Abstract 4652: The autophagy-senescence connection in chemotherapy of breast tumor cells; senescence accelerated by autophagy but not dependent on autophagy

Introduction: Previous studies from our laboratory have established the capacity of Adriamycin to promote accelerated senescence in MCF-7 cells, while a number of studies have indicated that Adriamycin promotes autophagy. Although senescence and autophagy are considered to be two distinct cellular events in response to genotoxic stress, recent reports have suggested that the two are functionally intertwined. Thus, the current work was designed to determine whether autophagy and senescence were related in response to treatment with Adriamycin (ADR) and Campthothecin (CPT) in MCF-7 cells in vitro. Experimental Procedure: Autophagy induction was measured by acridine orange staining/quantification by flow cytometry and RFP-LC3; autophagic flux was based on p62 western immunoblotting. Autophagy was inhibited pharmacologically using 5µM Chloroquine or 5mM 3-MA and genetically using shRNA against ATG5 and ATG7. Senescence was measured by β-galactosidase staining and C12FDG fluorescence by flow cytometry. To block senescence, shRNA against p21 and 53 were used. The senescence associated markers p21, pRb, and p53 were measured by western immunoblotting. To evaluate common signaling events, 20µM KU55933 or 2mM caffeine were used to downregulate ATM and 20µM N-acetyl cysteine or 20µM Glutathione were used for scavenging ROS. Results: Both ADR and CPT collaterally induced autophagy and senescence in a time-dependent manner. Downregulation of ATM by pharmacological inhibition or genetic ablation collaterally blocked both ADR-induced autophagy and senescence. Suppression of ROS generation collaterally interfered with ADR-induced senescence and autophagy. Moreover, shRNA against either p53 or p21 resulted in a marked reduction in ADR-induced autophagy. In contrast, autophagy blockade with chloroquine, 3-MA, or shRNA against ATG5 and ATG7 only delayed senescence. Finally, tissue and protein analysis from a 4T1 breast tumor model showed that both autophagy and senescence co-exist in vivo in response to ADR. Conclusions: Treatment of MCF-7 cells with either ADR or CPT induced both autophagy and senescence. Interference with ROS generation, ATM activation and induction of p53 or p21 suppressed both autophagy and senescence. However, these observations may indicate only that both responses are mediated by common DNA-damage induced signaling pathways. When autophagy was blocked either pharmacologically or genetically, senescence was temporally delayed, but the overall extent o