GLUT3 inhibition reduces metastatic potential of triple-negative breast cancer cells by modulating cell mechanics via the TEAD-VASP-Actin axis

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking hormone (estrogen and progesterone) receptors and human epidermal growth factor receptor 2 (HER2), limiting the development of targeted therapies and contributing to poor patient outcomes. Metastasis, a major contributor to TNBC mortality, is closely linked to cancer cell mechanics-such as deformability and contractility-yet the molecular signaling cues from the tumor microenvironment (TME) that regulate these mechanical properties remain poorly understood. This study investigates the role of glucose metabolism, particularly the role of glucose transporter 3 (GLUT3), in modulating TNBC cell mechanics and metastatic behavior. GLUT3, which has the highest glucose affinity among GLUT isoforms, is upregulated in various cancers including TNBC and is associated with poor prognosis and metastasis in TNBC patients. We demonstrate that GLUT3 inhibition by a selective inhibitor G3iA reduces glucose uptake, glycolysis and ATP production by 30% in TNBC cells, leading to AMP-activated protein kinase (AMPK) activation and suppression of myosin activity. The decreased myosin activity reduces cell contractility and ultimately cell invasion. AMPK activation also increased YAP phosphorylation resulting decreased YAP target gene expressions. We also identified VASP, an actin filament nucleator and polymerase, is a novel YAP-TEAD target gene that regulated by the glucose-AMPK signaling axis. Additionally, GLUT3 inhibition triggers the cAMP-Protein Kinase A (PKA) pathway, which impairs actin polymerization through vasodilator-stimulated phosphoprotein (VASP). The decreased F-actin levels correlates with reduced cell stiffness, which was measured by Atomic Force Microscope. Using pharmacological inhibitors that activate the AMPK signaling or the cAMP-PKA signaling pathways, we show that these altered cell mechanics-cell stiffness and contractility-diminishes cell motility in vitro. Furthermore, we confirmed our findings that GLUT3 null TNBC cells show reduced lung metastasis in vivo . In summary, we delineate molecular pathways that translate extracellular glucose signaling into regulation of cell mechanics and motility via actin rearrangement. For the actin rearrangement, we discovered that regulation of VASP in transcriptional level and post-translational modification is critical. Our findings reveal a novel mechanistic link between glucose metabolism and cancer cell mechanics with detailed molecular mechanisms, offering a promising therapeutic avenue for mitigating TNBC metastasis.