(CANCER RESEARCH 58. 1435-1443. April 1. 1998] Topical Retinoic Acid Reduces Skin Papilloma Formation but Resistant Papillomas Are at High Risk for Malignant Conversion1 Tamar Tennenbaum, David Lowry, Nadine Darwiche, David L. Morgan, Marina Gartsbein, Laura Hansen, Luigi M. De Luca, Henry Hennings, and Stuart H. Yuspa2 Department of Life Sciences, Bar ¡IanUniversity, Kamal-Can, 52 900 Israel ¡T.T., M. C.], and Laboratory of Cellular Carcinogenesis and Tunmr Promotion, Divisimi <ifBasil- Sciences. National Cancer Institute. NIH. Belhesda. Maryland 20892-4355 ¡D.L. N. D.. D. L M.. L H., L M. D. L. H. H.. S. H. K/ ABSTRACT Retinoic acid (RA) was topically applied to the skin of Sencar mice during the promotion phase of specific tumor induction protocols that produce papillomas at low (12-iMetradecanoylphorbol-13-acetate pro moted, TPA) or high (mezerein-promoted) risk for premalignant progres sion and malignant conversion. RA consistently reduced the yield of papillomas and carcinomas in both protocols, but the frequency of ma lignant conversion in papillomas that emerged during RA treatment was not reduced. When TPA was reapplied after cessation of RA treatment, the number of papillomas increased 2-fold, suggesting that RA had not eliminated initiated cells. In vitro, RA prevented the emergence of trans formed keratinocytes in an assay that mimics malignant conversion, sug gesting that RA can suppress conversion if applied during the stage of premalignant progression. Examination of tumor markers at weeks 14 and 22 of the tumor-induction experiments in vivo indicated that papillo mas evolving during RA treatment exhibited a phenotype of high pro gression risk, even in the TPA-promoted groups. In the majority of these tumors, the a6ß4 integrin and retinoid X receptor a transcripts were detected suprabasally, indicating an advanced state of premalignant pro gression. RA-treated tumors also expressed higher levels of transcripts for transforming growth factor (TGF)-ßl and localized TGF-/31 peptide in the basal portions of the tumor fronds. Because up-regulated expression of TGF-ßl suppresses papilloma formation, these studies suggest a mecha nism whereby RA can prevent papilloma eruption via a TGF-ßinterme diate, but papillomas resistant to RA may have altered 1(¡I-/> signaling and progress to carcinomas at an increased frequency. INTRODUCTION In experimental mouse skin carcinogenesis. the normal skin kera- tinocyte evolves through a well-differentiated squamous papilloma stage with progressively dysplastic foci, finally developing into inva sive squamous carcinoma. This phenotypic evolution is coincident with reproducible genetic and epigenetic changes ( 1). Premalignant progression is characterized by an increased proliferative compart ment, loss of normal differentiative function, acquisition of migratory potential, and aberrant cell-cell interactions. At the genetic level, progressing papillomas develop trisomies of chromosomes 6 and 7 and become aneuploid (2. 3). Where a mutation in a single alÃ-eleof the ra.vHa gene produced the papilloma phenotype, tumor clones undergoing premalignant progression often display duplication of the chromosome containing the mutant alÃ-ele,and carcinomas frequently are deleted of the chromosome containing the normal ras11"alÃ-ele(4). Experimentally produced genetic modifications can alter premalig nant progression. For example, papillomas generated by introducing an oncogenic ras"" gene into normal keratinocytes become malignant Received 9/18/97; accepted 1/26/98. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' T. T. is supported by a grant from the Smokeless Tobacco Research Council. : To whom requests for reprints should be addressed, at Laboratory of Cellular Carcinogenesis and Tumor Promotion. Division of Basic Sciences, National Cancer Institute. NIH. Building 37. Room 3B25. 37 Convent Drive MSC 4255. Bethesda. MD 20892-4255. Phone: (301)496-2162; Fax: (301)496-8709; E-mail: yuspas@dc37a. nci.nih.gov. when an oncogenic fits gene is added (5. 6). Conversely, genetic- deletion of the normal fos alÃ-eleprevents malignant progression of benign tumors induced by oncogenic ra.v, suggesting that AP-1 tran- scriptional activity is essential for progression (7). AP-I activity increases during premalignant progression of squamous tumors, sup porting a contributory role for this family of transcription factors in premalignant progression (8). Previous studies have demonstrated that squamous papillomas are at variable risk to undergo premalignant progression, and the high-risk phenotype is determined early in the neoplastic process (9). To ex plore the biological and molecular basis for the high-risk phenotype. we have developed both specific tumor induction protocols and de fined phenotypic markers to evaluate benign tumors that progress to malignancy at an accelerated rate (9, 10). Among the phenotypic changes in papillomas associated with premalignant progression are the loss of keratin 1 and increase in keratin 13 expression, an expan sion of both the proliferative cell compartment and the number of cells expressing the a6ß4integrin. and loss of TGF'-ßl in the basal cell compartment of the tumor papillae (9, 11, 12). The loss of TGF-/31 has been causally linked to the rapidly progressing squamous tumor phenotype in studies using keratinocytes from mice genetically de leted for the TGF-ßlalÃ-ele(12). Although these markers distinguish the high- and low-risk phenotypes early in the process of benign tumor formation, tumors generated from low-risk protocols acquire similar changes at later times. Thus, the molecular control of prema lignant progression is similar in low-risk tumors, but the process is accelerated in the high-risk lesions (9, 12). Nonmelanoma skin cancers are the most common malignancies in humans. Although the major carcinogen for human skin cancer is environmental exposure to sunlight, the evolution of squamous cell carcinoma on human skin has many similarities to the induction of squamous tumors by chemicals on mouse skin (I. 13). The precursor lesion to human squamous cell carcinoma, actinic keratosis, has variable risk for premalignant progression (14, 15). Although epithe lial skin cancer is rarely fatal, it is associated with pain, cosmetic complications, and substantial medical expenses. The high frequency of these tumors has promoted strategies to prevent tumor development on sun-damaged skin. Among the most discussed and tested agents for this purpose are the retinoids (16, 17). Systemic administration of 13-a.s'-RA has been effective in preventing skin cancer development in patients with xeroderma pigmentosum. but the treatment is limited by toxic side effects (18). Topical retinoids have met with mixed results in human trials and have also produced ambiguous findings in chemically initiated or UV light-treated mouse skin, preventing tumor formation in some studies and enhancing tumor formation or malig nant conversion in others (17, 19). In contrast, dietary RA has been effective in preventing carcinoma development from chemically in duced mouse skin papillomas using both low-risk and high-risk tumor induction protocols (20-23). 1The abbreviations used are: TGF. transforming growth factor; RA. all-rrÃ-Ã-/i.v-retinoic acid; RAR. RA receptor: RXR, retinoid X receptor; DMBA. 7.12-dimethylbenz(«(anthra cene: TPA. l2-O-tetradecanoylphorbol-13-acetate; MNNG. /V-methyl-iV'-nitro-A'- nitrosoguanidine. 1435 Research. on October 18, 2015. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from