Role of Oncogenes and Tumor Suppressor Genes in Multistage Carcinogenesis

GENETIC DETERMINANTS OF MALIGNANCY

Stuart H. Yuspa, Andrzej A. Dlugosz, Christina K. Cheng, Mitchell F. Denning, Tamar Tennenbaum, Adam B. Glick, and Wendy C. Weinberg Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland U.S.A.

The introduction of the techniques of molecular biol tiation and enhanced growth rate of initiated cells im ogy as tools to study skin carcinogenesis has provided parta growth advantage when the epidermis isexposed more precise localization of biochemical pathways to promoters. The frequencyof premalignant progres that regulate the tumor phenotype. This approach has sion varies among papillomas, and subpopulations at identified genetic changes that are characteristic of high risk for progression have been identified. These each of the specific stages of squamous cancer patho high-risk papillomas overexpress the a6p4 integrin genesis: initiation, exogenous promotion, premalig and are deficient in transforming growthfactor P1 and nant progression, and malignant conversion. Initiation fJ2 peptides, two changes associated with a very high can result from mutations in a single gene, and the proliferation rate in this subset of tumors. The intro Harvey allele of the rasgene family has been identified duction of an oncogenic rasH- gene into epidermal cells as a frequent site for initiating mutations. Heterozy derived from transgenic mice with a null mutation in gous activating mutations in c-rasH- are dominant, and the TGFp1 gene have an accelerated rate of malignant affected keratinocytes hyperproliferate and are resist progression when examined in .,ivo. Thus members of ant to signals for terminal differentiation. An impor the TGFp gene family contribute a tumor-suppressor tant pathway impacted by c-rasH- activation is the pro function in carcinogenesis. Accelerated malignant tein kinase C (PKC) pathway, a major regulator of progression is also found with v-rasH- transduced kera keratinocyte differentiation. Increased activity of tinocytesfrom skinof mice with a null mutation inthe PKCa and suppression ofPKC6by tyrosine phospho p53 gene. The similarities in risk for malignant con rylation contribute to the phenotypic consequences of version by initiated keratinocytes from TGp1 and p53 rasH- gene activation in keratinocytes. Tumor promot null geneotypes suggest that a common, growth-re ers disturb epidermal homeostasis and cause selective lated pathway may underly the tumor-suppressive clonal expansion of initiated cellsto produce multiple functions of these proteins in the skin carcinogenesis benign squamous papillomas. Resistance to differen- mode!.] Invest Dermatol103:90S-95S, 1994

he introduction of the techniques of molecular biol context of the intact epidermis. In cell culture, initiated keratino ogy as tools to study skin carcinogenesis has provided cytes display an altered response to signals for terminal differentia more precise localization of pathways that regulate tion, a characteristic that provides a selective growth advantage T the tumor phenotype. However, the application of under culture conditions favoring differentiation [2-4]. Exploita these tools to answer relevant questions has depended tion of this difference in vitro has been particularly helpful in isolat on the establishment of a conceptual framework that developed ing initiated keratinocytes of mouse and human origin [5-8]. over four decades of research on the biology and cellular physiology Exogenous tumor promotion causes the selective clonal expan of skin cancer induction by chemical exposures. These studies indi sion of initiated cells to produce multiple benign squamous cell cated that predictable and progressive stages occurred during the papillomas, each representing a clone of thousands of initiated cells. clonal evolution of a normal keratinocyte into a squamouscell car Potent exogenous promoters of the phorbol ester class activate, cinoma, and both genetic and epigenetic events contributed to these protein kinase C (PKC), and this enzyme activity accelerates epi changes [1]. Progress in understanding carcinogenesis wasenhanced dermalterminal differentiation [9-11]. Because initiated cells resist when certain properties associated with a particular stage of skin the induction of terminal differentiation by activators ofPKC [12], cancer pathogenesis were characterized [1]. Operational analyses, the differential response to phorbol esters favors the growth of the which defined the experimental requirements to produce a specific initiated subpopulation. This process recapitulates in vivo, the clonal neoplastic phenotype, have also been important for understanding selection of initiated cells by differentiation inducing agents in ke the process of tumor development (Fig 1). ratinocyte culture [13-15]. Squamous papillomas demonstrate a The earliest event documentedin skin carcinogenesis , initiation, �ign proliferation rate and delayed maturation, properties that are is carcinogen induced and mutational in nature. Initiated keratino analogous to the phenotype of individual initiated cells in vitro cytes express a subtle change in phenotype, unrecognizable in the [16,17]. Because most exogenous promoting agents are not muta genic [1], papillomas are diploid [18,19], and a single genetic change Reprint requests to: Dr. StuartYuspa, Building 37, Room 3B25, National in normal keratinocytes is sufficient to produce a papilloma pheno Cancer Institute, National Institutes of Health, Bethesda, MD 20892. type [20-22], the mechanism of exogenous promotion is likely to

90S VOL. 103, NO.5, SUPPLEMENT, NOVEMBER 1994 ONCOGENES AND TUMOR SUPPRESSORS IN CARCINOGENESIS 91S

Initiation Promotion Premalignant Progression Malignant Conversion pression of mRNA for K1 and K10 is blocked, loricrin and fiJaggrin (Mutation) (Epigenetic) (Chromosomal Deletion, (Mutation) Duplication) expression areenhanced, and TGK is expressed constitutively (Fig • 2). Similar changes in expression of these epidermal markers had been documented in normal keratinocytes treated withPKC activa tors such asphorbol esters and diacylglycerol(Fig 2) [11,12,47,50]. Furthermore,diacylglycerol levels are markedlyelevated in v-rasH. Promoter Promoter keratinocytes and in keratinocytes expressing a mutated c-rasHa, Dependent Independent when comparedto normalepidermal cells [51,52]. Diacylglycerol is the endogenous activatorofPKC [53,54]. WhenPKC activitywas H. Figure 1. Operational stages in experimental skin carcinogenesis. measured in v-ras keratinocytes and compared to activity in nor Each stage is ed fined by the biologic consequences of a specific experimental mal epidermal cells, PKC activity that is Ca++-dependentwas in protocol.This scheme serves as a framework for molecular analysis of multi- creased whereas PKC activity that is Ca++-independent was de s. i stage carcinogenesis. See text and [1] for detail creasedin the v-rasHainitiated cells (not shown). To examine if the PKC pathway could be responsible for the phenotypic alterations in v-rasHa keratinocytes, inhibitors of PKC were used to determine be epigenetic inmost cases. In the absence of exposure to exogenous their influence on the expression of epidermal markersin cells initi H promoters, initiated skin rarely develops tumors. Thus, exogenous ated by v-ras a transduction. Both constitutive and Ca++-induced promotion is a rate-limiting early event in carcinogenesis. expression ofTGK mRNA in v-rasHakeratinocytes was prevented by Premalignant progression is most often a spontaneous process, inactivating PKC with bryostatin or GF 109203X, selective PKC independentof exogenous promoters [23,24]. Genetic studies indi inhibitors. Bryostatin treatment also restored the Ca++-inducedex catethat non-random sequentialchromosomal aberrations are asso pression ofK1 andK10 to v-rasH. cells (not shown). These findings ciated with premalignant progression [19,25-28]. This stage of support the hypothesis that rasHa-induced alterations in keratino cancer pathogenesis constitutes the major time-dependent compo cyte differentiationare the result of chronic activation of a Ca++-de nent of carcinogenesis and must involve repeated episodes of cell pendent isoform of PKC and reduced activity of a Ca++-indepen selection since modal dominance of a specificchromosomal aberra dent isoform of PKC. Thus, pharmacologic modulation of this tionindicates clonal outgrowth. Thus, at least one functionof the pathway may provide an approach to reverse the neoplastic pheno relevant genetic events occurringduring premalignant progression type. must imparta growth advantage on the affectedcell. Since PKC activation results int erminaldifferentiation of normal Malignantconversion of benign tumorsis a relatively rare occur keratinocytes [9-11], the chronic activation ofPKC measured in rence; less than 5% of papillomas spontaneously convert to cancers v-rasHa keratinocytes that did not spontaneously undergo terminal [23,24]. The risk for malignant conversion is variable among benign differentiation presented a paradox. Neoplastic keratinocyte cell tumors,and subpopulations of papillomaswith a much higher risk lines expressing an activated c-rasH• gene also have elevated diacyl for malignant conversion have been identified[2 9 -31]. Because the glycerol levels [52], but are resistant to terminal differentiationin conversion frequency can be significantly increased by exposing duced by either Ca++or phorbol esters. This suggested that qualita- animals bearing benign tumors to mutagens, the crucial events in the conversion process appear to be genetic in nature [23,24]. How ever, even mutagen-induced malignant conversion varies in fre Ca2+(mM) 0.05 0. 12 0.05 0.12 quency, and the subclass of papillomas at high risk for spontaneous r-,. r----JI '" conversion is also at higher risk for mutagen-induced conversion "0... "0... "0... :r ...... e... CI) [30]. Thus, the factors that determine risk for conversion to malig c c t!) c c 0 0 0 0 � nancy susceptibility to spontaneous or induced ge <{ I must increase U U 0 U U > netic changes. The identification of these factors would be an im portantgoal in understanding cancer pathogenesis. K1 THE METABOLIC BASIS OF INITIATION INDUCED BY A ras ONCOGENE The mutational basisfor initiation of mouse epidermal carcinogen Ha esis was supported by the discovery that the c-ras gene was fre quently mutated in chemically-induced squamous papillomas, and Loricrin thesite of the mutation was determined by the initiating carcinogen [32-37]. A causal relationship for a rasmutation and initiation of skin carcinogenesis was established when an activatedv-rasHa onco gene was introduced into normal epidermis or culturedkeratino cytesand recipient cells developed into squamous papillomas in vivo Filaggrin 120,38]' Subsequent studies indicated that a significant number of human skin cancers also contained mutated c-ras genes [39-44]. We have used cultured keratinocytes into which the v-rasHagene was introduced via retroviral transduction to further characterize Figure 2. Protein kinaseC activation and the v-rasH- oncogene block the initiatedphenotype and to identify pathways affected by the expression of mRNA for spinous cell markers and enhance expres expressionof the v-rasHa p21 oncoprotein [45 -47]. sion of mRNA for granular cell markers. Mouse keratinocytes were Primary keratinocytes expressing the v-rasHa oncogene, intro cultured in Eagles medium containing 8% fetal bovine serum and a Ca++ duced by a defective retroviral vector, exhibit an altered response to concentration of 0.05 mM. Some cultures were transducedwith the v_rasH. signalsfor terminaldifferentiation in cell cultureand produce squa oncogene nsing a defective retroviral vector as described previously (20). a m u mous papillomas when grafted onto nude mice [20,45-47]. Thus, After 8 d in vitro subset of cultures was switched to edi m containing this single gene change can accomplish all the requirements for 0.12 mM Ca2+ for 24 h. In one group of cells the 0.12 mM Ca++ medium was supplemented with 125 pM 1-oleoyl-2-acetylglycerol (OAG), a syn initiation.In cultures of normal keratinocytes, increasingextracel thetic diacylglycerol that activates PKC [11]. Total RNAwas isolated, sepa mM lularCa++ from 0.05 to 0.12 induces both mRNA transcripts rated on a 1 % agarose gel containing0.6 M formaldehyde and Northernblot andprotein for severaldiff erentiationmarkers, includingkeratin 1 analysisperformed as describedpreviously (\ \, . A.tthe timepointexamined (Kl), KI0, loricrin, filaggrin, and keratinocyte transglutarninase (24 h), transcription of the later markers, loricrin and filaggrin, is low in (TGJ[48,49] (Fig 2). In v-rasHakeratinocytes, Ca++-mediated ex- normal keratinocytes (48). 9ZS YUSPA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

tive changes in PKC isoforms may also contribute to the initiated phenotype and could account for differences in Ca++-dependent and independent PKC activity in v-rasfh keratinocytes. PKC is a familyof enzymes, members of which are differentiallyexpressed in particular tissues and cell types [55]. Five PKC isozymes are ex pressed in keratinocytes (a, �, E, C, ,,) [56]. Of these, PKCa is the only Ca++-dependent isoform. In an analysis of modification of these isoforms, we found tyrosine phosphorylation of PKC� in v-rasfh keratinocytes [57]. PKC� was also tyrosine phosphorylated in neoplastic keratinocytes containing an activated allele of the c H. ras gene. The activity of tyrosine phosphorylated PKC� was con stitutively low and could not be stimulated significantly by phorbol ester treatment, indicating that tyrosine phosphorylationblocks ac tivation of PKC� [57]. This interaction between tyrosine kinases Figure 3. Immunohistochemicalmarkers distinguish high riskand and PKC permits signaling through specific PKC isozyme in cells low riskpapillomas. Papillomaswere generated on the skinof SEN CAR expressing an oncogenic rasfh gene and may represent a molecular mice by initiation with 10 pg of7,12-dimethylbenz[a]anthracene and pro block to differentiation in neoplastic keratinocytes. motion with 12-0-tetradecanoylphorbol-13-acetate (TPA, 2 JAg, once weekly) to produce papillomas at low risk for malignant progression or CHARACTERISTICS OF TUMORS AT HIGH RISK FOR mezerein (4 JAg,twice weekly) to produce papillomasat high riskfor malig MALIGNANT CONVERSION nant progression [31]. High risk papillomas (a,b,e) were excised at 8 weeks when they were first detected clinically and only about 1-2 mm in diame Variations in protocols used for inducing skin tumors yield sub ter. Lowrisk papillomas (d,ef) were excised at 11 weeks, also at firstdetec groups of papillomas with either low or high riskfor progression to tion. Frozen sections were processed for immunochemistry using specific malignancy [32]. High risk tumors represent a relatively small co antibodies for the 0:6 integrin subunit (a,d), keratin 1 (b,e) and keratin 13 hort of papillomas. They are selectively induced by limited expo (ef). Reagents and procedures for frozen sectious and immunochemistry sure to strong promoters or by more prolonged exposure to weak have been describedpreviously [59]. Each panel represents a 5-p.m frozen promoters [32] . Thus, they appear to be more sensitive to exogenous section through the entiretumor. Bar, 200 JAm. tumor promotion. In addition, high risk papillomas erupt early, grow larger, and do not regress when promoter exposure is discon tinued. Thus, high risk papillomas have unique growth properties. We have performed comparative phenotypic analyses of high and strata [17]. In low-risk tumors, 90% of the proliferating cells are low risk papillomas to identify differences that may suggest a mo confinedto the basal compartment. In contrast, the majorityof high lecular basis for the altered biologic potential of this class of benign risk papillomas are devoid of both TGF-p1 and TGF-fJ2 as soon as tumors. Several tumor induction protocols were utilized that pref they arise; these tumors have up to 40% of the proliferating cells in erentially produce each papilloma subtype. The tumors were evalu the suprabasal layers [17]. Squamous cell carcinomas are also devoid ated at the time they first erupted (8 -11 weeks) and sequentially of TGF-p [17,64], suggesting that they arise from the TGF-p during the course of tumor progression. Particular attention was deficient high-risk papillomas. In some high-risk papillomas, concentrated on expression of specific integrins and cytokeratins as TGF-p loss can occur first and correlates with basal cell hyperpro changes in these markers previously were identified with particular liferation, whereas TGF-fJ2 loss correlates with suprabasal hyper stages of skin tumor progression [58,59]. High-risk papillomas proliferation. In tumors, loss of TGF-p is controlled at the post demonstrated basal and suprabasal expression of the a6p4 integrin, transcriptional level [17,64]. These results show that TGF-p ex loss of keratin 1, and aberrant expression of keratin 13 when they pression and function are compartmentalized in epidermis and firsterupted at 8 weeks after promotion started (Fig 3a,b.c). In these epidermal tumors, and that loss ofTGF-p is an early, biologically tumors, a6p4 integrin expression coincided with an expansion of relevant risk factor for malignant progression. the proliferating compartment as indicated by suprabasal bromo The importance ofTGF-p loss in progression was demonstrated deoxyuridine labeling [60]. In contrast, a6p4 immunostaining was directly using transgenic mice with null mutations introduced into confined to basal cells in low risk tumors, keratin 1 was abundant, the TGF-p110cus [65]. Keratinocyte cultures were established from and keratin 13 was absent or focal in the majority of this group, epidermisof newbornmice either homozygous or heterozygous for whereas proliferating cells were largely in the basal compartment the TGF-p1 null mutation or from wildtype littermates. The v H. (Fig 3d,e!). By 33 weeks after promotion started, a6p4 suprabasal ras oncogene was introduced by a defective retrovirus into cells of expression continued to distinguish papillomas at higher risk for each genotype, and recipient cells were grafted onto nude mouse malignant conversion, but keratin 13 was expressed in all groups hosts. As expected, the v-rasH'-transduced wild type keratinocytes (Fig 4). At this time, high risk papillomas displayed focal expres produced benign papillomas in grafts. In contrast, the tumors that sion of keratin 8 [46,61,62] and y-glutamyltranspeptidase, markers developed from v-rasH'-transduced TGF-p1 null keratinocytes were also found in chemically-induced carcinomas but not low-risk either very dysplastic papillomas or squamous cell carcinomas. tumors (Fig 4). Keratin 13 was lost in most carcinomas. Keratin 8 These results provide strong genetic proof to indicatethat the loss of and y-glutamyltranspeptidase were only expressed in a6p4 positive TGF-p1 is a critical step in the progression to malignancy in the . cells [60]. These results indicate that expression of a6p4 integrinin e�idermis. suprabasal strata serves as an early predictive marker to identify THE P53 TUMOR SUPPRESSOR GENE AND benign squamous tumors at high risk for malignant progression and MALIGNANT CONVERSION that this marker is associated with a higher proliferation rate and suprabasal proliferation in high-risk tumors. The p53 gene is frequently mutated in human squamous cell and basal cell carcinomas of the skin [66-69]. This is likely to result GROWTH FACTORS AND THE RISK FOR from the direct mutational action of ultraviolet light since the most MALIGNANT CONVERSION frequent mutagenic change is a C -+ T transition commonly found The transforming growth factor-p's are expressed in the epidermis at C-C dinucleotides [66-69]. The E6 transforming gene of onco and are growth inhibitors for both normal and initiated mouse kera genic human papilloma virusestargets p53 protein to fonna rapidly tinocytes in vitro [63]; altered TGF-p expression could influencethe degraded complex. This action of E6 is essential for transforming growth properties of high risk papillomas. Normal epidermis, and activity of this virus for cultured keratinocytes [70-73]. Together skin papillomas at low risk for malignant conversion express TGF these results suggest an important role for p53 inepiderm al carcino P1 in the basal cell compartment and TGF-fJ2 in the suprabasal genesis. In experimental mouse skin carcinogenesis, p53 mutations VOL. 103, NO.5, SUPPLEMENT, NOVEMBER 1994 ONCOGENES AND TUMOR SUPPRESSORS IN CARCINOGENESIS 93S

,;-' .� . ., "

Figure S. Tumors induced on nude mice from grafts of keratino cytes expressing the v-rasHo oncogene. Skin keratinocytes were cultured from p53 null transgenic mice (e) or their wildtype (a) or heterozygous (b) Figure 4. Low-risk tumors are phenotypically distinct from carci littermates. In culture, cells of each genotype were transduced with the nomas after 33 weeks of tumor promotion. Tumors were induced by v-rasH> oncogene by a defective retroviral vector and removed fromculture the low-risk protocol described in the legend to Fig 3. After 33 weeks after 3 d for skin grafting as described [20]. Within 3-5 weeks, tumors papillomas were excised for immunohistochemical staining of frozen sec developed on recipient mice that had either the benign (a,b) or malignant (e) tions (a,b,c). Simultaneous staining was erformed on the few carcinomas p phenotype. See text for quantitative evaluation. Bar, 1 cm. that also develop fromthis protocol (d,eJ). The specificantibodies used were to recognize (a,d) keratin 13; (b,e) a6 integrin subunit; (en keratin 8. Re agents and procedures have been reported previously [60]. Bar, 100 Jlm.

tigation. In previous studies we have shown that a v-fos oncogene can cause malignant conversion of benign keratinocytes expressing are extremely rare in papillomas, but mutations are detected in car a ras oncogene [87,88]. Because thefos gene product is a transcrip cinomas [74-77]. This suggests that loss of p53 function may be tion factor known to regulate the expression of a number of genes relevant to malignant conversion [78]. To test this question, kerati that containAP-1 consensus sequences [89,90], the converting ac nocytes from skin of newborn p53 null transgenic mice [79] were tion of v-fos is likely to result from altered expression of normal H> cultured and transduced with the v-ras gene in experiments simi cellular genes. We are currently attempting to identify those cellu lar to those performed on TGF-fJ1 null mice. In all groups large lar genes that may be contributing to the malignant phenotype of tumors were apparent two weeks after grafting. 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