Abstract The SPROUTY (SPRY) proteins, key negative regulators of receptor tyrosine kinase (RTK) signaling, control developmental processes such as organ branching and maintain cellular homeostasis. The SPRY proteins inhibit critical signaling pathways that are downstream of RTKs, including the Ras-MAPK, PI3K-AKT, and the PLCα-PKC pathway. These pathways are highly deregulated in various cancers including HER2 overexpressing and PIK3CA mutant breast cancers. Importantly, the SPRY genes are significantly down-regulated in many cancers including 90% of human breast cancer cases, suggesting that they may function as putative tumor suppressors. To understand the role of Spry in normal mammary development, we examined the mammary glands of our Spry1 mutant mice at various developmental stages. In virgin Spry1-/- mice we observed a trend towards more branching of developing breast ducts. Upon pregnancy there was striking overgrowth of the mammary epithelium in Spry1+/− mice (Spry1-/- mice are sterile), suggesting that Spry regulates mammary development. To investigate the role of Spry in breast cancer we explored the consequence of Spry loss in vitro and in vivo. We found that Spry deficient cells possess accelerated cell growth, enhanced cell survival, and increased tumorigenic potential. In addition these cells were hypersensitive to a number of growth factors including EGF, showing enhanced and prolonged activation of the MAPK and PI3K-AKT pathways. To examine the role of Spry loss in promoting mammary tumorigenesis in vivo, we crossed Spry1 mutant mice with mice in which we can induce HER2/neu or PIK3CA expression. We found that Spry1 loss cooperates with oncogenic activation of HER2/neu or PIK3CA to increase cell proliferation and accelerate mammary hyperplasia. Breast tissues from the Spry1-/- mice with oncogenic activation of HER2/neu exhibited a trend towards increased levels of activated Mapk and Akt compared to their Spry1+/+ counterparts. These Spry1-/- breast tissues also accumulated significantly more DNA damage. Finally, microarray gene expression analysis of these tissues identified that genes differentially expressed in Spry1-/- breasts are involved in cell cycle regulation and the DNA damage response. Collectively, this data suggests that Spry functions as a tumor suppressor in breast cancer opposing HER2/neu and PIK3CA by suppressing MAPK and AKT activation to prevent DNA damage. Our work demonstrates that the misregulation of critical signaling pathways, through loss of Spry, leads to increased cell growth, enhanced cell survival, and accelerated tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2165. doi:1538-7445.AM2012-2165