Abstract

Simple SummaryTriple-negative breast cancer (TNBC) is often marked by very high Ki-67 expression—indicating rampant, uncontrolled cell division—but the drivers behind this fast proliferation remain poorly understood. Our study identifies the mitotic kinase TTK as a central factor: it is markedly overexpressed in TNBC (vs. non-TNBC), strongly correlates with elevated Ki-67 levels and worse patient survival, and functionally fuels tumor growth. We show that TTK overexpression is transcriptionally activated by the factor NFYA binding to the CCAAT box in the TTK promoter—a newly discovered regulatory mechanism. Importantly, inhibiting TTK induces G2/M arrest, disrupts the spindle assembly checkpoint (via BUB1B/MAD1L1 downregulation), triggers mitotic catastrophe, and potently suppresses TNBC growth in cells and animal models. These findings position TTK not only as a key biological driver and prognostic biomarker in TNBC, but also as a promising therapeutic target to halt its aberrantly accelerated cell cycle.Background/Objectives: Triple-negative breast cancer (TNBC) is frequently characterized by notably elevated Ki-67 expression, a hallmark of uncontrolled rapid cell-cycle progression. However, the underlying mechanisms remain unclear, leading to limited therapeutic options. Methods: In this study, hub gene was identified through integrated bioinformatic analysis of public datasets (TCGA-BRCA and METABRIC). Subsequent functional validation was performed both in vitro and in vivo using siRNA-mediated knockdown and small-molecule inhibitors. Phenotypic effects—including cell viability, cell cycle distribution, DNA synthesis, and clonogenic survival—were comprehensively assessed using MTT assays, flow cytometry, EdU, and colony formation assays. Protein-level changes were confirmed by Western blotting and immunohistochemistry (IHC). To dissect the transcriptional regulation of the key hub gene TTK, we first predicted potential upstream transcription factors using the J