P2-04-01: Extracellular PAI-1 – t-PA – IGFBP3 Cascade Mediates Chemotherapy-Induced Senescence of Breast Cancer Cells.

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.