Abstract 97: Microtubule dynamics in regulating bone metastasis of breast cancers: a novel function of tubulin β-2 and tubulin β-3

Introduction: Bone metastases (BM) represent a significant hurdle in advanced cancer management, with median survival rates of 12 to 50 months. Current treatment options, including surgical resection and microtubule (MT)-targeting chemotherapies like paclitaxel, often provide palliative rather than curative outcomes. MTs, composed of α- and β-tubulin heterodimers, are essential for cell division, migration, and trafficking. Dysregulated expression of β-tubulin isotypes, TUBβ2 and TUBβ3, has been implicated in chemoresistance, yet their specific roles in breast cancer bone metastases (BMBC) remain poorly understood. This study explores the novel functions of TUBβ2 and TUBβ3 and investigates their potential as therapeutic targets by disrupting their unique C-terminal domains. Methods: RNA-Seq datasets and patient survival data from publicly available databases were analyzed to evaluate the expression of TUBβ2 and TUBβ3. Immunohistochemistry (IHC) was performed on matched primary and BM tumor biopsies (n=16) from BC patients. CRISPR/Cas9-mediated knockdowns (KDs) in bone-derived MDA-MB-231 cells were employed to assess cell growth, survival, adhesion, migration, and metastatic properties. MT polymerization rates were evaluated using MT regrowth assays, subcellular trafficking was analyzed via immunofluorescence imaging. Focal adhesion turnover were assessed by western blotting, and IL6 secretion-analyzed by ELISA. Results: Elevated expression levels of TUBβ2 and TUBβ3 significantly correlated with poor survival in breast cancer patients and were markedly higher in BM tumors compared to primary and normal breast tissues. CRISPR KD revealed distinct morphological changes, with TUBβ2KD inducing giant cell formation and TUBβ3KD leading to elongated cytoplasmic extensions. •Metastatic properties: TUBβ2KD reduced adhesion but enhanced migration, while TUBβ3KD diminished migration but increased adhesion. Reduced focal adhesion kinase (FAK) levels were observed in both KDs, impairing adhesion and migration. •MT dynamics: KDs caused significant delays in MT repolymerization, indicating isotype-specific integration into MT polymers. •Cytokine secretion: Loss of TUBβ2 led to a 2-fold increase in IL6 production, whereas TUBβ3 loss resulted in a 70% reduction in IL6 secretion. •Subcellular trafficking: Loss of perinuclear endosomal puncta revealed novel roles for TUBβ2 and TUBβ3 in endosomal trafficking. Conclusions and Impact: This study identifies TUBβ2 and TUBβ3 as critical regulators of MT dynamics and metastatic properties in BMBC. Their roles in cell adhesion, migration, and trafficking underscore their potential as therapeutic targets. Current clinical approaches to BM focus largely on palliative care, but targeting TUBβ2 and TUBβ3 offers a promising strategy for more precise interventions that could improve patient survival and quality of life. Citation Format: Shreya Patel, Marcus Winogradzki, Waddell Holmes, Alan Blank, Niyati Patel, Jitesh Pratap. Microtubule dynamics in regulating bone metastasis of breast cancers: a novel function of tubulin β-2 and tubulin β-3 [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 97.