PO-224 consequences of FOXP1-mediated transcriptional repression of the mitotic checkpoint gene MAD2 in breast cancer

Abstract

IntroductionThe mitotic checkpoint, or spindle assembly checkpoint (SAC), is a safeguard mechanism that prevents chromosome missegregation during mitosis. It is well established that overexpression of the mitotic checkpoint component MAD2 leads to chromosome instability (CIN) and tumorigenesis. MAD2 expression is controlled by transcriptional activator and repressor E2Fs, as well as by cell cycle dependent element/cell cycle homology region (CDE/CHR) sequences in its promoter. However, precisely how MAD2 transcription is regulated has remained elusive. Focusing on breast cancer, we therefore aimed to identify novel MAD2 transcriptional regulators and to characterise how these might contribute to CIN and tumour formation.Material and methodsPotential MAD2 transcriptional regulators were identified using a DNA-protein pull-down assay and mass spectrometry. In-depth characterisation of protein-DNA interactions and transcriptional effects were studied using chromatin immunoprecipitation, promoter mutagenesis and reporter assays. Cell lines in which identified repressors were stably knocked-down by shRNAs were used to investigate changes in MAD2 protein levels, cell cycle progression and chromosome segregation fidelity. In silico analyses and immunohistochemistry on tissue microarrays were used to study associations between expression of the regulators and MAD2 at the mRNA and protein levels in breast cancer. Repressor expression levels were also assessed in the context of breast cancer patient prognosis and survival.Results and discussionsWe identified the forkhead box transcription factor FOXP1 as a main transcriptional repressor of MAD2 expression. FOXP1 reduces MAD2 promoter activity via direct binding to CDE/CHR elements. Furthermore, knockdown of FOXP1 expression increases the proportion of cells in G2/M phase. In addition, low FOXP1 expression strongly correlates with high MAD2 expression at the mRNA and protein levels, and with poor patient prognosis in breast cancer, especially triple-negative breast cancer.ConclusionWe identified FOXP1 as a novel transcriptional repressor of MAD2 expression. Our results suggest that reduced FOXP1 levels in breast cancer affect both MAD2 expression and cell cycle progression. This may promote CIN and tumour formation via upregulation of MAD2. Our results may have important implications for breast cancer diagnostics and, potentially, therapeutic targeting.