Up-Regulation of the Error-Prone DNA Polymerase K Promotes Pleiotropic Genetic Alterations and Tumorigenesis Clarisse Bavoux, 1 Andre ´ia Machado Leopoldino, 2 Vale ´rie Bergoglio, 1 Jiyang O-Wang, 3 Tomoo Ogi, 4 Anne Bieth, 1 Jean-Gabriel Judde, 5 Se ´rgio Danilo Junho Pena, 2 Marie-France Poupon, 5 Thomas Helleday, 6 Masatoshi Tagawa, 7 CarlosRenato Machado, 2 Jean-Se ´bastien Hoffmann, 1 and Christophe Cazaux 1 1 Laboratory V Genetic instability and cancer J, Institute of Pharmacology and Structural Biology, Centre National de la Recherche Scientifique, Toulouse, France; 2 Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 3 Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan; 4 Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, United Kingdom; 5 FRE2584 Centre National de la Recherche Scientifique, Section Recherche, Institut Curie-Centre National de la Recherche Scientifique, Paris, France; 6 Institute for Cancer Studies, University of Sheffield, Medical School, Sheffield, United Kingdom; and 7 Division of Pathology, Chiba Cancer Center Research Institute, Chiba, Japan Abstract It is currently widely accepted that genetic instability is key to cancer development. Many types of cancers arise as a consequence of a gradual accumulation of nucleotide aberra- tions, each mutation conferring growth and/or survival advantage. Genetic instability could also proceed in sudden bursts leading to a more drastic upheaval of structure and organization of the genome. Genetic instability, as an operative force, will produce genetic variants and the greater the instability, the larger the number of variants. We report here that the overexpression of human DNA polymerase K, an error-prone enzyme that is up-regulated in lung cancers, induces DNA breaks and stimulates DNA exchanges as well as aneuploidy. Probably as the result of so many perturbations, excess polymerase K favors the proliferation of competent tumor cells as observed in immunodeficient mice. These data suggest that altered regulation of DNA metabolism might be related to cancer-associated genetic changes and phenotype. (Cancer Res 2005; 65(1): 325-30) Introduction Genetic instability in cancer has a wide range of expression modes. It may affect nucleotide proofreading (leading to base sub- stitutions, deletions, or additions), chromosomal structure (pro- ducing translocations, sequence gains, or losses), or karyotypic integrity (resulting in aneuploidy). Whereas the impact of such ge- netic abnormalities in cancer progression remains controversial (1), it is now accepted that stochastic occurrence of genetic dis- orders and its gradual increase in frequency along the natural history of the disease is often promoted by either early mutations in genes that maintain genetic stability in normal cells and/or big- ger genetic changes such as gains or losses of chromosomes (2, 3). Together the highly conserved pathways involved in genome supervision act to limit cancer risk. This is clearly illustrated by pa- tients bearing a germ line mutation in genes encoding ‘‘guardians’’ of genome stability, who show vastly increased risk of cancer development. Hereditary forms of colon, breast, ovary and skin cancers are caused by mutations in mismatch repair (e.g., hMLH1), DNA break repair (e.g., BRCA1), nucleotide excision repair path- ways (e.g., XP proteins), or affecting the capacity to replicate through DNA damage (e.g., polD), respectively (4–7). In somatic cancers, such early mutations become ‘‘diluted’’ in the alterations that follow, making the relationship less obvious. However, it is very likely that genetic instability either induces or accelerates the proliferation of cancer cells by favoring the emergence of variant cells. Indeed, a controlled alteration of genes involved in genome maintenance promotes or favors carcinogenesis (8, 9). The accurate maintenance of undamaged genomic DNA re- quires the action of the error-free DNA polymerases poly and polq. When DNA is damaged, specialized error-prone DNA poly- merases, including pol~ , polD, polL, polh, and poln are believed to take part in the replication repair of damage that otherwise would not be tolerated (10). Poln, the product of the human dinB1 gene, bypasses in vitro thymine glycols (11) or benzo[a]- pyrene-N (2)-dG (12) lesions by preferentially incorporating correct nucleotides. These features suggest a specialized and adaptative role of Poln towards such lesions. Poln is targeted to the replication machinery probably because of its interaction with the proliferative cell nuclear antigen (13, 14) and the Rev1 protein (15). Because it lacks proofreading activity (16) and replicates DNA with limited processivity (17), Poln manifests an error rate of about 5  10 3 per nucleotide incorpo- rated when copying undamaged DNA in vitro (12, 17). In vivo this enzyme confers a mutator phenotype when overexpressed (14, 18). Besides its specialized role this enzyme could thus promote un- targeted mutagenesis when up-regulated. Here we wondered whether the human DNA polymerase Poln, which is overexpressed in non–small cell lung cancer (NSCLC, ref. 19), could be a candidate likely to play a role in genetic instability and cancer progression. We found that the ectopic expression of Poln promotes ho- mologous events, aneuploidy as well as tumorigenesis in nude mice. Moreover, we found that of eight poln NSCLC biopsies, seven displayed losses of heterozygosity (LOH) compared with adjacent nontumoral tissues. Taken together, these data suggest that misregulation of this error-prone enzyme directly promotes or accelerates the emergence of a large spectrum of genetic dis- orders associated with a malignant phenotype. Materials and Methods Cell Lines and Plasmids. MRC5 human fibroblasts (American Type Culture Collection, Rockville, MD), AA8 Chinese hamster ovary (CHO) Requests for reprints: Jean-Se ´bastien Hoffmann or Christophe Cazaux, Institute of Pharmacology and Structural Biology Centre National de la Recherche Scientifique UMR 5089, Supervision of the genome, de route de Narbonne, Toulouse, France, 31077. Phone: 356-117-5961; Fax: 356-117-5994; E-mail: jseb@ipbs.fr or cazaux@ipbs.fr. D2005 American Association for Cancer Research. www.aacrjournals.org 325 Cancer Res 2005; 65: (1). January 1, 2005 Research Article Research. on January 17, 2016. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from