[CANCER RESEARCH 64, 538 –546, January 15, 2004] E6 and E7 Oncoproteins Induce Distinct Patterns of Chromosomal Aneuploidy in Skin Tumors from Transgenic Mice Anthony J. Schaeffer, 1 Marie Nguyen, 2 Amy Liem, 2 Denis Lee, 2 Cristina Montagna, 1 Paul F. Lambert, 2 Thomas Ried, 1 and Michael J. Difilippantonio 1 1 Genetics Branch, Center for Cancer Research, National Cancer Institute/NIH, Bethesda, Maryland; and 2 McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin ABSTRACT Inactivation of the tumor suppressor genes p53 and Rb are two of the most common genetic alterations in cancer cells. We use a mouse model to dissect the consequences of compromising the function of either of these genes on the maintenance of genomic stability. Thirteen cell lines estab- lished from skin tumors of mice expressing either the E6 or E7 oncopro- tein of the human papillomavirus (HPV) type 16 under control of the keratin 14 promoter were analyzed by comparative genomic hybridiza- tion, spectral karyotyping and fluorescence in situ hybridization, reverse transcription-PCR, and mutation analysis. Deducing from the wealth of molecular cytogenetic data available from human cancers, we hypothe- sized that the more benign tumors in mice expressing E7 would be distinct from the more aggressive lesions in E6 transgenic mice. Tumorigenesis in E6-expressing mice required specifically the selection and maintenance of cells with extra copies of chromosome 6. Aneuploidy of chromosome 6 was independent of activating mutations in H-ras on chromosome 7. Expres- sion of either E6 or E7 resulted in centrosome aberrations, indicating that each viral oncoprotein interferes independently with the centrosome cycle. Although centrosome aberrations are consistent with development of aneuploidy, no direct correlation was evident between the degree of aneuploidy and the percentage of cells with aberrant centrosomes. Our results show that although aneuploidy and centrosome aberrations are present in tumor cells from mice expressing either E6 or E7, tumorigenesis via E6 requires copy number increases of mouse chromosome 6, which is partially orthologous to human chromosome 3q, a region gained in HPV- associated carcinomas. INTRODUCTION The retinoblastoma susceptibility gene (Rb) and p53 are tumor suppressor genes whose proper functioning is vital to maintaining genomic stability and normal cell growth and differentiation. Re- sponding to extracellular and intracellular signals, Rb is responsible for the correct timing of the G1-S transition and for regulating the S, G2, and M phases of the cell cycle. Inactivation of Rb leads to genomic instability by impaired growth control, imprecise timing of DNA synthesis, and chromosome mis-segregation (1). As an integral member of the DNA damage control pathway, p53 functions in the recognition and repair of damaged DNA and in programmed cell death. The loss of wild-type p53 eliminates a major roadblock in tumorigenesis, allowing cells with damaged DNA to proceed through the cell cycle, thereby propagating genomically unstable progeny containing random mutations, gene amplifications, chromosomal re- arrangements, and/or aneuploidy (2). Unfaithful chromosome segregation during mitosis can lead to aneuploidy, a genetic defect observed consistently in tumor cells. Proper chromosome segregation depends on attachment of mitotic spindles to the kinetochore of each chromosome. The centrosome, responsible for nucleating and organizing mitotic spindles, is an essential component of this process (3). As such, the centrosome duplication cycle is synchronized with the cell division cycle, and uncoupling these cycles results in centrosome numbers above the normal 1–2 per cell. Increased centrosome numbers can lead to multipolar mitoses, mis-segregation of chromosomes, and genomic instability (4). Numerical and/or structural centrosome abnormalities have been reported in a variety of cancers, including breast cancer (5), colon cancer (6), pancreatic cancer (7), head and neck cancer (8), and human papillomavirus (HPV)-associated squamous cell carcinoma (9), and is observed in a variety of mouse models of human cancers (10 –12). The respective roles that p53 and Rb play in centrosome duplication are unclear. Duensing et al. (9) found that cells expressing a mutant E7 protein unable to inactivate Rb had threefold-less centrosome amplification than cells expressing an Rb-inactivating E7 protein. The E7 mutant was equivalent roughly with respect to centrosome ampli- fication to the untransfected control, and thus their results linked Rb functionally to proper centrosome duplication. This is in contrast to other studies in which the absence or inhibition of Rb function had either no affect on centrosome number (8, 13) or depended on the mode of Rb inactivation (14). Likewise, there also has been debate as to whether centrosomes are affected by the absence of p53 function. Carroll et al. (8) and Fukusawa et al. (13) have shown that p53 deficiency induces directly increased centrosome numbers. In con- trast, centrosome amplification was observed in epidermal tumors from mice expressing a mutant form of p53 but not in p53 knockout mice after treatment with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (15, 16). Thus, more investigation into the role of Rb and p53 with respect to centrosome amplification, chromosome instability, and tumorigenesis is warranted. To address experimentally the independent effects of p53 and Rb inactivation with respect to centrosome abnormalities and genomic instability, we used a previously established mouse model of skin cancer (17). Our particular model system uses the human keratin 14 (K14) transcriptional promoter to express two HPV type 16 viral oncogenes, E6 and E7, in the basal epithelial cells of the epidermis. HPVs are DNA tumor viruses that cause benign tumors or warts in human skin. A subset of HPVs, including HPV types 16 and 18, are high-risk HPVs associated with cervical cancers (18, 19). In progress- ing from a low-grade squamous intraepithelial lesion to a high-grade squamous intraepithelial lesion, the high-risk HPV genomes are com- monly integrated into the host’s genome, and the E6 and E7 onco- genes become overexpressed (20). In binding to and degrading p53 and Rb, respectively, the E6 and E7 proteins confer transforming abilities to the cell, but their contributions to carcinogenesis are different. Molecular dissection of the respective pathways has shown specifically that the expression of E7 contributes primarily to early stages of carcinogenesis, leading to the formation of benign lesions. In contrast, E6 affects primarily later stages of carcinogenesis, leading to malignant conversion (20). Previous observations of cervical (21), colorectal (22), breast (11), and head and neck (23) tumors have revealed specific patterns of Received 01/22/03; revised 10/21/03; accepted 11/6/03. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Michael J. Difilippantonio, Section of Cancer Genomics, National Institutes of Health, 50 South Drive, Room 1306, Bethesda, MD 20892-8010. Phone: 301-435-3991; Fax: 301-402-1204; E-mail: difilipm@mail.nih.gov. 538 Research. on December 9, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from