Abstract 1667: Autophagy and unfolded protein response (UPR) signaling regulates progression of apoptosis in mammary gland involution.

Progression of local benign mammary lesions to invasive breast cancer is known to involve processes that are necessary for normal mammary gland development. Post-natal mammary gland is capable of undergoing repeated cycles of proliferation and cell death, most strikingly when fully differentiated (lactating) gland degenerates to a pre-lactation state during involution. Involution involves extensive cell death and tissue remodeling, which may temporarily increase breast cancer risk. Accumulation of milk proteins in the secretory epithelium creates the stress signal that triggers involution; however it is largely unknown whether the well-defined stress pathways, UPR and autophagy, are involved breast involution regulation. Using published gene expression array datasets from different phases of mouse mammary gland development we demonstrate that UPR, autophagy and apoptosis genes grouped in separate clusters. Next, in time-course experiments we show that autophagy and UPR signaling are tightly co-regulated during mammary involution. Early UPR signaling events such as GRP78 and phospho-eIF2α were elevated during early involution, i.e. the first 24 - 48 hours (h; reversible phase), while late UPR signaling events such as ATF4 and CHOP were upregulated during 72 h - 7 days of involution. An increase in AMPK was observed during early involution potentially preventing TORC1-mediated autophagy inhibition. Correlating with these observations, increased LC3-GFP punctate and decreased p62 expression were observed at 24 h when compared to 72 h post weaning, suggesting increased autophagy. Apoptosis was increased 48-72 h, peaking at 72 h involution, as measured by TUNEL. Increased apoptotic markers, such as cleaved caspase-7 and cleaved PARP were observed during these time points, with increased antiapoptotic BCL2 family members (BCL-XL and BCL-W) expressed from 24-48 h. To confirm the causal and inverse relationship between autophagy and apoptosis signaling, we performed involution time-course experiments using both low-dose drug interventions and an autophagy-related gene 7 (ATG7) deletion mouse model. Inhibition of autophagy by chloroquine, or genetic deletion of one ATG7 allele, enhanced the progression of mammary involution into an irreversible phase, characterized by earlier induction of apoptosis. In contrast, stimulation of autophagy by low dose tunicamycin treatment reduced apoptosis and extended the reversible phase of mammary involution by sustaining the secretory epithelium, suggesting a possible therapeutic use of autophagy stimulators in promoting lactation and nursing. Taken together these data indicate that UPR and autophagy play a key role in the regulation between survival and apoptosis during normal mammary gland involution and homeostasis, and, thus, if deregulated could contribute to breast cancer progression. Citation Format: Katherine L. Cook, Anni Warri, Rong Hu, Lu Jin, Alan Zwart, David R. Soto Pantoja, Jie Liu, Toren Finkel, Robert Clarke. Autophagy and unfolded protein response (UPR) signaling regulates progression of apoptosis in mammary gland involution. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2013-1667