A Novel Function of Tubulin β2a and Tubulin β3 in Regulating Metastatic Properties in Bone-Metastatic Breast Cancer Cells

Background/Objectives: Bone metastasis (BM) occurs in approximately 70% of patients with advanced breast cancer (BCa) and is associated with limited treatment options and a median survival of only 19–25 months. Although microtubule (MT)-targeting agents such as paclitaxel remain a standard therapy, their efficacy is often limited by chemoresistance linked to in part by altered β-tubulin isotype expression. Among these, tubulin β2a and tubulin β3 (encoded by TUBB2A and TUBB3) have been linked to poor clinical outcomes; however, their roles in breast cancer bone metastasis (BMBC) remain unclear. Here, we investigated the functional significance of TUBB2A and TUBB3 in regulating MT dynamics and metastatic behavior in bone-derived BCa cells. Methods: TUBB2A and TUBB3 expression levels were evaluated in primary breast tumors and bone metastases. CRISPR-mediated TUBB2A knockout and TUBB3 depletion were performed in bone-derived MDA-MB-231 cells to assess microtubule dynamics, trafficking, metastatic phenotypes, and gene expression. Results: Elevated expression of both TUBB2A and TUBB3 correlated with reduced patient survival. Immunohistochemical analyses demonstrated increased tubulin β2a abundance in BM lesions relative to matched primary breast tumors, while the proportion of moderate-to-strong tubulin-β3-positive cells was also increased in BM. Using CRISPR/Cas9-mediated knockout/knockdown in a bone-derived MDA-MB-231 cell model, we found that loss of TUBB2A or partial depletion of TUBB3 impaired MT regrowth, altered the cell cycle, and disrupted endosomal trafficking, characterized by enlarged perinuclear Rab5-positive vesicles. Functionally, depletion of either isotype reduced cell migration and invasion while enhancing adhesion, accompanied by increased expression of integrin β4, β5, α4, α5, and talin-1. Exploratory RNA-seq analysis suggests enrichment of cytokine–cytokine receptor signaling pathways, consistent with a potential link between MT remodeling and inflammatory/secretory programs. Conclusions: Collectively, these findings identify TUBB2A and TUBB3 as regulators of metastatic phenotypes and potential biomarkers and therapeutic targets in BMBC.