Insertional Mutagenesis To Identify Molecular Mechanisms Of Breast Cancer Dormancy And Metastasis

The PiggyBac transposon insertional mutagenesis system (PBTS) is a powerful genomic screen previously demonstrated to identify cancer-causing genes and which we have adapted to identify genes regulating breast cancer cell dormancy and metastases. Transposition takes places through a cut and paste mechanism catalyzed by the PiggyBac transposase, which excises and re-integrates the transposon into TTAA chromosomal sites. We have adapted the PBTS system using the well characterized D2.0R in vitro and in vivo models of breast cancer cell dormancy. D2.0R cells grow as primary tumors in the mammary fat pad, disseminate to the lungs, but remain dormant. The PBTS system induces random genome-wide gene activations or inactivations that we expect will induce dormant cells to proliferate and form metastases in vivo. Identification of these genes is performed using high throughput sequencing and existing computational algorithms. Candidate dormancy and metastasis genes will be functionally validated for their roles in dormancy and metastases. We have validated the performance of the PBTS in our model system. D2.0R cells were transfected with 20 copies of the PiggyBac transposon ATP-1. Single clones were obtained from the pooled population and expanded under selection. Pooled D2.0R cells and D2.0R single clones were subsequently transduced with lenti-hyPBase transposase and double-selected for the stable integration of the ATP-1 transposon and the lenti-hyPBase transposase. gDNA was extracted from these cells, sheared and amplified by splinkerette-PCR and sequenced using an Illumina platform. Our data show that there is a significantly higher variability in the integration sites of the ATP-1 transposon in D2.0R cells stably expressing hy-PBase as compared with non-hy-PBase expressing D2.0R cells carrying the ATP-1 transposons. This demonstrates that cells from each clone acquire new insertions sites upon ATP-1 re-mobilization due to hyPBase transposase activity. Likewise, we found a significantly higher variability in the ATP-1 insertions in cells from the pooled cultures than in the cells from individual cellular clones. We also observed that reads from both the 5’ and 3’ ends of the ATP-1 insertion sites of the transposon cluster together, showing a good correlation between reads from both ends. A complimentary approach will be performed by introducing the PBTS system into the poorly metastatic MMTV-Myc transgenic mammary cancer model to identify genes and pathways that induce metastases. Since Myc is overexpressed in ∼30% of human breast cancers, this is a relevant model to employ. These results demonstrate that our experimental protocols are able to induce transposon mobilization which should identify genes that regulate tumor dormancy and metastases. Identifying genes that promote dormancy and metastases will lead to novel strategies that could prevent disease recurrence or treat metastatic breast cancer. Citation Format: Laura Vera Ramirez, Sven Bilke, Robert Walker, Paul Meltzer, Tinghu Qiu, Roland Rad, Jeffrey E. Green. Insertional mutagenesis to identify molecular mechanisms of breast cancer dormancy and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1576.