Abstract 3968: Bacterial heat labile enterotoxin LT-IIc effects autophagy and cell death in human TNBC

Abstract

Microbes and microbial products are being re-evaluated as potential therapeutic agents for breast cancer treatment. We previously observed the rapid induction of cell death in the mouse TNBC cell line 4T1, and mesenchymal TM12T, but not epithelioid TM12 cells, upon exposure to LT-IIc, a member of the bacterial type II subfamily of ADP-ribosylating toxins that bind to specific ganglioside subtypes. To test the hypothesis that LT-IIc may be effective against human breast cancer, we tested a panel of human breast cancer cells. LT-IIc exposure induced cytotoxicity in TNBC, particularly basal B (mesenchymal) TNBC (BT549 and MDA-MB-231), in a dose dependent fashion. The triple negative non-transformed breast epithelial line MCF10A was unaffected. Two ER+ cell lines (T47D and MCF7), and HER2 overexpressing SKBR3 cells showed limited cytotoxic response. We observed a distinctive rapid accumulation of large cytoplasmic vacuoles in TNBC, but not other normal or transformed cell lines. Combined cytotoxicity and vacuolization were not induced by cholera toxin, LT-IIa holotoxin, or forskolin, but still occurred upon treatment with the LT-IIc isolated B pentamer (which lacks the toxin's catalytic A subunit), suggesting that the effects of LT-IIc were not due to intoxification of the adenylate cyclase pathway. These vacuoles were not intracellular lipid droplets, as determined by lack of staining with Oil Red O. Instead, vacuoles were positive for punctate staining with LC3B, a marker of autophagosome formation. Western blotting of MDA-MB-231 and BT549 cells revealed an increase in LC3B and p62 (sequestosome), a marker of autophagic flux. Bafilomycin, an inhibitor of lysosomal fusion, augmented the cell death effect of LT-IIc, but blocked the accumulation of distended autophagic vacuoles, suggesting a negative autophagy feedback response to bafilomycin. Both necrosis and apoptosis were activated in response to LT-IIc exposure, as indicated by increased annexin/AAD staining and caspase 3/7 activity. Knockdown of ATG5 by siRNA, although blocking ATG5 expression, did not block LC3B lipidation, cell death or increased caspase 3/7 activity in response to LT-IIc, suggesting a noncanonical autophagic response. These results suggest that LT-IIc binding or signaling may represent a novel target of therapy for TNBC. Current studies are directed towards defining the ganglioside content in LT-IIc sensitive cell lines and identifying potential downstream proteomic targets. Citation Format: Patricia A. Masso-Welch, Sofia Girald Berlingeri, Natalie D. King-Lyons, John C. Hu, Christopher Greene, Lorrie Mandell, Matthew Federowicz, Terry D. Connell, Yasser Heakal. Bacterial heat labile enterotoxin LT-IIc effects autophagy and cell death in human TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3968.