Mechanistic Insights into Threonine Tyrosine Kinase Mediated Cell Cycle Regulation in Triple-negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking targeted therapies, characterized by high heterogeneity and poor prognosis. Dysregulated cell cycle progression and aberrant Wnt/β-catenin signaling are critical drivers of TNBC proliferation. This study aimed to advance understanding of TTK driven oncogenesis and assess its promise as a prognostic biomarker and therapeutic target in TNBC.

We analyzed bulk and single-cell RNA sequencing datasets to assess TTK expression and clinical relevance in breast cancer. TNBC cell lines and normal breast epithelial cells were used for in vitro functional assays, including cell proliferation, colony formation, migration, and cell cycle analysis following TTK knockdown with siRNA. Immunofluorescence and Western blotting evaluated TTK expression and β-catenin pathway activity. Functional enrichment, protein-protein interaction, and pseudotime trajectory analyses elucidated TTK's mechanistic roles.

TTK was overexpressed in breast tumors versus normal tissues, with elevated levels correlating with worse overall and relapse-free survival. TTK knockdown impaired TNBC cell proliferation, colony formation, and migration, and induced G₁ arrest. Single-cell analyses demonstrated TTK enrichment in cancer cells, peaking during S and G₂/M phases. Pseudotime trajectory revealed dynamic TTK upregulation during G₁-S-G₂/M transition. Mechanistically, TTK maintained β-catenin signaling and downstream Cyclin D1 expression, facilitating G₁/S entry and supporting mitotic checkpoint fidelity. Pathway enrichment analyses further confirmed TTK's centrality in cell cycle regulation and proliferative programs.

TTK drives TNBC progression by orchestrating G₁/S and G₂/M transitions and sustaining β-catenin-Cyclin D1 signaling. Its restricted expression in normal tissues, combined with oncogenic effects, positions TTK as a promising prognostic biomarker and therapeutic target. Pharmacological inhibition of TTK, potentially combined with β-catenin pathway inhibitors, may offer an effective strategy for TNBC treatment.