Abstract 2324: The role of SERCA pump in cell death and autophagy

Abstract

Abstract The purpose of the study is to elucidate structural and molecular determinants of SERCA inhibition by Thapsigargin (Tg) and related analogs for their effects on intracellular calcium homeostasis, ER stress, cell death and autophagy. Tg specifically binds and blocks the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), which pumps Ca2+ from the cytosol to the ER. Sustained SERCA inhibition leads to calcium depletion from the ER causing ER stress and ultimately cell death. Recently we showed that Tg-induced calcium perturbation potently blocks autophagy (1). Tg is an attractive potential anti-tumor drug because it effectively kills both slow and fast proliferating cancer cells. However, since Tg is toxic also to normal cells, it must be targeted towards the cancer cells. Replacing a side chain with a linker connecting the Tg core to a peptide prevents Tg from entering cells. Two different linker-peptide sequences have been introduced in clinically tested Tg prodrugs; one is cleaved by PSA, secreted by prostate cancer cells, and the other is cleaved by PSMA, which is secreted by neovascular tissues of a broad range of tumors. The Tg analogs unmasked by the cleavage are able to enter cells and exert their toxic effects. Interestingly, however, in vitro experiments indicate that Tg analogs have different potencies and cellular effects depending on the terminal amino acid residue (2). Exploring why this is the case may lead not only to better Tg prodrug formulations, but also to a deeper understanding of the biological functions of SERCA pump activity. We compare a broad panel of Tg analogs in various cell types for their biological effects. Methods used so far are western blotting, real-time RT-PCR, live-cell imaging, flow cytometry, and assays that measure autophagic sequestration and degradation activity. Our unpublished data indicate the ER-stress sensors PERK, ATF4, CHOP, and IRE1 but not XBP1 and ATF6 to be involved in cell death signaling in LNCaP prostate cancer cells. Moreover, Tg-induced cell death required death receptor 5 and caspase-8. All Tg analogs tested displayed exactly the same molecular requirements for induction of cell death, although some required 5 to 10 times higher doses than Tg to evoke the same strength of death signal. Like Tg (1) the analogs inhibited autophagy before the closure of phagophores, but at higher doses and/or with slower kinetics. These results indicate that thapsigargin and its analogs evoke similar anti-autophagic and death signaling pathways. Further investigations are aimed at exploring the causes for differential potencies, and include measurements of cytosolic and compartmentalized calcium, as well as solving crystal structures of selected analog:SERCA complexes complemented with biophysical studies of analog:SERCA interactions.