Abstract C43: A Sleeping Beauty forward genetic screen identifies novel cancer drivers that cooperate with Pten in lung cancer
Authors: Dorr Casey , Weg Madison , Been Raha , O'Sullivan M. Gerard , Sarver Aaron , Largaespada David A. , Starr Timothy K. 
Published: August 2014

Abstract
Improved understanding of the genetic drivers of lung cancer will aid in the design of personalized therapy. To this end, we have conducted forward genetic screens in the lungs of mice using the DNA transposon Sleeping Beauty (SB), in order to identify novel cancer driver genes. We conducted the screens on four different backgrounds: wild-type, p19 deficient, p53 dominant negative, and Pten deficient mice. The Pten deficient background was the only screen that resulted in tumors developing at a rate significantly above controls. Experimental mice (SB/Pten-/-) harbored a concatamer of oncogenic transposons, a conditionally activated SB transposase and a floxed Pten gene. A Cre recombinase cDNA under control of the Surfactant Protein C promoter was introduced to simultaneously activate SB and inactivate Pten in alveolar epithelial cells. Two control groups were established. One control group (W/T) did not include the Cre recombinase allele and was essentially wild-type while the second control group (Pten-/-) expressed Cre recombinase but did not contain transposons, causing inactivation of Pten without SB activity. Survival of SB/Pten-/- and Pten-/- mice was similar and both were significantly reduced compared to W/T mice. Tumor penetrance was lowest for W/T mice (4%) followed by Pten-/- mice (11%) and was highest for SB/Pten-/- mice (29%). Histological analysis of tumors indicated the presence of adenomas, adenocarcinomas, and extensive alveolar epithelialization along with bronchiolar epithelial hyperplasia. The latter two findings were present in both SB/Pten-/- and Pten-/- mice. Transposon insertions in 24 tumors from 14 SB/Pten-/- mice were mapped to the mouse genome using linker-mediated PCR followed by amplicon sequencing using Illumina GAIIx. Common insertion sites (CISs) were defined using the TAPDANCE analysis pipeline and over 100 candidate cancer genes were identified. A majority of the CIS genes we discovered have documented mutations in human lung cancers based on recent large-scale genomic analyses. This overlap suggests our mouse data could be used to prioritize research into putative human oncogenes and tumor suppressors.
Cullin3 (Cul3) was highly ranked in our CIS list. The protein product of CUL3 is a core scaffolding protein in an E3 ubiquitin ligase complex that regulates ubiquitination of NRF2, IKBKB and other proteins. To determine the role of CUL3 in lung cancer we analyzed expression by western blot in human cancer cell lines and tumors and found CUL3 levels to be lower than normal bronchial epithelial cells. To directly test CUL3's tumor suppressor role we knocked down CUL3 using shRNA in stage II human lung cancer cell lines A549 and H522 either alone or in combination with an shRNA targeting PTEN. Reducing the level of CUL3 in these cell lines resulted in increased proliferation and an increased ability to grow in an anchorage-independent manner. These phenotypes were enhanced when both CUL3 and PTEN were coordinately knocked down. Western blot data indicate PTEN levels increase substantially, while mRNA levels maintain relatively static in CUL3 knockdown cells. In conclusion, we have performed studies that have identified a large number of candidate lung cancer genes and have confirmed a tumor suppressor role for CUL3. We are currently testing the effect of overexpressing CUL3 in both non-transformed and cancerous cell lines, as well as testing more of the candidate genes we have discovered. The results of these studies should lead to new treatment modalities for this deadly cancer.
Citation Format: Casey Dorr, Madison Weg, Raha Been, M. Gerard O'Sullivan, Aaron Sarver, David A. Largaespada, Timothy K. Starr. A Sleeping Beauty forward genetic screen identifies novel cancer drivers that cooperate with Pten in lung cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C43.