Abstract Upon successful chemotherapy, cancer cells undergo either apoptosis or senescence. Whereas the mechanism of cancer cell apoptosis is relatively well understood, there is only scant knowledge on how chemotherapy-induced cancer senescence occurs. Here we report that a cascade of secreted proteins, plasminogen activator inhibitor 1 (PAI-1) — tissue-type plasminogen activator (t-PA) — insulin-like growth factor-binding protein 3 (IGFBP3), mediates chemotherapy-induced senescence of breast cancer cells. MCF-7 breast cancer cells display robust senescence induction upon doxorubicin treatment and we found that the conditioned medium from senescent MCF-7 cells can induce senescence in non-senescent MCF-7 cells, which suggested the presence of secreted mediator(s) of senescence. To identify such mediator(s), we undertook a quantitative proteomic analysis of the protein secretion from senescent MCF-7 cells and observed significantly increased levels of secreted IGFBP3. Increased extracellular IGFBP3 levels were also observed upon doxorubicin-induced senescence of ZR75-1 breast cancer cells and primary mammary epithelial cells. We demonstrated that IGFBP3 induces senescence in breast cancer cells, which requires the Rb and p53 pathways. Conversely, shRNA-mediated knock-down of IGFBP3 alleviated doxorubicin-induced senescence of breast cancer cells. These results suggest that IGFBP3 functions as secreted mediator of chemotherapy-induced breast cancer senescence. To gain insight into the regulation of IGFBP3-induced senescence, we undertook a proteomic screening for IGFBP3 interactor(s) in the secretome and identified t-PA as candidate interactor. t-PA is a secreted protease that is known to cleave and activate plasminogen. We found that t-PA cleaves IGFBP3 and abolishes IGFBP3-induced or doxorubicin-induced senescence of breast cancer cells. The protease activity of t-PA toward plasminogen is inhibited by PAI-1 and we demonstrated that PAI-1 also protects IGFBP3 from cleavage by t-PA. Interestingly, PAI-1 was previously identified as an inducer of senescence that acts downstream of p53 (Nature Cell Biology 8:877–84, 2006). We showed that IGFBP3 knock-down by shRNAs abolishes senescence induction by PAI-1, suggesting that IGFBP3 is a critical downstream mediator of PAI-1-induced senescence. We also observed dramatically increased extracellular PAI-1 levels upon doxorubicin-induced senescence of breast cancer cells. Importantly, RNAi suppression of PAI-1 in breast cancer cells resulted in concomitant suppression of extracellular IGFBP3 levels upon doxorubicin treatment and abrogation of senescence induction. Taken together, these results suggest a role for extracellular PAI-1 — t-PA — IGFBP3 cascade in mediating chemotherapy-induced senescence of breast cancer cells. This study uncovered the extracellular components of senescence signaling for chemotherapy-treated breast cancer cells. Senescence mediators secreted from chemotherapy-treated breast cancer cells may amplify the senescence response and provide a non-cell autonomous tumor suppression mechanism. These extracellular senescence mediators could be exploited to increase the efficacy of breast cancer chemotherapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-04-01.