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Role of Oncogenes and Tumor Suppressor Genes in Multistage Carcinogenesis
GENETIC DETERMINANTS OF
MALIGNANCY
Role of Oncogenes and Tumor Suppressor Genes in Multistage Carcinogenesis 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 ogy as tools to study skin carcinogenesis has provided more precise localization of biochemical pathways that regulate the tumor phenotype. This approach has identified genetic changes that are characteristic of each of the specific stages of squamous cancer patho genesis: initiation, exogenous promotion, premalig nant progression, and malignant conversion. Initiation can result from mutations in a single gene, and the Harvey allele of the ras gene family has been identified as a frequent site for initiating mutations. Heterozy gous activating mutations in c-rasH- are dominant, and affected keratinocytes hyperproliferate and are resist ant to signals for terminal differentiation. An impor tant pathway impacted by c-rasH- activation is the pro tein kinase C (PKC) pathway, a major regulator of keratinocyte differentiation. Increased activity of PKCa and suppression ofPKC6by tyrosine phospho rylation contribute to the phenotypic consequences of rasH- gene activation in keratinocytes. Tumor promot ers disturb epidermal homeostasis and cause selective clonal expansion of initiated cells to produce multiple benign squamous papillomas. Resistance to differen-
tiation and enhanced growth rate of initiated cells im part a growth advantage when the epidermis is exposed to promoters. The frequency of premalignant progres sion varies among papillomas, and subpopulations at high risk for progression have been identified. These high-risk papillomas overexpress the a6p4 integrin and are deficient in transforming growth factor P1 and fJ2 peptides, two changes associated with a very high proliferation rate in this subset of tumors. The intro duction of an oncogenic rasH- gene into epidermal cells derived from transgenic mice with a null mutation in the TGFp1 gene have an accelerated rate of malignant progression when examined in .,ivo. Thus members of the TGFp gene family contribute a tumor-suppressor function in carcinogenesis. Accelerated malignant progression is also found with v-rasH- transduced kera tinocytes from skin of mice with a null mutation in the p53 gene. The similarities in risk for malignant con version by initiated keratinocytes from TGp1 and p53 null geneotypes suggest that a common, growth-re lated pathway may underly the tumor-suppressive functions of these proteins in the skin carcinogenesis mode!.] Invest Dermatol103:90S-95S, 1994
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context of the intact epidermis. In cell culture, initiated keratino cytes dis play an altered response to signals for terminal differentia tion, a characteristic that provides a selective growth advantage under culture conditions favoring diffe rentiation [2-4]. Exploita tion of this differen ce in vitro h as been particularly hel pful in isolat ing initiated keratinocytes of mouse and human origin [5-8]. Exogenous tumor promotion causes the selective clonal expan sion of initiated cells to produce multiple benign squamous cell papillomas, each representing a clone of thousands of initiated cells. Potent exog enous promoters of the phorbol ester class activate, protein kinase C (PKC), a nd this enzyme activity accelerates epi dermal terminal differentiation [9-11]. Because initiated cells resist the induction of terminal differentiation by activators ofPKC [12], the differential response to phorbol esters favors the growth of the initiated subpopulation. This process recapitulates in vivo, the clonal selection of initiated cells by differentiation inducing agents in ke ratinocyte culture [13-15]. Squamous papillomas demonstrate a �ign proliferation rate and delayed maturation, properties that are analog ous to the phenotype of individual initiated cells in vitro [16,17]. Because most exogenous promoting agents are not muta genic [1], papillomas are d iploid [18,19], and a single genetic change in normal keratinocytes is sufficient to produce a papilloma pheno type [20-22], the mechanism of exogenous promotion is likely to
he introduction of the techniques of molecular biol ogy as tools to study skin carcinogenesis has provided more precise localization of pathway s that regulate the tumor phenotype However, the application of these tools to answer relevant questions has depended on the establishment of a conceptual framework that developed over four decades of research on the biology and cellular physiology of s kin cancer induction by chemical exposures. These studies indi cated that p redictable and progressive stages occurred during the clonal evolution of a normal keratinocyte into a squamous cell car cinoma, and both genetic an d epigenetic events contributed to these changes [1]. Progress in understanding carcinogen esis was enhanced when certain p roperties associated with a particular stag e of skin cancer pathogenesis were characterized [1]. Operational analyses, which defined the experimental requirements to produce a s pecific neoplastic phenotype, have also been important for understanding the process of tumor development (Fig 1). The earliest event documented in skin carcinogenesis initiation, is carcinogen induced and mutational in nature. Initiated keratino cytes express a subtle change in phenoty pe unrecognizable in the .
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Reprint requests to: Dr. Stuart Yuspa, Building 37, Room 3B25, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
0022-202Xj94jS07.00
Copyright © 1994 by The Society for Investigative Dermatology, Inc.
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Initiation
(Mutation)
Promotion
Premalignant Progression
(Epigenetic)
(Chromosomal Deletion,
ONCOGENES AND TUMOR SUPPRESSORS IN CARCINOGENESIS
Malignant Conversion (Mutation)
Duplication)
• Promoter Dependent
Promoter Independent
Figure 1. Operational stages in experimental skin carcinogenesis. Each stage is defined by the biologic consequences of a specific experimental protocol. This scheme serves as a framework for molecular analysis of multii stage carcinogenesis. See text and [1] for details.
be epigenetic in most cases. In the absence ofexposure to exogenous
promoters, initiated skin rarely develops tumors. Thus, exogenous promotion is a rate-limiting early event in carcinogenesis. Premalignant progression is most often a spontaneous process, independent of exogenous promoters [23,24]. Genetic studies indi cate that non-random sequential chromosomal aberrations are asso ciated with premalignant progression [19,25-28]. This stage of cancer pathogenesis constitutes the major time-dependent compo nent of carcinogenesis and must involve repeated episodes of cell selection since modal dominance of a specific chromosomal aberra tion indicates clonal outgrowth. Thus, at least one function of the relevant genetic events occurring during premalignant progression must impart a growth advantage on the affected cell. Malignant conversion of benign tumors is a relatively rare occur rence; less than 5% of papillomas spontaneously convert to cancers [23,24]. The risk for malignant conversion is variable among benign tumors, and subpopulations of papillomas with a much higher risk for malignant conversion have been identified [29 - 31]. Because the conversion frequency can be significantly increased by exposing 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 quency, and the subclass of papillomas at high risk for spontaneous conversion is also at higher risk for mutagen-induced conversion [30]. Thus, the factors that determine risk for conversion to malig nancy must increase susceptibility to spontaneous or induced ge netic changes. The identification of these factors would be an im portant goal in understanding cancer pathogenesis. THE
METABOLIC BASIS OF INITIATION INDUCED BY A ras ONCOGENE
The mutational basis for initiation of mouse epidermal carcinogen esis was supported by the discovery that the c-rasHa gene was fre quently mutated in chemically-induced squamous papillomas, and the site of the mutation was determined by the initiating carcinogen [32-37]. A causal relationship for a ras mutation and initiation of skin carcinogenesis was established when an activated v-rasHa onco gene was introduced into normal epidermis or cultured keratino cytes and recipient cells developed into squamous papillomas in vivo 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-rasHa gene was introduced via retroviral transduction to further characterize the initiated phenotype and to identify pathways affected by the expression of the v-rasHa p21 oncoprotein [45 -47]. Primary keratinocytes expressing the v-rasHa oncogene, intro duced by a defective retroviral vector, exhibit an altered response to signals for terminal differentiation in cell culture and produce squa mous papillomas when grafted onto nude mice [20,45-47]. Thus, this single gene change can accomplish all the requirements for initiation. In cultures of normal keratinocytes, increasing extracel lular Ca++ from 0.05 to 0.12 mM induces both mRNA transcripts and protein for several differentiation markers, including keratin 1 (Kl), KI0, loricrin, filaggrin, and keratinocyte transglutarninase (TGJ [48,49] (Fig 2). In v-rasHa keratinocytes, Ca++-mediated ex-
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pression of mRNA for K1 and K10 is blocked, loricrin and fiJaggrin expression are enhanced, 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 as phorbol esters and diacylglycerol (Fig 2) [11,12,47,50]. Furthermore, diacylglycerol levels are markedly elevated in v-rasH. keratinocytes and in keratinocytes expressing a mutated c-rasHa, when compared to normal epidermal cells [51,52]. Diacylglycerol is the endogenous activator ofPKC [53,54]. WhenPKC activity was measured in v-rasH. keratinocytes and compared to activity in nor mal epidermal cells, PKC activity that is Ca++-dependent was in creased whereas PKC activity that is Ca++-independent was de creased in the v-rasHa initiated 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 their influence on the expression of epidermal markers in cells initi ated by v-rasHa transduction. Both constitutive and Ca++-induced expression ofTGK mRNA in v-rasHa keratinocytes was prevented by inactivating PKC with bryostatin or GF 109203X, selective PKC inhibitors. Bryostatin treatment also restored the Ca++-induced ex pression ofK1 andK10 to v-rasH. cells (not shown). These findings support the hypothesis that rasHa-induced alterations in keratino cyte differentiation are the result of chronic activation of a Ca++-de pendent isoform of PKC and reduced activity of a Ca++-indepen dent isoform of PKC. Thus, pharmacologic modulation of this pathway may provide an approach to reverse the neoplastic pheno type. Since PKC activation results in terminal differentiation of normal keratinocytes [9-11], the chronic activation ofPKC measured in v-rasHa keratinocytes that did not spontaneously undergo terminal differentiation presented a paradox. Neoplastic keratinocyte cell lines expressing an activated c-rasH• gene also have elevated diacyl glycerol levels [52] , but are resistant to terminal differentiation in duced by either Ca++ or phorbol esters. This suggested that qualitaCa2+(mM) 0.05
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Figure 2. Protein kinase C activation and the v-rasH- oncogene block expression of mRNA for spinous cell markers and enhance expres sion of mRNA for granular cell markers. Mouse keratinocytes were cultured in Eagles medium containing 8% fetal bovine serum and a Ca++ concentration of 0.05 mM. Some cultures were transduced with the v_rasH. oncogene nsing a defective retroviral vector as described previously (20). After 8 d in vitro a subset of cultures was switched to medium containing 0.12 mM Ca2+ for 24 h. In one group of cells the 0.12 mM Ca++ medium was supplemen ted with 125 pM 1-oleoyl-2-acetylglycerol (OAG), a syn thetic diacylglycerol that activa tes PKC [11]. Total RNA was isolated, sepa rated on a 1 % agarose gel containing 0.6 M formaldehyde and Northern blot
analysis performed as described previously (\ \,. A.t the timepoint examined
(24 h), transcription of the later markers, loricrin and filaggrin, is low in normal keratinocytes (48).
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YUSPA ET AL
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 family ofenzymes, members of which are differentially expressed 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 phosphorylation blocks ac tivation of PKC� [57]. This interaction between tyrosine kinases and PKC permits signaling through specific PKC isozyme in cells expressing an oncogenic rasfh gene and may represent a molecular block to differentiation in neoplastic keratinocytes. CHARACTERISTICS OF TUMORS AT HIGH RISK FOR MALIGNANT CONVERSION Variations in protocols used for inducing skin tumors yield sub groups of papillomas with either low or high risk for progression to malignancy [32] . High risk tumors represent a relatively small co hort of papillomas. They are selectively induced by limited expo sure to strong promoters or by more prolonged exposure to weak promoters [32] . Thus, they appear to be more sensitive to exogenous 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 low risk papillomas to identify differences that may suggest a mo lecular basis for the altered biologic potential of this class of benign tumors. Several tumor induction protocols were utilized that pref erentially produce each papilloma subtype. The tumors were evalu ated at the time they first erupted (8 -11 weeks) and sequentially during the course of tumor progression. Particular attention was concentrated on expression of specific integrins and cytokeratins as changes in these markers previously were identified with particular stages of skin tumor progression [58,59]. High-risk papillomas demonstrated basal and suprabasal expression of the a6p4 integrin, loss of keratin 1, and aberrant expression of keratin 13 when they first erupted at 8 weeks after promotion started (Fig 3a,b.c). In these tumors, a6p4 integrin expression coincided with an expansion of the proliferating compartment as indicated by suprabasal bromo deoxyuridine labeling [60]. In contrast, a6p4 immunostaining was confined to basal cells in low risk tumors, keratin 1 was abundant, and keratin 13 was absent or focal in the majority of this group, whereas proliferating cells were largely in the basal compartment (Fig 3d,e!). By 33 weeks after promotion started, a6p4 suprabasal expression continued to distinguish papillomas at higher risk for malignant conversion, but keratin 13 was expressed in all groups (Fig 4). At this time, high risk papillomas displayed focal expres sion of keratin 8 [46,61,62] and y-glutamyltranspeptidase, markers also found in chemically -induced carcinomas but not low-risk tumors (Fig 4). Keratin 13 was lost in most carcinomas. Keratin 8 and y-glutamyltranspeptidase were only expressed in a6p4 positive cells [60]. These results indicate that expression of a6p4 integrin in suprabasal strata serves as an early predictive marker to identify benign squamous tumors at high risk for malignant progression and that this marker is associated with a higher proliferation rate and suprabasal proliferation in high-risk tumors. GROWTH FACTORS AND THE RISK FOR MALIGNANT CONVERSION The transforming growth factor-p's are expressed in the epidermis and are growth inhibitors for both normal and initiated mouse kera tinocytes in vitro [63] ; altered TGF-p expression could influence the growth properties of high risk papillomas. Normal epidermis, and skin papillomas at low risk for malignant conversion express TGF P1 in the basal cell compartment and TGF-fJ2 in the suprabasal
THE
JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 3. Immunohistochemical markers distinguish high risk and low risk papillomas. Papillomas were generated on the skin of SEN CAR
mice by initiation with 10 pg of7,12-dimethylbenz[a]anthracene and pro motion with 12-0-tetradecanoylphorbol-13-acetate (TPA, 2 JAg, once weekly) to produce papillomas at low risk for malignant progression or mezerein (4 JAg, twice weekly) to produce papillomas at high risk for malig 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 ter. Low risk papillomas (d,ef) were excised at 11 weeks, also at first detec tion. Frozen sections were processed for immunochemistry using specific antibodies for the 0:6 integrin subunit (a,d), keratin 1 (b,e) and keratin 13 (ef). Reagents and procedures for frozen sectious and immunochemistry have been described previously [59]. Each panel represents a 5-p.m frozen section through the entire tumor. Bar, 200 JAm.
strata [1 7] . In low-risk tumors, 90% of the proliferating cells are confined to the basal compartment. In contrast, the majority of high risk papillomas are devoid of both TGF-p1 and TGF-fJ2 as soon as they arise; these tumors have up to 40% of the proliferating cells in the suprabasal layers [17]. Squamous cell carcinomas are also devoid of TGF-p [17,64], suggesting that they arise from the TGF-p deficient high-risk papillomas. In some high-risk papillomas, TGF-p loss can occur first and correlates with basal cell hyperpro liferation, whereas TGF-fJ2 loss correlates with suprabasal hyper proliferation. In tumors, loss of TGF-p is controlled at the post transcriptional level [17,64]. These results show that TGF-p ex pression and function are compartmentalized in epidermis and epidermal tumors, and that loss ofTGF-p is an early, biologically relevant risk factor for malignant progression. The importance ofTGF-p loss in progression was demonstrated directly using transgenic mice with null mutations introduced into the TGF-p110cus [65]. Keratinocyte cultures were established from epidermis of newborn mice either homozygous or heterozygous for the TGF-p1 null mutation or from wildtype littermates. The v H ras . oncogene was introduced by a defective retrovirus into cells of each genotype, and recipient cells were grafted onto nude mouse hosts. As expected, the v-rasH'-transduced wild type keratinocytes produced benign papillomas in grafts. In contrast, the tumors that developed from v-rasH'-transduced TGF-p1 null keratinocytes were either very dysplastic papillomas or squamous cell carcinomas. These results provide strong genetic proof to indicate that the loss of TGF-p1 is a critical step in the progression to malignancy in the . e�idermis. THE P 5 3 TUMOR SUPPRESSOR GENE AND MALIGNANT CONVERSION
The p53 gene is frequently mutated in human squamous cell and basal cell carcinomas of the skin [66-69]. This is likely to result from the direct mutational action of ultraviolet light since the most frequent mutagenic change is a C -+ T transition commonly found at C-C dinucleotides [66-69]. The E6 transforming gene of onco genic human papilloma viruses targets p53 protein to fonn a rapidly degraded complex. This action of E6 is essential for transforming activity of this virus for cultured keratinocytes [70-73]. Together these results suggest an important role for p53 in epidermal carcino genesis. In experimental mouse skin carcinogenesis, p53 mutations
VOL. 103, NO.5, SUPPLEMENT, NOVEMBER 1994
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ONCOGENES AND TUMOR SUPPRESSORS IN CARCINOGENESIS
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.,
Figure 4. Low-risk tumors are phenotypically distinct from carci nomas after 33 weeks of tumor promotion. Tumors were induced by the low-risk protocol described in the legend to Fig 3. After 33 weeks papillomas were excised for immunohistochemical staining of frozen sec tions (a,b,c). Simultaneous staining was p erformed on the few carcinomas that also develop from this protocol (d,eJ). The specific antibodies 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.
are extremely rare in papillomas, but mutations are detected in car cinomas [74-77]. This suggests that loss of p53 func tion may be relevant to malig nant conversion [78]. To test this question, kerati nocytes from skin of newborn p53 nul l transgenic mice [79] were culture d and transduced with the v-rasH> gene in experiments simi lar to those performed on TGF-fJ1 null mice. In all groups large tumors were apparent two weeks after grafting. Mice grafted with p53(+j+) keratinocytes developed papill omas (eigh t of eight), whereas all mice grafted with p53(-j-) keratinocytes developed squamous cell carcinomas (eight of eight) . p53(+j-) cells produced rapidly growing papillomas; 10 of 19 of these papillomas progressed to carcinomas within five weeks (Fig 5). Thus, loss of the p53 p rotein in combination with a mutant rasH> gene is associated with the malignant conversion stage of ep idermal carcinogenesis .
CONCLUSIONS The use of molecular approaches to anal yze established biologic concepts has provided insights into functional changes in cell physi ology during carcinogenesis. We now recognize that the initiated phenotype results from intrinsic changes in intracellular signa ling pathways, particula rly those related to terminal differentiation. When activation of the c-rasH> gene is responsible for initiation of keratinocyte neoplasia, a critical target is the PKC pathway. The five PKC isoforms in keratinocytes contribute to specific compo nents of the differentiation response. Presently, the analysis suggests that chronic activ ation of P KCa through increased cellular diacyl glycerol and functional inhibition ofPKCa due to phosphorylation on tyrosine residue(s) contribute to the altered differentiation re sponse characteristic of initiated epidennal cells. Extrinsic tumor promotion and premalignant progression are as sociated with changes in extrinsic signaling pathways. The control of prol iferation of neo lastic epidermal cells seems central to these stages. Se lective clona expansion of the neoplastic population is a requirement . The accumulation of genetic and chromosomal ch anges associated with rogression may be a consequence of a l tered proliferation contro . We now know that loss of expression or localization of the TGF-fJ class of growth factors is a common pathway through which premalignant progress ion proceeds in skin carcinogenesis. Loss of response by neoplastic cells to negative growth factors such as TGFfJ be an alternative pathway to achieve the same en dpoint [80-84. Loss ofTGF-fJ responsiveness has been associated with mutation and functional inactivation in p53 [85 ,86] and could account for the accelerated skin tumor pro gression observed for both p53 and TGF-fJ null mice transduced with the v-rasH> gene. The molecular ,basis for malignant conversion is still under inves-
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mar
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) littermates. In culture, ce lls of each genotype were transduced with the v-rasH> oncogene by a defective retroviral vector and removed from culture after 3 d for skin grafting as described [20]. Within 3-5 weeks, tumors developed on recipient mice that had either the benign (a,b) or malignant (e) phenotype. See text for quantitative evaluation. Bar, 1 cm.
tigation. In previous studies we have shown that a v-fos oncogene can cause malignant conversion of benign keratinocytes expressing a ras oncogene [87,88]. Because thefos gene product is a transcrip tion factor known to re gulate the expres sion of a number of genes that contain AP-1 consensus sequences [89,90], the converting ac tion of v-fos is likely to result from altered expression of normal cellular genes. We are currently attempting to identify those cellu lar genes that may be contributing to the malignant phenotype of keratinocytes. REFERENCES 1. YuspaS H, D)'u gosz AA: Cutaneous carcinogenesis: natural and experimental. In: Goldsmith L (ed.). PhYSiology, Biochonistry and Molecular Biology of The Skin. Oxford University Press, New York , 1991, pp 1365-1402 2. Yuspa S H, Morgan DL: Mouse skin cells resistant to terminal differentiation associated with initiation of carcino genesis. Nature 293:72-74,1981 3. Kulesz-Martin MF, Koehler B, Hennings H, Yuspa SH: Quantitative assay for carcinogen altered differentiation in mouse epidermal cells. Carcinogenesis 1:995-1006, 1980 4. Kilkenny AB, Morgan D, Spangler EF, Yuspa SH: Correlation of initiating po tency of skin carcinogens with potency to induce resistance to terminal differ entiation in cultured mouse keratinocytes. Cancer Res 45:2219-2225, 1985 5. YuspaS H, Morgan D, Lichti U, Spangler EF, MichaelD, Kilkenny A, Hennings H: Cultivation and characterization of cells derived from mouse skin papillo mas induced by an initiation-promotion protocol. Carcinogenesis 7:949 -958, 1986 6. Strickland]E, Greenhalgh DA, Koceva-Chyla A, Hennings H, Restrepo C, Ba laschak M, Yuspa SH: Development of murine epidermal cell lines which con tain an activated rasH. oncogene and form papillomas in skin grafts on athymic nude mouse hosts. Cancer Res 48:165-169,1988 7. Hennings H, Michael D, Lichti U, Yuspa SH: Response of carcinogen-altered mOuse epidermal cells to phorbol ester tumor promoters and calcium.] Invest Derm4toI88:60-65, 1987 8. Kulesz-Martin M, Kilkenny AE, Holbrook KA. Digernes V, Yuspa S H: Proper ties of carcinogen altered mouse epidermal cells resistant to calcium-induced terminal diKerentiation. CarcilUlgmesis 4:1367-1377,1983 9. Yuspa SH, Ben T, Hennin gs H. Lichti U: Phorbol ester rumor promoters induce epidermal tTansglutaminase activity. Biochon Bwphys Res Co",,,,"n 97:700708, 1980 10. Yuspa SH, Ben T, He nnings H, Lichti U: Divergent responses in epidermal basal cells exposed to the tumor promoter 12-0-tetradecanoylphorbol-13-acetate. Cancer Res 42:2344-2349, 1982 )' 11. D ugosz AA, Yuspa SH: Coordinate changes in gene expression which mark the spinous to granular cell tTansition in epidermis are regulated by protein kinase C.] Cell BioI 120:217-225, 1993 12. Yuspa SH, Ben T, Hennings H: The induction of epidermal transglutaminase and terminal differentiation by tumor promoters in cultured epidermal cells. Carci nogenesis 4:1413-1418, 1983 13. Hennings H, Robinson VA, Michael DM, Pernt GR,Jung R, YuspaS H: Devel opment of an in vitro analogue of initiated mouse epidermis to study tumor promoters and antipromoters. Cancer Res 50:4794-4800.1990 14. Hennin gs H, Lowry DT. Robinson VA. Morgan DL, Fujiki H. YuspaSH: Activ ity of diverse tumor promoters in a keratinocyte co-culture model of initiated epidermis. Carcinogenesis 13:2145-2151,1992 15. S trickland JE. Ueda M, Hennings H, Yuspa SH: A model for initiated mouse skin: suppression of papilloma but not carcinoma formation by normal epidermal cells in grafts on athymic nu de mice. Cancer Res 52:1439-1444, 1992
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Harper JR, Reynolds SH, Greenhalgh DA, S trickiand JE, Lacal JC, Yuspa SH: Analysis of the rasH oncogene and its p21 product in chemically induced skin tumors and tumor-derived cell lines. Carcinogenesis 8: 1821 - 1 825, 1987 Brown K, Quintanilla M, Ramsden M. Kerr m , Young S. Balmain A: v-ras genes from Harvey and BALB murine sarcoma viruses can act as initiators oftwo stage mouse skin carcinogenesis. Cell 46:447 - 456, 1986 Leon J, Kamino H, S teinberg JJ, Pellicer A: H-ras activation in benign and s elf-regressing skin tumors (keratoacanthomas) in both humans and an animal model system. Mol Cell Bioi 8:786- 793, 1988 Corominas M. Karnino H, Leon J, Pellicer A: Oncogene activation in human benign tumors of the skin (keratoacanthomas): is HRAS involved in dilferen tiation as well as proliferation? Proc Natl Acod Sci USA 86:6372- 6376, 1989 Ananthaswamy HN, Price JE, Goldberg LH , Bales ES: Detection and identifica tion of activated oncogenes in human skin cancers occurring on sun-exposed body sites . Cancer Res 48:3341 - 3346. 1 988 Ananthaswamy HN, Applegate LA, Goldb erg LH, Bales ES: Deletion of the c-Ha-ras-1 allele in human skin cancers. Mol Carcinog 2:298 -301, 1989 Suarez HG, Daya-Grosjean L, Schlaifer D, Nardeux P, Renault H, BosjL, Sarasin A: Activated oncogenes in human skin tumors from a repair-deficient syn drome, xeroderma pigmentosum. Cancer Res 49: 1 223 - 1228. 1989
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D!fferentiation 35: 1 43 - 150, 1987 Yuspa SH, Kilkenny AE, Steinert PM, Roop DR: Express ion ofmurine epidermal dilferentiation markers is tigh dy regulated by restricted extracellular calcium concentration s in vitro.J Cell Bioi 109:1207 - 1217, 1 989 Lichti U, Yuspa SH: Modulation of tissue and epidermal transglutaminases in mouse epidermal cells after treatment with 12-0-tetradecanoylphorbol-13-ac etate and/or retinoic acid in vivo and in culture. Cancer Res 48:74- 8 1 , 1 988 Tofrgard R, Yuspa SH, Roop DR: Keratin gene expre ssion in mouse skin tumors . and in mouse skin treated with 12-0-tetradecanoylphorbol-13-acetate. Can cer Res 45:5845 -5850, 1985 Lee E, Punnonen K, Cheng C, Glick A, D'ugosz A, Yuspa SH: Analysis of phospholipid metabolism in murine keratinocy tes transformed by the v-ras oncogene: rel ationship of phosphatidylinositol turnover and cytokine stimula tion to the transformed phen otype . Carcinogenesis 13:2367 - 2373, 1992 Lee E. Yuspa SH: Changes in inositol phosphate metabolism are associated with terminal dilferentiation and neoplasia in mouse keratinocyte s. Carcinogenesis 12:165 1 - 1 658, 1991 B lumberg PM, Pe ttit GR. Warren BS, Szallasi A, Schuman LD, S harkey NA, Nakakuma H, Dell 'Aquila ML, de Vries DJ : The protein kinase C pathway in tumor p romotion. Prog Clin Bioi res 298:20 1 - 212, 1989 Blumberg PM: Complexities of the protein kinase C pathway. Mol Carcinog 4:339 - 344, 1991 Stabel S, Parker PJ: Protein kinase C. Pharmacol Ther 51 :7 1 - 95, 199 1 Dlugosz AA, Mischak H , Mushinski JF, Yuspa SH: Transcripts encoding protein kinase C alpha. delta, epsilon, zeta, and eta are expressed in basal and dilferen tiating mouse keratinocytes in vitro and exhibit quantitative changes in neo plastic cells. Mol Carcinog 5:286- 292, 1992 Denning MF, D'ugosz AA, Howett MK, Yuspa SH: Expression of an oncogenic raslla gene in murine keratinocytes induces tyrosine phosphorylation and re duced activity of protein kinase C 6.J Bioi Chem 268:26079- 26081 , 1 993 Nischt R, Roop DR, Mehrel T, Yuspa SH, Rentrop M, Winter H, Schweizer J: Aberrant expression during two-stage mouse skin carcinogenesis of type I 47kDa Keratin, K 13 . normally associated with terminal differentiation ofinternal stratified epithelia. Mol Carcinog 1 :96- 1 08, 1988 Tennenbaum T, Yuspa SH , Grover A, Castronovo V, Sobel ME, Yamada Y, De Luca LM: Extracellular matrix receptors and mouse skin carcinogenesis: altered expression linked to appearance of early markers of tumor progression. Cancer Res 52:2966-2976, 1992 Tennenbaum T, Weiner AK, Belanger AJ , Glick AB, Hennings H, Yuspa SH: The suprabasal expression of o.6P4 integtin is associated with a hig h risk for malignant progression in mouse skin carcinogenesis. Cancer Res 53:48034810, 1993 Larcher F, Bauluz C, Diaz-Guerra M. Quintanilla M, Conti CJ, Ballestin C. Jorcano JL: Aberrant expression of the simple epithelial type II keratin 8 by mouse skin carcinomas but not papillomas . Mol Carcinog 6:1 12 - 1 21, 1992 Diaz-Guerra M, Haddow S, Bauluz C,JorcanoJL, Cano A, Balmain A, Quintan illa M: Expression of simple epithelial cytokeratius in mouse epidermal kerati nocytes harboring Harvey ras gene alterations. Cancer Res 52:680 - 687, 1992 Glick AB, Sporn MB, Yuspa SH: Altered regulation of TGF-p 1 and TGF-o. in primary keratinocytes and papillomas expressing v-Ha-ras. Mol Carcinog 4:2 1 0 - 219, 1991 Fowlis DJ, Flanders KC, Dullie E, Balmain A, Althurst RJ: Discordant trans forming growth factor P1 RNA and protein localization during chemical carci nogenesis of the skin. Cell Growth Differ 3:8 1 - 9 1 , 1992 Kulkarni AB. Huh GG, Becker D, Geiser A, Lyght M, Flanders KC, Roberts AB. Sporn MB, Ward JM. Karlsson S: Transforming growth bctor-p1 null muta tion in mice caus es excessive inflammatory response and early death. Pro, Nail Acad Sci USA 90:770 - 774, 1992 Brash DE, RudolphJA, Simon JA, Lin A, McKenua GJ, Baden HP , Halperin AJ, Ponten J: A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Pro, Natl Acad Sci USA 88:10124 - 10128, 1991 Rady p. Scinicariello F, Wagner RF Jr, Tyring SK: p53 mutations in basal cell carcinomas. Cancer Res 52:3804 - 3806, 1992 Ziegler A, Keffell DJ, Kunaia S. Sharma HW. Gailani M. SimonJA, Halperin AJ, Baden HP, Shapiro PE, Bale AE: Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers. Proc Natl Acad Sci USA 90:4216- 4220, 1 993 Kanjilal S, Pierceall WE, Cummings K, Kripke ML, Ananthaswamy HN: High frequency of p53 mutations in ultraviolet radiation-induced murine skin tumors: evidence for strand bias and tumor he terogeneity. Cancer Res 53:2961 2964, 1 993 Werness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with p 53 . Science 248:76 -79, 1990 Schdfner M. Werness BA, Huibregtse JM, Levine AJ, Howley PM: The B6 oncoprotein encoded by human papillomavirus types 1 6 and 1 8 promotes the degradation of p53. Cell 63: 1 1 29 - 1 1 36, 1990
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gen-specific mutational pattern in the p53 gene in ultraviolet B radiation-in duced squamous cell carcinomas of mouse skin. CtJnar Res 52:6400-6403, 1992 Ruggeri B, DiRado M, Zhang SY, Haner B, Goodrow T, Klein-SZanto AJ: Benzo[a]pyrene-indua:d murine skin tumon exhibit frequent and characteris tic G to T mutations in the p53 gene. ProcNatl Acad Sci USA 90: 1 0 1 3 - 10 17, 1993 Kemp C], Donehower LA, Bradley A, BalmaiD. A: Reduction of p53 gene dosage docs not increase initiation or promotion but enhances malignant progression or chemically induced skin rumors. CJl 74:8 1 3 - 822, 1993 Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CAJr, Butel Js, Bradley A: Mice cldicicnt for p53 are developmental ly normal but suscepti ble to spontaneous rumours. Nature 356:215 - 221, 1992 Sipes NJ, Lyons RM, Moses HL: Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor beta. Mol Carcinog 3:1 2 - 19, 1990
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during epidermal hyperproliferation and multistage tumorigenesis. Mol Car cinog 4: 129 - 137, 1991 Missero C, Ramon C, Dotto GP: Escape from transforming growth factor P conrrol and oncogene cooperation in skin tornor devel opment. p"" NIIll ACtUl Sd USA 88:96 1 3 - 9617, 1991 Reiss M, Sartomli AC: Regulation of growth and differentiation ofhuman kera tinocytes by Type B transforming growth factor and epidermal growth factor. Cancer Res 47:6705 - 6709, 1 987 Gerwin BI, Spil1are E, Forrester K, Lehman TA, KispertJ, WelshJA, Pfeifer AM, Lechner JF, Baker SJ. Vogelstein B: Mutant p53 can induce tumorigenic con version of human bronchial epithelial cells and reduce their responsiveness to a negative growth factor, transforming growth factor beta, . Proc Natl ACtUl Sci USA 89:2759 - 2763, 1992 Reiss M, vellucci VF, ZhOll ZL: Mutant p53 tornor suppressor gene causes resist ance to transforming growth factor P, in murine keratinoeytcs. CtJnar .Ra 53:899 - 904, 1993 Greenhalgh DA, Yuspa SH: Malignant conversion of murine squamous papil loma cell lines by transfection with the fos oncogene. Mol Carclnog 1: 1 34 - 143, 1988 Greenhal gh DA, Welty DJ, Player A, Yuspa SH: Two oncogenes, v-fos and v-ras, cooperate to convert normal keratinocytes to squamous cell carcinomas. Proc Natl Acad Sd USA 87:643 -647, 1990 Curran T, FranzaBR: Fos andjun: The AP-1 connection. CtlI 55:395 - 397, 1988 Rauscher FJ, Sambucerti LC, Curran T, Distel RJ, Spiegelman BM: Common DNA binding site for fos protein complexes and transcription factor APt . Cell 52:471 - 480, 1 988
MALIGNANCY
Role of Oncogenes and Tumor Suppressor Genes in Multistage Carcinogenesis 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 ogy as tools to study skin carcinogenesis has provided more precise localization of biochemical pathways that regulate the tumor phenotype. This approach has identified genetic changes that are characteristic of each of the specific stages of squamous cancer patho genesis: initiation, exogenous promotion, premalig nant progression, and malignant conversion. Initiation can result from mutations in a single gene, and the Harvey allele of the ras gene family has been identified as a frequent site for initiating mutations. Heterozy gous activating mutations in c-rasH- are dominant, and affected keratinocytes hyperproliferate and are resist ant to signals for terminal differentiation. An impor tant pathway impacted by c-rasH- activation is the pro tein kinase C (PKC) pathway, a major regulator of keratinocyte differentiation. Increased activity of PKCa and suppression ofPKC6by tyrosine phospho rylation contribute to the phenotypic consequences of rasH- gene activation in keratinocytes. Tumor promot ers disturb epidermal homeostasis and cause selective clonal expansion of initiated cells to produce multiple benign squamous papillomas. Resistance to differen-
tiation and enhanced growth rate of initiated cells im part a growth advantage when the epidermis is exposed to promoters. The frequency of premalignant progres sion varies among papillomas, and subpopulations at high risk for progression have been identified. These high-risk papillomas overexpress the a6p4 integrin and are deficient in transforming growth factor P1 and fJ2 peptides, two changes associated with a very high proliferation rate in this subset of tumors. The intro duction of an oncogenic rasH- gene into epidermal cells derived from transgenic mice with a null mutation in the TGFp1 gene have an accelerated rate of malignant progression when examined in .,ivo. Thus members of the TGFp gene family contribute a tumor-suppressor function in carcinogenesis. Accelerated malignant progression is also found with v-rasH- transduced kera tinocytes from skin of mice with a null mutation in the p53 gene. The similarities in risk for malignant con version by initiated keratinocytes from TGp1 and p53 null geneotypes suggest that a common, growth-re lated pathway may underly the tumor-suppressive functions of these proteins in the skin carcinogenesis mode!.] Invest Dermatol103:90S-95S, 1994
T
context of the intact epidermis. In cell culture, initiated keratino cytes dis play an altered response to signals for terminal differentia tion, a characteristic that provides a selective growth advantage under culture conditions favoring diffe rentiation [2-4]. Exploita tion of this differen ce in vitro h as been particularly hel pful in isolat ing initiated keratinocytes of mouse and human origin [5-8]. Exogenous tumor promotion causes the selective clonal expan sion of initiated cells to produce multiple benign squamous cell papillomas, each representing a clone of thousands of initiated cells. Potent exog enous promoters of the phorbol ester class activate, protein kinase C (PKC), a nd this enzyme activity accelerates epi dermal terminal differentiation [9-11]. Because initiated cells resist the induction of terminal differentiation by activators ofPKC [12], the differential response to phorbol esters favors the growth of the initiated subpopulation. This process recapitulates in vivo, the clonal selection of initiated cells by differentiation inducing agents in ke ratinocyte culture [13-15]. Squamous papillomas demonstrate a �ign proliferation rate and delayed maturation, properties that are analog ous to the phenotype of individual initiated cells in vitro [16,17]. Because most exogenous promoting agents are not muta genic [1], papillomas are d iploid [18,19], and a single genetic change in normal keratinocytes is sufficient to produce a papilloma pheno type [20-22], the mechanism of exogenous promotion is likely to
he introduction of the techniques of molecular biol ogy as tools to study skin carcinogenesis has provided more precise localization of pathway s that regulate the tumor phenotype However, the application of these tools to answer relevant questions has depended on the establishment of a conceptual framework that developed over four decades of research on the biology and cellular physiology of s kin cancer induction by chemical exposures. These studies indi cated that p redictable and progressive stages occurred during the clonal evolution of a normal keratinocyte into a squamous cell car cinoma, and both genetic an d epigenetic events contributed to these changes [1]. Progress in understanding carcinogen esis was enhanced when certain p roperties associated with a particular stag e of skin cancer pathogenesis were characterized [1]. Operational analyses, which defined the experimental requirements to produce a s pecific neoplastic phenotype, have also been important for understanding the process of tumor development (Fig 1). The earliest event documented in skin carcinogenesis initiation, is carcinogen induced and mutational in nature. Initiated keratino cytes express a subtle change in phenoty pe unrecognizable in the .
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Reprint requests to: Dr. Stuart Yuspa, Building 37, Room 3B25, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
0022-202Xj94jS07.00
Copyright © 1994 by The Society for Investigative Dermatology, Inc.
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Initiation
(Mutation)
Promotion
Premalignant Progression
(Epigenetic)
(Chromosomal Deletion,
ONCOGENES AND TUMOR SUPPRESSORS IN CARCINOGENESIS
Malignant Conversion (Mutation)
Duplication)
• Promoter Dependent
Promoter Independent
Figure 1. Operational stages in experimental skin carcinogenesis. Each stage is defined by the biologic consequences of a specific experimental protocol. This scheme serves as a framework for molecular analysis of multii stage carcinogenesis. See text and [1] for details.
be epigenetic in most cases. In the absence ofexposure to exogenous
promoters, initiated skin rarely develops tumors. Thus, exogenous promotion is a rate-limiting early event in carcinogenesis. Premalignant progression is most often a spontaneous process, independent of exogenous promoters [23,24]. Genetic studies indi cate that non-random sequential chromosomal aberrations are asso ciated with premalignant progression [19,25-28]. This stage of cancer pathogenesis constitutes the major time-dependent compo nent of carcinogenesis and must involve repeated episodes of cell selection since modal dominance of a specific chromosomal aberra tion indicates clonal outgrowth. Thus, at least one function of the relevant genetic events occurring during premalignant progression must impart a growth advantage on the affected cell. Malignant conversion of benign tumors is a relatively rare occur rence; less than 5% of papillomas spontaneously convert to cancers [23,24]. The risk for malignant conversion is variable among benign tumors, and subpopulations of papillomas with a much higher risk for malignant conversion have been identified [29 - 31]. Because the conversion frequency can be significantly increased by exposing 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 quency, and the subclass of papillomas at high risk for spontaneous conversion is also at higher risk for mutagen-induced conversion [30]. Thus, the factors that determine risk for conversion to malig nancy must increase susceptibility to spontaneous or induced ge netic changes. The identification of these factors would be an im portant goal in understanding cancer pathogenesis. THE
METABOLIC BASIS OF INITIATION INDUCED BY A ras ONCOGENE
The mutational basis for initiation of mouse epidermal carcinogen esis was supported by the discovery that the c-rasHa gene was fre quently mutated in chemically-induced squamous papillomas, and the site of the mutation was determined by the initiating carcinogen [32-37]. A causal relationship for a ras mutation and initiation of skin carcinogenesis was established when an activated v-rasHa onco gene was introduced into normal epidermis or cultured keratino cytes and recipient cells developed into squamous papillomas in vivo 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-rasHa gene was introduced via retroviral transduction to further characterize the initiated phenotype and to identify pathways affected by the expression of the v-rasHa p21 oncoprotein [45 -47]. Primary keratinocytes expressing the v-rasHa oncogene, intro duced by a defective retroviral vector, exhibit an altered response to signals for terminal differentiation in cell culture and produce squa mous papillomas when grafted onto nude mice [20,45-47]. Thus, this single gene change can accomplish all the requirements for initiation. In cultures of normal keratinocytes, increasing extracel lular Ca++ from 0.05 to 0.12 mM induces both mRNA transcripts and protein for several differentiation markers, including keratin 1 (Kl), KI0, loricrin, filaggrin, and keratinocyte transglutarninase (TGJ [48,49] (Fig 2). In v-rasHa keratinocytes, Ca++-mediated ex-
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pression of mRNA for K1 and K10 is blocked, loricrin and fiJaggrin expression are enhanced, 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 as phorbol esters and diacylglycerol (Fig 2) [11,12,47,50]. Furthermore, diacylglycerol levels are markedly elevated in v-rasH. keratinocytes and in keratinocytes expressing a mutated c-rasHa, when compared to normal epidermal cells [51,52]. Diacylglycerol is the endogenous activator ofPKC [53,54]. WhenPKC activity was measured in v-rasH. keratinocytes and compared to activity in nor mal epidermal cells, PKC activity that is Ca++-dependent was in creased whereas PKC activity that is Ca++-independent was de creased in the v-rasHa initiated 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 their influence on the expression of epidermal markers in cells initi ated by v-rasHa transduction. Both constitutive and Ca++-induced expression ofTGK mRNA in v-rasHa keratinocytes was prevented by inactivating PKC with bryostatin or GF 109203X, selective PKC inhibitors. Bryostatin treatment also restored the Ca++-induced ex pression ofK1 andK10 to v-rasH. cells (not shown). These findings support the hypothesis that rasHa-induced alterations in keratino cyte differentiation are the result of chronic activation of a Ca++-de pendent isoform of PKC and reduced activity of a Ca++-indepen dent isoform of PKC. Thus, pharmacologic modulation of this pathway may provide an approach to reverse the neoplastic pheno type. Since PKC activation results in terminal differentiation of normal keratinocytes [9-11], the chronic activation ofPKC measured in v-rasHa keratinocytes that did not spontaneously undergo terminal differentiation presented a paradox. Neoplastic keratinocyte cell lines expressing an activated c-rasH• gene also have elevated diacyl glycerol levels [52] , but are resistant to terminal differentiation in duced by either Ca++ or phorbol esters. This suggested that qualitaCa2+(mM) 0.05
0. 1 2
r-,. "0 "0 ... ... ... ... c 0
U
c 0
U
t!) <{ 0
0.05
0.12
r----JI "0 ... e ... ... c 0
U
c 0
U
'"
:r CI)
�
> I
K1
Loricrin
Filaggrin
Figure 2. Protein kinase C activation and the v-rasH- oncogene block expression of mRNA for spinous cell markers and enhance expres sion of mRNA for granular cell markers. Mouse keratinocytes were cultured in Eagles medium containing 8% fetal bovine serum and a Ca++ concentration of 0.05 mM. Some cultures were transduced with the v_rasH. oncogene nsing a defective retroviral vector as described previously (20). After 8 d in vitro a subset of cultures was switched to medium containing 0.12 mM Ca2+ for 24 h. In one group of cells the 0.12 mM Ca++ medium was supplemen ted with 125 pM 1-oleoyl-2-acetylglycerol (OAG), a syn thetic diacylglycerol that activa tes PKC [11]. Total RNA was isolated, sepa rated on a 1 % agarose gel containing 0.6 M formaldehyde and Northern blot
analysis performed as described previously (\ \,. A.t the timepoint examined
(24 h), transcription of the later markers, loricrin and filaggrin, is low in normal keratinocytes (48).
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YUSPA ET AL
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 family ofenzymes, members of which are differentially expressed 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 phosphorylation blocks ac tivation of PKC� [57]. This interaction between tyrosine kinases and PKC permits signaling through specific PKC isozyme in cells expressing an oncogenic rasfh gene and may represent a molecular block to differentiation in neoplastic keratinocytes. CHARACTERISTICS OF TUMORS AT HIGH RISK FOR MALIGNANT CONVERSION Variations in protocols used for inducing skin tumors yield sub groups of papillomas with either low or high risk for progression to malignancy [32] . High risk tumors represent a relatively small co hort of papillomas. They are selectively induced by limited expo sure to strong promoters or by more prolonged exposure to weak promoters [32] . Thus, they appear to be more sensitive to exogenous 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 low risk papillomas to identify differences that may suggest a mo lecular basis for the altered biologic potential of this class of benign tumors. Several tumor induction protocols were utilized that pref erentially produce each papilloma subtype. The tumors were evalu ated at the time they first erupted (8 -11 weeks) and sequentially during the course of tumor progression. Particular attention was concentrated on expression of specific integrins and cytokeratins as changes in these markers previously were identified with particular stages of skin tumor progression [58,59]. High-risk papillomas demonstrated basal and suprabasal expression of the a6p4 integrin, loss of keratin 1, and aberrant expression of keratin 13 when they first erupted at 8 weeks after promotion started (Fig 3a,b.c). In these tumors, a6p4 integrin expression coincided with an expansion of the proliferating compartment as indicated by suprabasal bromo deoxyuridine labeling [60]. In contrast, a6p4 immunostaining was confined to basal cells in low risk tumors, keratin 1 was abundant, and keratin 13 was absent or focal in the majority of this group, whereas proliferating cells were largely in the basal compartment (Fig 3d,e!). By 33 weeks after promotion started, a6p4 suprabasal expression continued to distinguish papillomas at higher risk for malignant conversion, but keratin 13 was expressed in all groups (Fig 4). At this time, high risk papillomas displayed focal expres sion of keratin 8 [46,61,62] and y-glutamyltranspeptidase, markers also found in chemically -induced carcinomas but not low-risk tumors (Fig 4). Keratin 13 was lost in most carcinomas. Keratin 8 and y-glutamyltranspeptidase were only expressed in a6p4 positive cells [60]. These results indicate that expression of a6p4 integrin in suprabasal strata serves as an early predictive marker to identify benign squamous tumors at high risk for malignant progression and that this marker is associated with a higher proliferation rate and suprabasal proliferation in high-risk tumors. GROWTH FACTORS AND THE RISK FOR MALIGNANT CONVERSION The transforming growth factor-p's are expressed in the epidermis and are growth inhibitors for both normal and initiated mouse kera tinocytes in vitro [63] ; altered TGF-p expression could influence the growth properties of high risk papillomas. Normal epidermis, and skin papillomas at low risk for malignant conversion express TGF P1 in the basal cell compartment and TGF-fJ2 in the suprabasal
THE
JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 3. Immunohistochemical markers distinguish high risk and low risk papillomas. Papillomas were generated on the skin of SEN CAR
mice by initiation with 10 pg of7,12-dimethylbenz[a]anthracene and pro motion with 12-0-tetradecanoylphorbol-13-acetate (TPA, 2 JAg, once weekly) to produce papillomas at low risk for malignant progression or mezerein (4 JAg, twice weekly) to produce papillomas at high risk for malig 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 ter. Low risk papillomas (d,ef) were excised at 11 weeks, also at first detec tion. Frozen sections were processed for immunochemistry using specific antibodies for the 0:6 integrin subunit (a,d), keratin 1 (b,e) and keratin 13 (ef). Reagents and procedures for frozen sectious and immunochemistry have been described previously [59]. Each panel represents a 5-p.m frozen section through the entire tumor. Bar, 200 JAm.
strata [1 7] . In low-risk tumors, 90% of the proliferating cells are confined to the basal compartment. In contrast, the majority of high risk papillomas are devoid of both TGF-p1 and TGF-fJ2 as soon as they arise; these tumors have up to 40% of the proliferating cells in the suprabasal layers [17]. Squamous cell carcinomas are also devoid of TGF-p [17,64], suggesting that they arise from the TGF-p deficient high-risk papillomas. In some high-risk papillomas, TGF-p loss can occur first and correlates with basal cell hyperpro liferation, whereas TGF-fJ2 loss correlates with suprabasal hyper proliferation. In tumors, loss of TGF-p is controlled at the post transcriptional level [17,64]. These results show that TGF-p ex pression and function are compartmentalized in epidermis and epidermal tumors, and that loss ofTGF-p is an early, biologically relevant risk factor for malignant progression. The importance ofTGF-p loss in progression was demonstrated directly using transgenic mice with null mutations introduced into the TGF-p110cus [65]. Keratinocyte cultures were established from epidermis of newborn mice either homozygous or heterozygous for the TGF-p1 null mutation or from wildtype littermates. The v H ras . oncogene was introduced by a defective retrovirus into cells of each genotype, and recipient cells were grafted onto nude mouse hosts. As expected, the v-rasH'-transduced wild type keratinocytes produced benign papillomas in grafts. In contrast, the tumors that developed from v-rasH'-transduced TGF-p1 null keratinocytes were either very dysplastic papillomas or squamous cell carcinomas. These results provide strong genetic proof to indicate that the loss of TGF-p1 is a critical step in the progression to malignancy in the . e�idermis. THE P 5 3 TUMOR SUPPRESSOR GENE AND MALIGNANT CONVERSION
The p53 gene is frequently mutated in human squamous cell and basal cell carcinomas of the skin [66-69]. This is likely to result from the direct mutational action of ultraviolet light since the most frequent mutagenic change is a C -+ T transition commonly found at C-C dinucleotides [66-69]. The E6 transforming gene of onco genic human papilloma viruses targets p53 protein to fonn a rapidly degraded complex. This action of E6 is essential for transforming activity of this virus for cultured keratinocytes [70-73]. Together these results suggest an important role for p53 in epidermal carcino genesis. In experimental mouse skin carcinogenesis, p53 mutations
VOL. 103, NO.5, SUPPLEMENT, NOVEMBER 1994
.�
"
,;-. '
ONCOGENES AND TUMOR SUPPRESSORS IN CARCINOGENESIS
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Figure 4. Low-risk tumors are phenotypically distinct from carci nomas after 33 weeks of tumor promotion. Tumors were induced by the low-risk protocol described in the legend to Fig 3. After 33 weeks papillomas were excised for immunohistochemical staining of frozen sec tions (a,b,c). Simultaneous staining was p erformed on the few carcinomas that also develop from this protocol (d,eJ). The specific antibodies 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.
are extremely rare in papillomas, but mutations are detected in car cinomas [74-77]. This suggests that loss of p53 func tion may be relevant to malig nant conversion [78]. To test this question, kerati nocytes from skin of newborn p53 nul l transgenic mice [79] were culture d and transduced with the v-rasH> gene in experiments simi lar to those performed on TGF-fJ1 null mice. In all groups large tumors were apparent two weeks after grafting. Mice grafted with p53(+j+) keratinocytes developed papill omas (eigh t of eight), whereas all mice grafted with p53(-j-) keratinocytes developed squamous cell carcinomas (eight of eight) . p53(+j-) cells produced rapidly growing papillomas; 10 of 19 of these papillomas progressed to carcinomas within five weeks (Fig 5). Thus, loss of the p53 p rotein in combination with a mutant rasH> gene is associated with the malignant conversion stage of ep idermal carcinogenesis .
CONCLUSIONS The use of molecular approaches to anal yze established biologic concepts has provided insights into functional changes in cell physi ology during carcinogenesis. We now recognize that the initiated phenotype results from intrinsic changes in intracellular signa ling pathways, particula rly those related to terminal differentiation. When activation of the c-rasH> gene is responsible for initiation of keratinocyte neoplasia, a critical target is the PKC pathway. The five PKC isoforms in keratinocytes contribute to specific compo nents of the differentiation response. Presently, the analysis suggests that chronic activ ation of P KCa through increased cellular diacyl glycerol and functional inhibition ofPKCa due to phosphorylation on tyrosine residue(s) contribute to the altered differentiation re sponse characteristic of initiated epidennal cells. Extrinsic tumor promotion and premalignant progression are as sociated with changes in extrinsic signaling pathways. The control of prol iferation of neo lastic epidermal cells seems central to these stages. Se lective clona expansion of the neoplastic population is a requirement . The accumulation of genetic and chromosomal ch anges associated with rogression may be a consequence of a l tered proliferation contro . We now know that loss of expression or localization of the TGF-fJ class of growth factors is a common pathway through which premalignant progress ion proceeds in skin carcinogenesis. Loss of response by neoplastic cells to negative growth factors such as TGFfJ be an alternative pathway to achieve the same en dpoint [80-84. Loss ofTGF-fJ responsiveness has been associated with mutation and functional inactivation in p53 [85 ,86] and could account for the accelerated skin tumor pro gression observed for both p53 and TGF-fJ null mice transduced with the v-rasH> gene. The molecular ,basis for malignant conversion is still under inves-
r
f
mar
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) littermates. In culture, ce lls of each genotype were transduced with the v-rasH> oncogene by a defective retroviral vector and removed from culture after 3 d for skin grafting as described [20]. Within 3-5 weeks, tumors developed on recipient mice that had either the benign (a,b) or malignant (e) phenotype. See text for quantitative evaluation. Bar, 1 cm.
tigation. In previous studies we have shown that a v-fos oncogene can cause malignant conversion of benign keratinocytes expressing a ras oncogene [87,88]. Because thefos gene product is a transcrip tion factor known to re gulate the expres sion of a number of genes that contain AP-1 consensus sequences [89,90], the converting ac tion of v-fos is likely to result from altered expression of normal cellular genes. We are currently attempting to identify those cellu lar genes that may be contributing to the malignant phenotype of keratinocytes. REFERENCES 1. YuspaS H, D)'u gosz AA: Cutaneous carcinogenesis: natural and experimental. In: Goldsmith L (ed.). PhYSiology, Biochonistry and Molecular Biology of The Skin. Oxford University Press, New York , 1991, pp 1365-1402 2. Yuspa S H, Morgan DL: Mouse skin cells resistant to terminal differentiation associated with initiation of carcino genesis. Nature 293:72-74,1981 3. Kulesz-Martin MF, Koehler B, Hennings H, Yuspa SH: Quantitative assay for carcinogen altered differentiation in mouse epidermal cells. Carcinogenesis 1:995-1006, 1980 4. Kilkenny AB, Morgan D, Spangler EF, Yuspa SH: Correlation of initiating po tency of skin carcinogens with potency to induce resistance to terminal differ entiation in cultured mouse keratinocytes. Cancer Res 45:2219-2225, 1985 5. YuspaS H, Morgan D, Lichti U, Spangler EF, MichaelD, Kilkenny A, Hennings H: Cultivation and characterization of cells derived from mouse skin papillo mas induced by an initiation-promotion protocol. Carcinogenesis 7:949 -958, 1986 6. Strickland]E, Greenhalgh DA, Koceva-Chyla A, Hennings H, Restrepo C, Ba laschak M, Yuspa SH: Development of murine epidermal cell lines which con tain an activated rasH. oncogene and form papillomas in skin grafts on athymic nude mouse hosts. Cancer Res 48:165-169,1988 7. Hennings H, Michael D, Lichti U, Yuspa SH: Response of carcinogen-altered mOuse epidermal cells to phorbol ester tumor promoters and calcium.] Invest Derm4toI88:60-65, 1987 8. Kulesz-Martin M, Kilkenny AE, Holbrook KA. Digernes V, Yuspa S H: Proper ties of carcinogen altered mouse epidermal cells resistant to calcium-induced terminal diKerentiation. CarcilUlgmesis 4:1367-1377,1983 9. Yuspa SH, Ben T, Hennin gs H. Lichti U: Phorbol ester rumor promoters induce epidermal tTansglutaminase activity. Biochon Bwphys Res Co",,,,"n 97:700708, 1980 10. Yuspa SH, Ben T, He nnings H, Lichti U: Divergent responses in epidermal basal cells exposed to the tumor promoter 12-0-tetradecanoylphorbol-13-acetate. Cancer Res 42:2344-2349, 1982 )' 11. D ugosz AA, Yuspa SH: Coordinate changes in gene expression which mark the spinous to granular cell tTansition in epidermis are regulated by protein kinase C.] Cell BioI 120:217-225, 1993 12. Yuspa SH, Ben T, Hennings H: The induction of epidermal transglutaminase and terminal differentiation by tumor promoters in cultured epidermal cells. Carci nogenesis 4:1413-1418, 1983 13. Hennings H, Robinson VA, Michael DM, Pernt GR,Jung R, YuspaS H: Devel opment of an in vitro analogue of initiated mouse epidermis to study tumor promoters and antipromoters. Cancer Res 50:4794-4800.1990 14. Hennin gs H, Lowry DT. Robinson VA. Morgan DL, Fujiki H. YuspaSH: Activ ity of diverse tumor promoters in a keratinocyte co-culture model of initiated epidermis. Carcinogenesis 13:2145-2151,1992 15. S trickland JE. Ueda M, Hennings H, Yuspa SH: A model for initiated mouse skin: suppression of papilloma but not carcinoma formation by normal epidermal cells in grafts on athymic nu de mice. Cancer Res 52:1439-1444, 1992
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gen-specific mutational pattern in the p53 gene in ultraviolet B radiation-in duced squamous cell carcinomas of mouse skin. CtJnar Res 52:6400-6403, 1992 Ruggeri B, DiRado M, Zhang SY, Haner B, Goodrow T, Klein-SZanto AJ: Benzo[a]pyrene-indua:d murine skin tumon exhibit frequent and characteris tic G to T mutations in the p53 gene. ProcNatl Acad Sci USA 90: 1 0 1 3 - 10 17, 1993 Kemp C], Donehower LA, Bradley A, BalmaiD. A: Reduction of p53 gene dosage docs not increase initiation or promotion but enhances malignant progression or chemically induced skin rumors. CJl 74:8 1 3 - 822, 1993 Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CAJr, Butel Js, Bradley A: Mice cldicicnt for p53 are developmental ly normal but suscepti ble to spontaneous rumours. Nature 356:215 - 221, 1992 Sipes NJ, Lyons RM, Moses HL: Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor beta. Mol Carcinog 3:1 2 - 19, 1990
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