[CANCER RESEARCH 55. 1883-1893, May 1, 1995] Autocrine Transforming Growth Factor a Is Dispensible for v-rasHa-induced Epidermal Neoplasia: Potential Involvement of Alternate Epidermal Growth Factor Receptor Ligands1 Andrzej A. Dlugosz,2 Christina Cheng, Erin K. Williams, Nadine Darwiche, Peter J. Dempsey, Bruce Mann, Ashley R. Dunn, Robert J. Coffey, Jr., and Stuart H. Yuspa Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892 [A. A. D., C. C„E. K. W., N. D., S. H. YJ; Departments of Medicine and Cell Biology, Vanderbill University School of Medicine, Nashville, Tennessee 37232 ¡P.J. D., R. J. C.¡;and Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria 3050, Australia [B. M., A. R. DJ ABSTRACT Autocrine epidermal growth factor receptor activation by transforming growth factor a (TGFa) has been implicated in growth stimulation during epithelial neoplasia. Using keratinocytes isolated from mice with genetic defects in TGFa expression, we tested whether TGFa is required for transformation by the v-ras"" oncogene. Introduction of v-ras"" into primary epidermal cultures using a retroviral vector stimulated growth of both control (TGFa +/+, BALB/c) and TGFa-deficient (TGFa -/-, ira-I i keratinocytes. Moreover, v-ra.v"1' elicited characteristic changes in marker expression (keratin 1 was suppressed; keratin 8 was induced), previously shown to be associated with epidermal growth factor (EGF) receptor activation, in both TGFa +/+ and TGFa -/- keratinocytes. v-ra.v"" markedly increased secreted o 10-fold) and cell-associated (2-3- fold) TGFa levels in keratinocytes from TGFa +/+ and BALB/c mice, but not TGFa —/— or wa-l mice. Based on Northern blot analysis, \-ras"" induced striking up-regulation of transcripts encoding the additional EGF family members amphiregulin, heparin-binding EGF-like growth factor, and betacellulin in cultured keratinocytes from all four mouse strains. Interestingly, in addition to the normal 4.5-kilobase TGFa transcript, wa-l keratinocytes expressed two additional TGFa transcripts, 4.7 and 5.2 kilobases long. All three transcripts were up-regulated in response to \-ras"", as well as exogenous TGFa or keratinocyte growth factor treat ment, and were also detected in RNA isolated from wa-l brain and skin. In vivo, v-rasH" keratinocytes from control as well as TGFa-deficient mice produced squamous tumors when grafted onto nude mice, and these lesions expressed high levels of amphiregulin, heparin-binding EGF-like growth factor, and betacellulin mRNA, regardless of their TGFa status. These findings indicate that TGFa is not essential for epidermal neoplasia induced by the v-ra.v"1'oncogene and suggest that another EGF family member(s) may contribute to autocrine growth stimulation of ra.v-trans- formed keratinocytes. INTRODUCTION The majority of malignancies affecting humans arise in the epithe lial component of organs including the skin, breast, lung, and gastro intestinal and genitourinary tracts. Both clinical data and results of studies using animal models indicate that epithelial neoplasia is a stepwise process driven by the accumulation of multiple genetic and epigenetic changes (reviewed in Ref. l). Chemical carcinogenesis in mouse skin has provided a powerful experimental model for studying both early changes involved in the development of premalignant lesions, as well as later events important in neoplastic progression (2). Application of a subtumorigenic dose of the chemical carcinogen Received 12/1/94; accepted 2/24/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance wilh 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by a Thomas B. Fitzpatrick-Kao Corporation Research Award (A. A. D.), and by Grant MCICA46413 and by the Veterans Adminis tration (R. J. C.). R. J. C. is a Veterans Administration Clinical Investigator. 2 To whom requests for reprints should be addressed, at LCCTP/NCI, Bldg 37/Rm 3B08, 37 Convent DR MSC 4255, Bethesda, MD 20892-4255. DMBA3 to mouse skin results in mutation of the c-ra.vHa gene (3). Repeated treatment with the phorbol ester 12-O-tetradecanoylphor- bol-13-acetate promotes the clonal expansion of cells expressing the mutant ras gene to form a benign tumor capable of autonomous growth. The central role of ras in the pathogenesis of benign papil- lomas was corroborated in studies where introduction of oncogenic ras into wounded skin could replace chemical initiation by DMBA (4), as well as nude mouse experiments in which primary keratino cytes transduced with v-rasHa in vitro produced tumors when grafted as a reconstituted skin (5). In addition to its involvement in early stages of cutaneous neoplasia, raÃ-has also been implicated in malig nant progression of skin tumors (reviewed in Ref. l). Since mutation of cellular raÃ-genes has been detected in a variety of human cancers (6, 7), understanding how activated Ras influences keratinocyte biology is likely to provide insights relevant to human disease. Numerous studies have documented alterations in growth factor signaling pathways during epithelial neoplasia, suggesting that these changes may be important in the development and/or maintenance of the neoplastic phenotype. TGFa and its receptor, EGFR, appear to play a particularly prominent role in epithelial neoplasia (reviewed in Refs. 8 and 9). Expression of v-raiHa in cultured mouse keratinocytes induces a large increase in TGFa mRNA and protein expression (10, 11). In addition, high levels of TGFa have been detected in papillo- mas produced either by grafting v-raiHa keratinocytes onto nude mice (10), or generated using a two-stage chemical carcinogenesis protocol (12-14). Treatment of normal keratinocytes in vitro with exogenous TGFa elicits many of the same phenotypic alterations as v-raÃ-Ha(11), suggesting that oncogenic raÃ-may alter keratinocyte behavior, at least in part, via activation of the TGFa/EGFR signaling pathway. Con sistent with this concept, transgenic mice in which TGFa is targeted to the epidermis using keratin 14 (K14) or keratin 1 (Kl) promoters develop squamous papillomas following tumor promotion by wound ing or 12-O-tetradecanoylphorbol-13-acetate treatment (15, 16), sug gesting that overexpression of TGFa can substitute for raÃ-Hagene activation as an initiator in two-stage cutaneous carcinogenesis. It has also been suggested that TGFa can function as a tumor-promoting stimulus, based on the results of two-stage chemical carcinogenesis studies (13) and grafting experiments using papilloma cell lines in an environment where TGFa is overexpressed (17). The development of cutaneous papillomas and carcinomas following DMBA treatment of a TGFa transgenic mouse strain (18) supports the concept that TGFa can function as a tumor promoter. In addition to TGFa and EGF, several other structurally related proteins bind and activate the EGFR, including amphiregulin (19), HB-EGF (20), and betacellulin (21). The role of these "alternate" EGFR ligands in the physiology of normal or neoplastic keratinocytes has not yet been defined. 3 The abbreviations used are: DMBA, 7,12-dimethylbenz[a]anthracene; EGFR, epi dermal growth factor receptor; TGFa, transforming growth factor a; HB-EGF, heparin- binding epidermal growth factor-like growth factor; KGF, keratinocyte growth factor; CM, conditioned medium; TBS, Tris-buffered saline (20 mM Tris, pH 7.4-500 IHMNaCI); poly(A)+ RNA, polyadenylated RNA; PL-7, ribosomal protein L7. 1883 Research. on September 1, 2021. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from