[CANCER RESEARCH 64, 2406 –2410, April 1, 2004] Oncogene Expression and Genetic Background Influence the Frequency of DNA Copy Number Abnormalities in Mouse Pancreatic Islet Cell Carcinomas Jeffrey H. Hager, 1 J. Graeme Hodgson, 2,3 Jane Fridlyand, 3 Sujatmi Hariono, 2 Joe W. Gray, 2,3 and Douglas Hanahan 1 Departments of 1 Biochemistry, University of California at San Francisco Diabetes and Comprehensive Cancer Center, and 2 Laboratory Medicine and 3 University of California at San Francisco Comprehensive Cancer Centers, University of California at San Francisco, San Francisco, California ABSTRACT Quantitative measurements of tumor genome composition show re- markable heterogeneity in tumors arising from the same anatomical location and/or histopathological class and stage. The factors that con- tribute to genomic heterogeneity are not clear, but germ-line allelic var- iation and timing of initiating oncogenic events are likely candidates. We investigated these factors by using array comparative genomic hybridiza- tion to measure genomic aberrations in genetically engineered mouse models of pancreatic islet cell carcinoma, in which oncogenic transforma- tion is elicited by the SV40 T antigens expressed under the control of the rat insulin promoter (RIP-Tag). Two distinct transgenic RIP-Tag lines, and three polymorphic sublines of one, enabled us to investigate the effects of genetic background and differing age of oncogene induction. Both parameters were found to bias the spectrum of genomic copy number abnormalities. Specifically, the frequency of losing portions of chromo- somes 9 and 16 was significantly modulated by genetic background, with the former being lost at highest rates in the FVB/N background and the latter being lost to greatest extent in both FVB/N and C57Bl/6 tumors compared with C3HeB/Fe tumors. The frequency of losing a region of chromosome 6 varied according to the age when tumorigenesis was initi- ated; loss of chromosome 6 was significantly higher when oncogene ex- pression was first activated in adulthood. These studies illustrate the utility of transgenic animal models for investigation of factors influencing genomic heterogeneity despite the commonalty of target cell type and initiating oncogene. INTRODUCTION Human solid tumors are remarkably heterogeneous in genome abnormalities (1, 2). Variation in the spectrum of recurrent genomic abnormalities between tumors arising in different anatomical loca- tions is not surprising given that different organ microenvironments likely pose distinct barriers to incipient neoplasias as a consequence of their particular biological functions and homeostasis. By contrast, the degree of genomic heterogeneity observed in histologically similar tumors arising in the same anatomical site is surprising. One inter- pretation is that ostensibly similar tumors in an organ arise from cells that subtly differ in maturity, location, or function and, as such, have distinctive genome-wide expression profiles that are benefited by distinct genomic alterations. This notion is to some extent supported by gene expression microarray analyses showing that cancers origi- nating in the same anatomical site can cluster into distinct groups (3, 4); however, tumors within a distinctive expression cluster can still show considerable genomic heterogeneity (5). One source of such heterogeneity may be passage through telomere crisis, which intro- duces a substantial stochastic component to the genomic aberration composition (6, 7). In addition, the particular genomic aberrations selected in tumors of ostensibly similar histological type may also be influenced by polymorphic variation of modifier genes between indi- viduals and/or variation between individuals in the timing and type of carcinogenic insult. Our goal in this study was to test these latter possibilities. We used the RIP-Tag model of pancreatic islet cell carcinogenesis (8) because our earlier studies indicated that the com- position of recurrent aberrations in these tumors was influenced by genetic background and carcinogen exposure (9). RIP-Tag mice carry the SV40 early region encoding the large T (Tag) and small t oncoproteins under the control of the rat insulin gene regulatory region (RIP; Ref. 8). Tag abrogates the functions of the retinoblastoma and p53 tumor suppressors (10) in the pancreatic islet  cells, leading to the formation of  cell tumors (insulinomas) in every mouse inheriting the hybrid oncogene. We used two different RIP-Tag lines in these studies. In one line, designated RIP1-Tag2, transgene expression begins at about E8.5 in progenitor cells of the endocrine pancreas and rare cells of the thymus, and expression is maintained in their descendants thereafter. Mice in this class develop islet tumors by 10 weeks of age, preceded by neoplastic lesions that are inferred to be stages on a multistep pathway to solid tumors. In the other line, designated RIP1-Tag5, the transgene is not expressed during prenatal pancreatic development or at any time in the thymus. Rather, Tag expression is sporadically activated in individual islet  cells beginning 9 –11 weeks after birth (11). These delayed onset RIP-Tag mice develop islet tumors at 20 –22 weeks of age (12). In the present study, we compared recurrent aberrations in the delayed onset RIP1-Tag5 model and early onset RIP1-Tag2 model, both bred into the C3H background, to determine whether the timing of oncogene activation influences the tumor genome aberration pat- tern. We also compared three sublines of RIP1-Tag2, inbred into three different genetic backgrounds, C57Bl/6 (B6), C3Heb/Fe (C3H), and FVB/N (FVB), to determine whether genetic background influences the tumor genotype. MATERIALS AND METHODS Mouse Genetics. The RIP1-Tag2;C57B6/J and RIP1Tag2C3HeFeb/J were derived from the same founder animal and subsequently backcrossed to either C57B6 or C3HeFeb/J for 40 generations (n  40). The RIP1Tag2;FVB/N line was derived by backcrossing the RIP1-Tag2;C57B6/J line (n  40) to FVBN/J mice for three generations (N3). The RIP1-Tag5 line harbors the same transgene as that of the RIP1-Tag2 lines but was derived independently via injection of C3HebFe/J embryos; the founder animal was bred and line subsequently maintained in the C3HebFe/J strain background. Histology. Pancreases were removed and fixed overnight in zinc-buffered formalin. Fixed tissue was rinsed in 1 PBS, subsequently dehydrated in 35, 50, and 70% ethanol, and embedded in paraffin using a Leica TP1050 tissue processor. Sections (5 M) were deparrafinized in xylene and rehydrated in 100, 95, 70, and 50% ethanol and 1 PBS. H&E staining of sections was carried out using standard reagents (Surgipath) and protocols. Array Comparative Genomic Hybridization (CGH). Array CGH was performed as described (14) Gained and deleted clones were identified in each tumor as those with fluorescence ratios above and below the tumor-specific threshold (see Supplemental Information for additional details). For compari- Received 11/10/03; revised 12/23/03; accepted 1/26/04. Grant support: National Cancer Institute grants (J. W. Gray and D. Hanahan). J. G. Hodgson was supported by a grant from United States Department of Defense Breast Cancer Research Program. 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. Note: J. H. Hager and J. G. Hodgson contributed equally to this work. Requests for reprints: Douglas Hanahan, Department of Biochemistry, University of California at San Francisco Diabetes and Comprehensive Cancer Center, University of California at San Francisco, San Francisco, CA 94143. Phone: (415) 476-9209; E-mail: dh@biochem.ucsf.edu. 2406 Research. on January 2, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from