STUB1 downregulates TOP2A through a dual mechanism of ubiquitination and FOXM1-mediated transcription repression, suppressing breast cancer growth and enhancing sensitivity to chemotherapy

Abstract Background DNA topoisomerase IIɑ (TOP2A) is crucial for maintaining genomic stability and is an important target for genotoxic chemotherapeutic drugs. STIP1 homology and U-box-containing protein 1 (STUB1) is a U-box containing E3 ubiquitin ligase that participates in the degradation of specific oncogenic proteins. This research examined the potential regulatory function of STUB1 in relation to TOP2A, and explored its functional implications. Methods To identify interactions between STUB1 and TOP2A, coimmunoprecipitation, Glutathione S-transferases (GST) pull-down, and immunofluorescence assays were performed. Cycloheximide (CHX) pulse-chase assay, in vivo and in vitro ubiquitination, quantitative RT-PCR, chromatin immunoprecipitation (CHIP), and luciferase assays were performed to determine how STUB1 interacts with TOP2A. In addition, TOP2A catalytic activity, colony formation, WST-1, and flow cytometry assays were performed and a xenograft model was further developed to explore whether STUB1 could downregulate the catalytic activity of TOP2A, reduce the growth of breast cancer, and increase its sensitivity to doxorubicin. Moreover, immunohistochemical staining was conducted to assess STUB1 and TOP2A expression levels, as well as their predictive roles in the efficacy of neoadjuvant chemotherapy in individuals diagnosed with breast cancer. Results STUB1 enhanced TOP2A translocation to the cytoplasm, downregulating its expression through increased ubiquitination and degradation. Forkhead box M1 (FOXM1), another substrate of STUB1, served as a transcription factor for TOP2A, playing a role in STUB1-mediated downregulation of TOP2A at the transcriptional level. STUB1 inhibited TOP2A’s activity, reduced cancer cell proliferation, increased doxorubicin-induced apoptosis, and promoted cell cycle arrest. In a breast cancer xenograft model, STUB1 suppressed tumor growth and improved doxorubicin sensitivity. A positive correlation between FOXM1 and TOP2A expression was found in patients with breast cancer undergoing EC-T chemotherapy, both negatively correlated with STUB1, whose higher expression levels were linked to increased pathologic complete response (pCR) rates. STUB1 was evaluated as an independent predictor of pCR through univariate and multivariate analyses. Conclusions This study proposes a novel function of STUB1 in the downregulation of TOP2A, which directly enhances sensitivity to chemotherapy.