Abstract 1393: Functional analysis of mitochondria-targeting Wnt-inhibitors on metabolically distinct breast cancer cell types

Abstract Mitochondria are essential organelles responsible for energy production and survival. In cancer cells, mitochondrial function is often altered through metabolic reprogramming, which enhances their growth, survival, and resistance to therapy. Recent findings suggest that bi-directional crosstalk between Wnt signaling and mitochondrial function plays a crucial role in tumorigenesis, as well as in the differentiation and self-renewal of stem cells. This study investigates the potential of targeting the interplay between Wnt signaling and mitochondrial function in breast cancer cells. We screened 71 agents known to alter Wnt signaling and assessed their impact on mitochondrial function and cell viability in BT474 and Hs578T breast cancer cell lines. The Mito Tox Index (MTI) obtained using the Agilent Seahorse XF MitoTox Assay was used as the parameter to evaluate the impact of test compounds on mitochondrial function. The screening identified Pyrvinium, Niclosamide, and Hexachlorophene as the most potent mitochondrial modulators. Pyrvinium and Niclosamide inhibited the mitochondrial electron transport chain, whereas Hexachlorophene acted as an uncoupler when the cells were exposed to the drugs for 24 hours. The mechanisms of mitochondrial perturbation induced by these drugs were further investigated using BT474 cells permeabilized with the Seahorse XF Plasma Membrane Permeabilizer (PMP), which selectively permeabilizes the cellular plasma membrane without affecting mitochondria integrity. This approach enabled detailed characterization of key components in mitochondrial function, including transporters, enzymes, and electron transport chain complexes. Our results indicated that Niclosamide inhibited complex I/II, either directly or indirectly, while Pyrvinium appeared to suppress mitochondrial function indirectly. Despite the different bioenergetic phenotypes of BT474 (highly oxidative) and Hs578T (highly glycolytic) cells, there was no significant difference in mitochondrial susceptibility to the three drugs. However, significant differential effects on ATP production rates were detected using the Agilent Seahorse XF Real-Time ATP Rate, BT474 cells exhibited a significant metabolic energy crisis in the presence of all three compounds, whereas Hs578T cells maintained stable total ATP production despite mitochondrial inhibition. This differential impact on ATP production rates corresponded directly to differences in cell viability, with BT474 cells showing higher susceptibility to these compounds. These findings suggest that the anti-cancer efficacy of mitochondrial modulators can vary depending on the metabolic phenotype of the target cell and should be considered in therapeutic design. Targeting mitochondrial function through Wnt signaling inhibitors presents a promising strategy for developing effective cancer therapies. Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. Functional analysis of mitochondria-targeting Wnt-inhibitors on metabolically distinct breast cancer cell types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1393.