Triple negative breast cancer (TNBC) remains a serious health problem with poor prognosis and limited therapeutic options. To discover novel approaches to treat TNBC, we screened cholera toxin (CT) and the members of the bacterial type II heat-labile enterotoxin family (LT-IIa, LT-IIb, and LT-IIc) for cytotoxicity in TNBC cells. Only LT-IIc significantly reduced viability of the TNBC cell lines BT549 and MDA-MB-231 (IC50 = 82.32 nM). LT-IIc had no significant cytotoxic effect on MCF10A (IC50 = 2600 nM), a non-tumorigenic breast epithelial cell line, and minimal effects on MCF7 and T47D, ER+ cells, or SKBR-3 cells, HER2+ cells. LT-IIc stimulated autophagy through inhibition of the mTOR pathway, while simultaneously inhibiting autophagic progression, as seen by accumulation of LC3B-II and p62. Morphologically, LT-IIc induced the formation of enlarged LAMP2+ autolysosomes, which was blocked by co-treatment with bafilomycin A1. LT-IIc induced apoptosis as demonstrated by the increase in caspase 3/7 activity and Annexin V staining. Co-treatment with necrostatin-1, however, demonstrated that the lethal response of LT-IIc is elicited, in part, by concomitant induction of necroptosis. Knockdown of ATG-5 failed to rescue LT-IIc-induced cytotoxicity, suggesting LT-IIc can exert its cytotoxic effects downstream or independently of autophagophore initiation. Collectively, these experiments demonstrate that LT-IIc acts bifunctionally, inducing autophagy, while simultaneously blocking autolysosomal progression in TNBC cells, inducing a specific cytotoxicity in this breast cancer subtype.

Despite the recent advances in breast cancer treatment, triple negative breast cancer (TNBC) remains a serious health problem with limited treatment options. Poor prognosis is due to the development of chemoresistance. To discover novel therapeutic approaches to treat TNBC, we screened cholera toxin (CT) and the members of the bacterial type II heat-labile enterotoxin family (LT-IIa, LT-IIb, and LT-IIc) for their capacity to induce cell death in TNBC cells. LT-IIc, but not the other toxins, significantly reduced viability of the TNBC cell lines BT549 and MDA-MB-231 (IC50 = 82.32 nM). LT-IIc had no significant cytotoxic effects on MCF10A (IC50 = 2600 nM), a non-tumorigenic breast epithelial cell line, and minimal effects on MCF7 and T47D, ER+ breast cancer cells, or SKBR-3, HER2+ cells. LT-IIc stimulated autophagy through inhibition of the mTOR pathway, while simultaneously inhibiting autophagic progression, as observed by the accumulation of LC3B-II and p62 proteins. Morphologically, LT-IIc induced accumulation of enlarged LAMP2+ autolysosomes selectively in TNBC cells. Bafilomycin A1, an inhibitor of autophagic flux, blocked the formation and/or retention of these autolysosomes. The increase in caspase 3, 7 activity and annexin V staining indicated that LT-IIc induced an apoptotic response. Co-treatment with necrostatin-1, however, demonstrated that the lethal response to LT-IIc is elicited, at least in part, by concomitant induction of necroptosis. Collectively, these experiments demonstrate that LT-IIc acts bifunctionally, inducing autophagy while simultaneously blocking autolysosomal progression in TNBC cells, resulting in specific cytotoxicity in this breast cancer subtype. Citation Format: Patricia A. Masso-Welch, Sofia Girald Berlingeri, Natalie D. King-Lyons, Lorrie Mandell, John C. Hu, Christopher Greene, Matthew Federowicz, Peter Cao, Yasser Heakal. LT-IIc, a bacterial type II heat-labile enterotoxin, induces specific lethality in triple negative breast cancer cells by modulaton of autophagy and induction of apoptosis and necroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4281.

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.

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