Background: Proliferation, migration, and stem cell (SC) activity have all been implicated in the development and progression of breast cancer. Growing evidence suggests a relationship between these cellular characteristics, which are tightly regulated by complex signalling pathways. The oncogene cyclin D1 is an essential cell cycle protein that regulates G1 to S phase transition and is overexpressed in many breast cancers. Despite the proliferation activating properties of cyclin D1 it has been linked to a less malignant phenotype. Aim: The aim is to investigate the relationship between migration, proliferation and SC activity in breast cancer and to determine the molecular mechanisms that control these processes focusing on cyclin D1. Methods and Results: To establish a link between proliferation and migration two breast cancer cell lines (MDAMB231 and MDAMB468) were quiesced and proliferation assessed by Ki67 and migration by transwell migration assays. G 0 /G 1 cells displayed an increased migratory capacity (2.9 ±0.15 fold) compared to cycling cells. Next we downregulated cyclin D1 in actively cycling cells using siRNA which resulted in significantly decreased proliferation and the novel observation of increased migration (1.7±0. 19 fold). Having established a link between proliferation and migration the next aim was to investigate relationships with SC activity. To achieve this, cells were sorted following Hoechst 33342 and 7AAD labelling into three populations G 0 /G 1, S, and G2/M. SC-like activity was assessed using a non-adherent mammosphere culture system where single cells are seeded at a density of 500 cells per cm2 for cultured for 7 days. The data clearly demonstrated that G 0 /G 1 cells had increased migratory capacity and also SC-like activity (1.5±0.2 fold) compared to actively cycling cells. Interestingly, down-regulation of cyclin D1 increased both migratory and SC-like activity with most significant effects observed in G 0 /G 1 and S phase of the cell cycle indicating that cyclin D1 is a key protein in the regulation of these cellular processes. When downregulating the cyclin D1 associated kinases CDK4/6 no effect on migration or SC activity was observed suggesting that cyclin D1 affects these cellular processes independent of CDK activation. Finally, when analysing a large set of premenopausal breast cancers we observed an inverse, proliferation-independent link between cyclin D1 and cancer recurrence as well as tumour size (p=0.03), validating that the protein might abrogate infiltrative and malignant behaviour in vivo. Conclusion: These novel functions of cyclin D1 illustrates the interplay between tumor proliferation and migration and SC activity and may explain the lessened malignant behaviour in cyclin D1 high breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-01.