Role of Oncogenes and Tumor Suppressor Genes in Multistage Carcinogenesis
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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 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 0022-202Xj94jS07.00 Copyright © 1994 by The Society for Investigative Dermatology, Inc. 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