BUB1 Inhibition Induces Ferroptosis in Triple- Negative Breast Cancer Cell Lines

Background: Triple-negative breast cancer (TNBC) is a highly aggressive subtype with limited effective treatments available, including targeted therapies, often leading to poor prognosis. Mitotic checkpoint kinase BUB1 is frequently overexpressed in TNBC and correlates with poor survival outcomes suggesting its potential as a therapeutic target. This study explores the cytotoxicity of TNBC cells to BUB1 inhibition, alone or in combination with radiation and demonstrates that ferroptosis, an iron-dependent form of programmed cell death, has a role. Methods: TNBC cell lines (SUM159, MDA-MB-231, and BT-549) were treated with a BUB1 inhibitor BAY1816032 (BUB1i) alone or in combination with the ferroptosis activator RSL3 with or without 4 Gy irradiation. Cell viability assays were conducted to assess treatment effects, and qPCR analyses measured expression of key ferroptosis markers including ACSL4 and PTGS2, and GPX4 and SLC7A11 expression. Ferroptosis specificity was confirmed through co-treatment with the ferroptosis inhibitor Ferrostatin-1 (F-1). Results: In all TNBC cell lines studied, BUB1 inhibition significantly induced ferroptosis, marked by increased expression of ACSL4 and PTGS2 and decreased expression of GPX4 and SLC7A11. The combination of BUB1i with RSL3 further amplified these ferroptosis markers, suggesting at least an additive effect, which was not present with the combination of BUB1i and radiation. Co-treatment with Ferrostatin-1 reversed the expression of ferroptosis markers, suggesting that BUB1i mediated cell death may involve ferroptosis signaling in TNBC cell lines. Conclusions: This study demonstrates that BUB1 inhibition may independently induce ferroptosis in TNBC cell lines which is enhanced when combined with a ferroptosis activator. Further research is warranted to delineate molecular mechanism of BUB1 mediated ferroptosis in TNBC.