascular, do not express Kl or K10, but synthesize keratin 8,demonstrate elevated 7-glutamyltranspeptidase activity, and have dis

seminated expression of the a(,ÃŸ4integrin throughout the tumor mass(23). All of these changes are characteristics common to chemicallyinduced squamous cell carcinomas. Other oncogenes which modifygene transcription, such as c-myc, EIA, or v-jun, do not cause phe-

notypic progression when transduced into papilloma cells suggestingthat /iw-related changes in genetic regulation are specific for malig

nant conversion.

Among the characteristics associated with premalignant progression and malignant conversion, changes in integrin distribution couldhave profound effects on the neoplastic phenotype since integrins areimportant mediators of cell-cell and cell-extracellular matrix interactions (141, 142). In fact, changes in cell-cell interactions are characteristic of malignant conversion. When the papilloma cell line SP-1 is

grafted to nude mice in combination with normal keratinocytes, tumorformation is suppressed. However, normal keratinocytes cannot suppress the growth of the v-/os-converted malignant derivative of SP-1

(69). Changes in integrins have been associated with alterations incell-cell and cell-matrix interactions in several neoplastic cell types,

and these are particularly important for invasion and metastasis(143-146).

Integrins are heterodimeric transmembrane glycoproteins, assembled among members of two large multigene families (141, 142).The expression of a particular integrin complex is tissue and cell typespecific. In skin, redistribution of the a,,ÃŸ4integrin to the suprabasalcompartment serves as a marker of high risk for premalignant progression (123), and the a,,ÃŸ4complex is the predominant integrin ofsquamous cell cancers (23, 147-149) (Fig. 4). The suprabasal redis

tribution of a6ÃŸ4positive cells in papillomas concomitantly deficientin TGFÃŸmay reflect an enhanced population of proliferating cells inthe suprabasal compartment (123). Redistribution of the a^ÃŸjintegrinis associated with an increase in a6 mRNA, both in papillomas undergoing premalignant progression and in carcinomas. Furthermore,the quantitative increase in a,, mRNA coincides with the expression ofa splice variant of the a,, transcript (150, 151) exclusively in carcinomas.6 This qualitative change in ah transcript processing is not

detected in benign tumors or normal skin. When malignant conversionof SP-1 cells is induced by transduction of the v-fos oncogene, the

splice variant of ah mRNA is detected. Nuclear oncogenes which donot cause malignant conversion (c-myc, v-jun) do not induce a splic

ing variant of a,, mRNA. These results, in concert with similar studieson the cell surface matrix receptor CD44 (152, 153), suggest thatqualitative and quantitative changes in cell surface matrix receptorsare linked to malignant conversion and may contribute to the alteredinteraction of carcinoma cells with surrounding normal cells andstroma. Thus, epigenetic changes in benign tumor cells may mediatethe emergence of characteristics generally attributed to the malignantphenotype. Additional studies are required to determine if these epigenetic alterations in integrin expression are causally related to thephenotypic conversion of squamous papillomas to carcinomas.

Integration of New Data in the Analysis of Multistage SkinCarcinogenesis

For almost 20 years, my laboratory has focused on the elucidationof biochemical pathways which contribute to the evolution of squamous cancer, a malignancy both common and lethal in human populations. Some of this effort has been devoted to refining the skin modelsystem itself and to understanding the physiology of the epidermis asa surrogate for all squamous lining epithelia. A well defined biologicalmodel is fundamental for the interpretation of molecular data in thecontext of a disease process. Model systems are required for establishing causal relationships among basic research discoveries andclinical observations, and they are essential for testing new ideas inearly diagnosis, cancer prevention, and cancer therapy.

We now have an opportunity to extend an operationally designedscheme of multistage skin carcinogenesis to a more function-based

model (Fig. 5). The initiated phenotype results from mutational eventsproducing intrinsic changes in intracellular signaling pathways, par-

ftT. Tennenbaum, A. Belanger. R. Jamura, V. Quaranta, and S. H. Yuspa. manuscript5 W. Weinberg, C. G. Azzoli, N. Kadiwar, and S. H. Yuspa, manuscript in preparation. in preparation.

1184




ticularly those related to terminal differentiation. However, initiatedcells remain responsive to extrinsic restraints imposed by surroundingnormal cells and the microenvironment. Extrinsic tumor promotersalter tissue homeostasis by a variety of nonmutagenic pathways andmodify the signaling environment in the tissue to enhance the outgrowth of initiated cells. At a critical clone size, initiated cells escapethe constraints imposed by normal neighbors and display their neo-

plastic phenotype. It is likely that several classes of initiated cellsresult from exposure to initiators. This could result from initiation ofdifferent target cell types or a single cell type with multiple carcinogen-induced changes. We are currently exploring experimental ap

proaches to distinguish among these possibilities.When activation of the c-ras"" gene is responsible for initiation of

keratinocyte neoplasia, a critical target is the PKC pathway. Sincepapillomas arising from initiation with carcinogens that do not induceras mutations (51) share a common phenotype with papillomas causedby a mutant ras"" gene, the PKC pathway may be a target for many

initiating events. Presently, our analysis suggests that the hyperpro-liferative component of the initiated phenotype is caused by overex-

pression of TGFa while chronic activation of PKCa through increased cellular diacylglycerol and functional inhibition of PKC8 dueto phosphorylation on tyrosine residue(s) contribute to the altereddifferentiation response characteristic of initiated epidermal cells.Confirmation of this concept is essential since these changes wouldprovide important targets to reverse early neoplastic changes in squa-

mous epithelia where terminal differentiation can eliminate incipienttumor cells.

Further genomic changes in neoplastic epidermal cells seem centralto the stage of premalignant progression, and selective clonal expansion of the progressing neoplastic subpopulation is a requirement. Themechanistic basis for premalignant progression is not clear, but theaccumulation of genetic and chromosomal changes associated withprogression may be a consequence of altered control of proliferation(154). Loss of expression or localization of the TGFÃŸclass of growthfactors is a common pathway through which enhanced proliferationand premalignant progression proceeds spontaneously in experimentalskin carcinogenesis. This is also likely to be the cause of enhancedsensitivity to mutagen-induced premalignant progression in high risk

papillomas. Loss of response to negative growth regulation by neoplastic cells may be an alternative pathway to achieve the same endpoint in many tumor types. It is at the stage of premalignant progression that inactivation of tumor suppressor genes exerts its greatestinfluence, and many suppressor genes identified thus far impact ongrowth restraint (155). Loss of TGFÃŸresponsiveness has been associated with mutation in thep53 gene (105, 136) and could contribute

to the accelerated skin tumor progression observed for both p53 andTGFÃŸnull mice. As tested thus far, most tumor promoters have littleinfluence on premalignant progression suggesting that intrinsicchanges in tumor cells or their microenvironment are sufficient forselective outgrowth and spontaneous accumulation of geneticchanges. This is consistent with previous data indicating that theinfluence of promoters on the normal cells of the target tissue isfundamental to their action at an early stage of carcinogenesis (156).However, certain exogenous exposures with nongenotoxic carcinogens and cytotoxic agents that stimulate proliferation may also influence the rate of premalignant progression (154). This may explain theactivity of benzoyl peroxide for enhancing premalignant progressionof skin papillomas (157).

Elucidating the molecular basis for malignant conversion is a particular challenge since prevention of this change would have the mostprofound effect on host survival. Malignant conversion of skin tumorsmust involve more than changes in tumor cell growth, since thegrowth rate of high risk papillomas and malignant tumors is similar.Previous studies, in which either exposure to mutagens enhanced theconversion from benign to malignant tumors or oncogene transductioncaused malignant conversion of papilloma cell lines, had suggestedthat malignant conversion is a discrete step in carcinogenesis with amutational basis. The homozygosity of mutant ras"" alÃelesand mu

tations in the p53 gene frequently found in carcinomas suggest thatstructural genetic changes are required for conversion. However, certain properties of the process in mouse skin carcinogenesis now suggest otherwise. The phenotypic changes associated with premalignantprogression and malignant conversion are a continuum, and markersof conversion arise in a multifocal pattern at a frequency too great tobe mutational in origin. The potent activity of the v-fos gene as a

mediator of squamous malignant conversion, even in keratinocytesdiffering from normal only by the expression of a ras oncogene (139),suggests that changes in gene expression are sufficient to cause themalignant phenotype. Of course gene expression changes can have abasis in structural genetic alterations, and loss or mutation of a tran-

scriptionally active suppressor gene could influence the phenotype ofa tumor cell in addition to its growth potential. Furthermore, stage-

specific overexpression of a mutant form of a heterozygous tumorsuppressor gene, as recently documented for the wild-type p53 gene in

skin cancer cell lines (158), could alter the tumor cell phenotype in aprogressive manner and influence clonal selection. While the documentation of changes in gene expression or mRNA processing associated with malignant conversion of squamous tumors is limited(159), those identified include the c-ras"" gene (160), secreted pro

teases (161), and cell surface receptors including integrins (23, 152).

GENOTYPE:

Fig. 5. Functional stages in experimental skincarcinogenesis. Stages are defined by specific biological and biochemical alterations and linked togenetic changes. Horizontal axis, an approximatetime line with maximum carcinoma incidence generally reached by 50 weeks after initiation in mouseskin studies. The thickness of horizontal lines correlates with the approximate frequency of progression from one stage to another. This serves as aframework for additional biochemical studies in thecontext of stage-specific consequences of a particular metabolic alteration.

STAGE:

BIOLOGICALALTERATION:

BIOCHEMICALALTERATION:

Normal1NORMAL1rasâ„¢mutation1

1LOW

RISKPAPILLOMAHIGH

RISKPAPILLOMAInitiation,PromotionIntrinsicSignalingTGFct?

SuppressorlossPremalignantProgressionGenomicInstabilityTGFÃŸPLC/diacyglycerolPKC

Â¡soformsTGFÃŸras1*â„¢1

homozCARCINOMA1MalignantConversionCell-cell/matrixInteractionslntegrins/CD44ProteasesAP-1

Activity

1185



PATHOOENESIS OF SQUAMOUS CELL CANCER

Changes in cell-cell or cell-matrix interactions or modification of the

tumor cell microenvironment by secreted proteases could have profound effects on the behavior of the progressing tumor cells and theirneighbors and could contribute to a multifocal evolution of a cancerphenotype. The distinction between permanent structural geneticchanges and epigcnetic alterations in cancer cells is not trivial. In thelatter case, intervention by pharmaceuticals could be effective in reversing the behavior that we now consider typical of cancer cells. Themouse skin model of squamous cancer development seems well suitedfor testing these ideas.

The Challenge Ahead

1 have tried to emphasize the contributions that model systems canprovide as a component of our efforts to understand carcinogenesis inhumans and develop rational preventive and therapeutic strategies. Ihave also presented examples of advances in our understanding of thebiochemical alterations contributing to specific stages in squamouscell cancer development in the mouse skin model. Of course theseinsights provide only a small view of the overall process, but theyindicate directions for further investigation and highlight the substantial progress that has been made. Rapidly accumulating informationdefining the regulation of growth and differentiation of the epidermalkeratinocyte, increasing sophistication in genetic and biochemicaltechniques, and sharing of resources across the cancer research community should enhance the pace of future research in this model. Themouse skin model is also particularly well suited for targeting oftransgenes, and efforts in this area should yield important data onmechanisms of cancer pathogenesis and enhance screening efforts forrecognizing exogenous agents that contribute to human cancer incidence (20, 162, 163). Experimental animal models have been underutilized for identifying the genetic basis for susceptibility to initiation,promotion, and premalignant progression. Marked, target site-specific

susceptibility differences among inbred animal strains provide anideal setting for finding cancer susceptibility genes. In addition to skincancer, enhanced susceptibility to breast, liver, colon, kidney, andpancreatic cancers are well defined in animal models and could facilitate the identification of genes responsible for enhanced susceptibility of humans at these target sites.

I have highlighted the insights into multistage carcinogenesis andthe molecular pathogenesis of squamous cancer that have been contributed by studies in the mouse skin model. As we learn more aboutthe molecular details of cancer development in other systems, thevalidity of these concepts receives powerful support. Great progresshas been made recently in defining the biological and genetic alterations involved in human colon cancer (164). Development of humancolon cancer frequently involves mutations in the c-ras*' gene, chro-

mosomal changes leading to an alteration in a cell adhesion molecule(DCC), p53 gene mutations, and other genetic changes producingclonal selection that occur in a relatively predictable manner. Furthermore, changes in PKC activity and diacylglycerol levels have beendocumented in colonie adenomas (165, 166), proliferation of adenomacells is selectively stimulated by activators of PKC (167), and colonieflora produce lipids which are PKC activators (168). A major riskfactor for progression from colonie adenoma to adenocarcinoma is ashift in the proliferative zone of adenomatous crypts to a more superficial compartment, indicating that a change in growth restraint isimportant in premalignant progession in this tissue (169). Further-more, novel expression of splice variants of the cell surface glyco-protein CD44, a major matrix receptor and mediator of cell-cell in-

teructions in several cell types, has been associated with premalignantprogression and malignant conversion in both squamous cell cancersand colon adenocarcinomas of humans (152, 170, 171). These remark -

able parallels suggest that the patterns defined by studies in skincarcinogenesis may be more generally characteristic of epithelialcancers.

The evolution of a normal cell into a malignant tumor is an enormously complex change, and it is remarkable that we can definestages of this process in a predictable manner. Perhaps equally remarkable is the emergence of patterns of genetic and biochemicalchanges within tumors and tumor cells of a particular organ which arecommon across species. These patterns have only been recognizedrecently as the result of data gathered from the multifaceted approaches of a dedicated international cancer research community. Asin the making of a quilt, each patch contributes a better view of theoverall pattern, and in the end both the background and the designmerge to provide comfort. I am confident that within a few years themomentum currently experienced in basic cancer research will blendwith the efforts of our clinical colleagues to provide the comfort ofbetter diagnosis and rational preventive and therapeutic approachesfor human cancer.

Acknowledgments

I would like to thank my current postdoctoral fellows Andrzcj Dlugosz,Adam Click, Tamar Tennenbaum, and Wendy Weinberg tor sharing theirunpublished data for this paper and Mitchell Denning for creating the designof Fig. 1. I am grateful to Henry Hennings. Peter Blumbcrg. and Luigi De Lucafor critical reading of the manuscript. The editorial assistance of MargaretTaylor is also deeply appreciated.

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1994;54:1178-1189. Cancer Res Stuart H. Yuspa Clowes Memorial Award Lecture

Thirty-third G. H. A.−−from Studies of Skin Carcinogenesis The Pathogenesis of Squamous Cell Cancer: Lessons Learned

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