Mechanistic studies on the role of GABA type A (GABA A ) ion channel receptor in tumor invasion and metastasis in triple negative breast cancer

Triple-negative breast cancer (TNBC) represents 15-20 percent of breast cancer cases and is associated with aggressive progression, metastasis, and limited treatment options. TNBC treatment relies on surgery, radiation, and chemotherapy, but these approaches are non-selective and highly toxic, emphasizing the need for novel molecular targets. Traditionally known for mediating inhibitory neurotransmission through chloride ion conductance, recent evidence implicates gamma-aminobutyric acid type A ion channel-coupled receptors (GABA A R) in cancer cell proliferation, migration, and metastasis. Our previous studies demonstrated that beta3 and alpha1 GABA A R subunits are overexpressed in TNBC cells, promoting chloride influx and cell survival. We also showed that pharmacological inhibition or shRNA-mediated GABA A R beta3 subunit knockdown reduces proliferation and induces cell cycle arrest of TNBC cell lines. Epithelial-mesenchymal transition (EMT) drives cancer metastasis by enabling cells to lose adhesion, reorganize the cytoskeleton, degrade the extracellular matrix, and gain migratory properties. The steroid receptor coactivator and focal adhesion kinase (Src-FAK) axis plays a central role in these processes by regulating cell remodeling, promoting cell proliferation, and migration. Src-FAK activation also stimulates the PI3K/AKT pathway, enhancing cell survival and resistance to apoptosis. In addition, FAK activates Rho-GTPases, which regulate actin cytoskeleton reorganization and cell motility. In this study, we investigated whether GABA A R beta3 subunit knockdown and pharmacological inhibition alter the invasive potential and EMT in TNBC. Our results showed that inhibiting GABA A R genetically and pharmacologically significantly reduced the invasive potential and reversed the EMT, as evidenced by decreased expression of mesenchymal markers (vimentin and N-cadherin) and increased expression of the epithelial marker E-cadherin as determined by Western blot analysis. Furthermore, beta3 subunit knockdown decreased the expression of total Src and reduced the phosphorylation of both Src and FAK. Morphological assessment further demonstrated that beta3 subunit knockdown reduced cell size, disrupted actin stress fibers, and altered actin distribution, consistent with impaired migratory capacity. To further define the molecular mechanisms downstream of GABA A R signaling, we will perform RNA sequencing on control and beta3 knockdown TNBC cells to identify transcriptional pathways involved in invasion and metastasis. Collectively, these findings reveal that GABA A R beta3 promotes TNBC cell proliferation, EMT, and invasion through Src-FAK signaling, highlighting GABAergic signaling as a promising therapeutic target in metastatic breast cancer.