Targeting overexpressed TTK and NEK2 decreases early metastatic potential of triple-negative breast cancer cell lines

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer lacking estrogen, progesterone, and HER2 receptors, making it unresponsive to hormonal or anti-HER2 therapies. Non-Hispanic Black (NHB) and Hispanic/Latino (H/L) women have lower survival rates, partly due to later-stage tumor detection and higher TNBC prevalence. Higher African ancestry has been associated with increased expression of mitotic kinases, such as TTK and NEK2, which regulate cell division and tumor progression. Our research confirms that TTK overexpression is associated with TNBC growth and epithelial-to-mesenchymal transition (EMT). Preliminary analyses indicate that molecular functions and processes, such as beta-catenin binding, are associated with TTK and NEK2 expression.Therefore, this study aims to investigate, via in vitro assays, the expression levels of EMT markers and transcription factors, as well as changes in the invasive capacity and motility of metastatic TNBC upon downregulation of TTK, NEK2, and beta-catenin. To assess this, TNBC cell lines, MDA-MB-231 (NHW) or MDA-MB-157 (NHB), were cultured, followed by TTK/NEK2 or beta-catenin siRNA-mediated knockdown. Western blotting was used to measure EMT-related proteins and transcription factors. Immunocytochemistry evaluated beta-catenin co-localization. Invasion and migration assays used Matrigel-coated chambers and wound-healing models, respectively. In vitro assays showed a significant decrease in Vimentin protein levels in all TTK-, NEK2-, and double-knockdown treatment groups of the MDA-MB-231 cell line. TTK/NEK2 double knockdown, specifically, reduced N-cadherin, beta-Catenin, and SNAIL transcription factor.​ In contrast, results from MDA-MB-157 cell lines showed a significant reduction in beta-catenin protein levels upon NEK2 knockdown. Immunocytochemistry results also showed a decrease in cytoplasmic, nuclear, and membrane levels of beta-catenin in the MDA-MB-157 cell line. Furthermore, downregulation of TTK, NEK2, and beta-catenin reduces the invasive capacity and motility of both TNBC cell lines.These findings support a pivotal role for TTK and NEK2 in beta-Catenin regulation, EMT, and the invasive behavior of TNBC cells. Dual targeting of these mitotic kinases may be a promising strategy to reduce the expression of key proteins involved in TNBC progression and metastasis.