Effects of ergosterol peroxide on proteostasis disruption in triple negative breast cancer cells

Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and HER2 expression, known for its high malignancy, invasiveness, and propensity for metastasis. Due to the lack of targeted therapies, TNBC patients typically undergo multimodality chemotherapy with cytotoxic agents like taxanes, anthracyclines, and cyclophosphamide. Therefore, there is an urgent need for selective therapies for TNBC. Natural products have gained significant attention for their potential in cancer therapy, as around 60% of clinically approved anticancer drugs were inspired by secondary metabolites found in nature. In our laboratory, we work with Ergosterol Peroxide (EP), a bioactive compound extracted from the Ganoderma lucidum mushroom. We discovered that the natural product EP exhibits selective cytotoxicity against TNBC models, demonstrating activity in the low micromolar range while sparing normal cells. Our studies indicate that EP exerts its anticancer effects by disrupting critical cellular processes including proteostasis, protein synthesis, and protein degradation pathways. We hypothesize that EP compromises TNBC cell viability by disrupting proteostatic balance. Mechanistically, we propose that EP inhibits protein synthesis and induces mitochondrial dysfunction, which collectively impair the ability of TNBC cells to restore protein homeostasis, ultimately resulting in cell death . To validate our hypothesis, we performed protein synthesis assays in two TNBC cell models, SUM149 and MDA-MB-231. Veh (0.2% DMSO), EP (20μM), or cyclophosphamide (1μM, positive control) were administered for 6 or 24h. EP effects on protein aggregation, under Veh, EP, or MG132 (positive control) in SUM149 or MDA-MB-231 TNBC cells and MCF10A non-cancerous cells, we used an aggresome assay. Finally, to assess protein degradation we performed western blots probing for ubiquitin. We previously established that EP increases ROS levels in TNBC cells. EP affects protein synthesis, compared to negative vehicle control treated cells, and in reduced capacity when compared to cyclophosphamide. Moreover, EP significantly increases protein aggregation, although the signal detected for MG132 was greater. Finally, EP decreases cancer cell degradation, as seen by decreased levels of ubiquitin when treated after 24h. In summary, EP demonstrates the capacity to modulate proteostasis in TNBC cells. Further investigation is warranted to comprehensively characterize the intricate mechanistic pathways by which EP exerts its anticancer effects in this malignancy.