Abstract 330: Utilization of Sleeping Beauty mutagenesis for the identification of potential driver genes of ovarian cancer.

Abstract

Abstract Background: The goal of this project is to increase our understanding of the molecular aetiology of ovarian cancer by developing a mouse model of this tumor. Inherited mutation of BRCA1 is the best known risk factor for the most common subtype of ovarian cancer, i.e. serous epithelial ovarian cancer (SEOC); however, loss of Brca1 alone is insufficient for SEOC development in the mouse. Somatic mutation of TP53 is the most common molecular change in SEOC, but does not result in SEOC in the mouse, either alone or in combination with loss of Brca1. These findings suggest that additional currently unknown genetic factors are required for ovarian carcinogenesis. We hypothesized that random mutagenesis would activate these genetic factors and utilized Sleeping Beauty (SB) insertional mutagenesis to initiate ovarian tumors in mice that we have sequenced to determine potential genetic drivers of these tumors. Methods: Breeding colonies of the following genetically engineered mice were established and cross bred: homozygous floxed SB (STOCK Rosa26-LsL-D-SB11;T2/Onc2,TG6113), homozygous floxed Brca1 knock-out (C57BL/6.Brca1tm2Brn) and Tp53 mutant (C57BL/6-Trp53tm1Tyj/J). CRE recombinase packaged into an adenoviral vector (AdCreM2, MicroBix Biosystems Inc, CA) was surgically injected under the ovarian bursal membrane of mature female mice to delete Brca1 and activate SB mutagenesis in the ovarian surface epithelium. Mice were monitored and sacrificed at ethically defined endpoints or a maximum of 15 months post-surgery. Tumors were assessed for SB transposase activity by immunohistochemical staining. DNA was extracted from paraffin embedded sections of ovarian tumors and underwent high-throughput sequencing for T2/Onc2 insertion sites (Illumina, University of Iowa). Results: Ovarian tumors were observed at low penetrance starting at 30 weeks post-surgery in SBflox/+Tp53mut/+ mice (6%, 3/48) and SBflox/+Brca1flox/flox p53mut/+ mice (8%, 4/50). No ovarian tumors were observed in SBflox/+Brca1flox/flox mice (n=38) or in SBflox/+Brca1flox/+ mice (n=26). Sequencing of the insertion sites identified a number of genes of interest including kinases and tumor suppressors. Conclusions: While ovarian cancer had low penetrance in these models, a number of potential driver genes were identified which may provide insights into the pathogenesis of these tumors. The low penetrance of tumor from targeted epithelial cells with SB T2/Onc2 is similar to that seen for pancreatic cancer. For this reason, mice with SB T2/Onc3 which is reported to preferentially induce epithelial tumors are currently being monitored for the development of ovarian tumors. This work was supported by Cancer Institute NSW, Cancer Council NSW and The Northern Translational Cancer Research Unit, NSW, Australia. Citation Format: Emily K. Colvin, Emily Fuller, Jizhou Cheng, Anthony Gill, Deborah J. Marsh, Viive M. Howell. Utilization of Sleeping Beauty mutagenesis for the identification of potential driver genes of ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 330. doi:10.1158/1538-7445.AM2013-330