NFYA-Mediated TTK Up-Regulation Drives Fast Cell Cycle Progression and Its Inhibition Leads to Mitotic Catastrophe in Triple Negative Breast Cancer

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 JASPAR database; binding specificity was then validated through in silico motif analysis, luciferase reporter assays, and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Results: The mitotic kinase TTK is significantly overexpressed in TNBC compared with non-TNBC breast cancers. Notably, TTK overexpression exhibited a strong positive correlation with elevated Ki-67 indices and reduced overall survival in TNBC patients. Functional validation demonstrated that pharmacological or genetic inhibition of TTK effectively induced G2/M cell-cycle arrest and potently suppressed TNBC proliferation in both in vitro cell cultures and in vivo xenograft models. Mechanistically, TTK overexpression stems from enhanced transcriptional initiation driven by the transcription factor NFYA binding to the CCAAT box in the TTK promoter—an interaction newly identified here. Concurrently, TTK blockade disrupted spindle assembly checkpoint (SAC) signaling via BUB1B/MAD1L1 downregulation, triggering mitotic arrest and catastrophe. Conclusions: Collectively, these findings establish TTK as a key cell-cycle regulator driving TNBC proliferation. More importantly, targeting mitotic control through TTK inhibition represents an efficient strategy to impede the aberrantly fast cell cycle progression in TNBC.