Abstract PO4-06-10: Targeting necroptosis in breast cancer induced by Ophiobolin A

Abstract

Abstract Abstract Necroptosis or programmed necrosis is a form of cell death distinct from apoptosis. Necroptotic cells display the character of rounding of the cell, gain in cell volume, rupture of plasma membrane and release of intra-cellular contents. Although few key necroptosis regulators including receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like pseudokinase (MLKL) has been identified, their role/mechanism in inducing cell death in triple-negative breast cancer (TNBC) which lack RIPK3 expression remains elusive. Ophiobolin A is a compound naturally made by fungus and potently kills cancer cells especially TNBC. Our current study investigated the cell death mechanism(s) induced by Ophiobolin A (natural product) in breast cancer cell lines. The mechanism of cell death elicited by OpA was analyzed by applying inhibitors for specific pathways including apoptosis, necroptosis, ferroptosis and paraptosis, by analyzing markers of cell death using flow cytometry and western blotting. The formation of necrosomes was analyzed with exogeneous expression of RIPK3 tagged with green fluorescence protein (GFP) in HeLa, MDA-MB-231, MDA-MB-468 cell lines which lacks endogenous RIPK3 expression and in MCF7 estrogen receptor (ER) positive cell line. OpA induced necrosome formation was identified by formation of puncta in TNBC and HeLa cells which lack RIP3 expression, however, necrosomes were not detectable in ER positive cells. Clinical relevance of RIPK3 and MLKL has been analyzed using bioinformatic tools. Effect of OpA In vivo has been analyzed using Immune compromised SCID mice. The data obtained in the present study indicates that OpA has an increased anticancer effect in TNBC compared to the estrogen receptor (ER) positive breast cancer cells. Cells having undergone EMT were shown to have more sensitivity towards OpA when compared to uninduced cell lines, which is sensitive to Necrostatin-1, an inhibitor of necroptosis. However, apoptosis, ferroptosis and paraptosis inhibitors failed to rescue cell death which clearly confirms that OpA induces necroptosis cell death pathway in EMT undergone cells. OpA treatment significantly increased puncta in TNBC compared non-TNBC cells, further confirming the necroptotic cell death in TNBC. Bioinformatic analysis revealed that RIPK3 is negatively corelated with breast cancer and has least over-all survival in patients who lack RIPK3 expression. OpA treatment showed a significant tumor regression in tumor bearing SCID mice. All together our findings identify that OpA induce necroptosis in EMT-enriched TNBC. The study further needs an in-depth analysis of molecular mechanism induced by OpA and involvement of EMT in necroptotic cell death. Citation Format: Tolulope Ojo, Santhalakshmi Ranganathan. Targeting necroptosis in breast cancer induced by Ophiobolin A [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-10.