Abstract 6949: Downregulating the mitotic kinases TTK and NEK2 as a promising approach for the reduction of metastasis in triple-negative breast cancer cell lines

Abstract Triple-negative breast cancer (TNBC) lacks estrogen, progesterone, and HER2 receptors, making it the most aggressive form of breast cancer. TNBC with distant metastases is nearly untreatable with anti-hormonal or anti-HER2 therapies, leaving chemotherapy as the primary option. TNBC is characterized by uncontrolled cell growth, increased centrosome amplification, genetic instability, and high invasive potential. Non-Hispanic Black (NHB) and Hispanic/Latino (H/L) women have lower 5-year survival rates compared to non-Hispanic White (NHW) women, partly due to later-stage tumor detection and higher TNBC prevalence. Women with a greater proportion of African ancestry exhibit elevated expression of several mitotic kinases, such as TTK and NEK2. TTK helps maintain genomic stability by regulating the spindle assembly checkpoint, while NEK2 regulates centrosome separation and microtubule organization. However, overexpression of these kinases in breast cancer is linked to increased cell growth, chromosomal instability, aneuploidy, and epithelial-to-mesenchymal transition (EMT). Previous data from a tissue microarray confirmed the link between TTK overexpression and markers of proliferation, EMT, and the TNBC subtype. Thus, we aim to measure expression levels of EMT markers and transcription factors, as well as changes in the invasive capacity of metastatic TNBC cell lines, upon downregulation of TTK and/or NEK2. To address this, TNBC cell lines, MDA-MB-231 (NHW) or MDA-MB-157 (NHB), were cultured, followed by TTK and/or NEK2 siRNA-mediated knockdown. Western blot and qPCR analyses measured protein and mRNA expression levels of EMT markers and transcription factors. Invasion assays were performed using Matrigel matrix invasion chambers. Preliminary results indicate that silencing of TTK, NEK2, or both resulted in a reduction of the mRNA expression levels of EMT markers, Vimentin, N-cadherin, or β-Catenin in MDA-MB-231 and MDA-MB-157 cell lines. Western blot results showed a significant decrease in Vimentin protein levels in all treatment groups of MDA-MB-231 cell lines, while TTK/NEK2 knockdown also reduced N-cadherin, β-Catenin, and SNAIL transcription factor.​ Comparably, results from MDA-MB-157 cell lines showed a significant reduction of β-Catenin protein levels upon NEK2 knockdown. Furthermore, TTK/NEK2 knockdown leads to a decrease in the invasive capacity of both TNBC cell lines. These preliminary data suggest that dual silencing of mitotic kinases TTK and NEK2 may be a promising approach for reducing key proteins involved in EMT processes in TNBC cell lines. Due to their increased expression in women of African descent (e.g. African American and Caribbean Hispanics), targeting mitotic kinases could represent a promising therapeutic strategy, particularly in Afro-descendants with TNBC by limiting tumor growth and metastasis. Citation Format: Alexandra N. Aquino-Acevedo, Ángel D. Colón-Burgos, Marileana Rodríguez-Ruiz, Gabriel Rosario-Ortiz, Elliott R. Rodríguez-López, Esther M. Irizarry-Quintana, Gretchen M. Albarrán-Acosta, Joel A. Orengo-Orengo, Melanie E. Cruz-Robles, Harold I. Saavedra. Downregulating the mitotic kinases TTK and NEK2 as a promising approach for the reduction of metastasis in triple-negative breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6949.