Mechanistic studies on the role of GABA type A ion channel (GABAA) receptors in triple negative breast cancer - Abstract Triple-negative breast cancer (TNBC) is aggressive, metastasizes quickly and has a poor clinical outcome, highlighting the urgent need for targeted treatments beyond traditional chemotherapy. An ideal druggable target for TNBC would be at the membrane, allowing selective targeting with a small molecule or antibody. Emerging evidence suggests that γ-amino butyric acid (GABA), a major inhibitory neurotransmitter plays a tumor-supporting role in some solid cancers. GABA signals via membrane bound GABA type A ion channels (GABAAR) and GABAB G-protein coupled receptors. The role(s) of GABAAR signaling in TNBC remains unclear and this multidisciplinary proposal aims to decipher these roles. Our preliminary results show that GABAAR, which are permeable to chloride (Cl-) ions, are significantly upregulated in several human TNBC cell lines. GABA is also produced by these cells. Cl- flux alterations may be critical for TNBC growth, since intracellular Cl- ion levels alter cell division in other cancers. Moreover, we observe morphology changes and decrease in actin stress fibers after GABAA β3 subunit knockdown, implicating the involvement of cytoskeletal proteins, which support cell adhesion and migration. Our two-pronged strategy is to dissect the involvement of GABAAR mediated Cl- flux and/or cytoskeletal architecture changes in TNBC progression. Our preliminary study demonstrates that GABAAR inhibition decreases TNBC migration in vitro and causes cell-cycle arrest. We hypothesize that knocking down/blocking GABAAR in TNBC cell lines will decrease their proliferation, migration and invasion via a decrease in Cl- ion influx and/or disruption of cytoskeletal architecture. We propose a joint effort between the PI’s group (specializing in GABAAR signaling), and Co-I’s group (expertise in cancer signaling). Specifically, Aim1 will determine the impact of GABAAR inhibition (genetic and pharmacological) and overexpression in vivo in mouse orthotopic models. Aim 2 will determine the mechanisms of GABAAR inhibition (in TNBC cells) and overexpression (in MCF10As). Aim2a will use an automated benchtop patch clamp assay to measure Cl- ion influx in TNBC cells after GABAAR knockdown and inhibition. Aim2b will examine actin stress fiber organization and employ RNA sequencing in GABAAR knockdown cells to identify important EMT markers. To tease out the relative contribution of GABAAR mediated Cl- flux and actin cytoskeletal changes in TNBC growth, we will overexpress loss-of-function GABAA β3 subunit mutants in MCF10A cells and examine the effect on actin stress fiber organization. Successful completion of this project will not only advance understanding of the role of GABAAR in TNBC growth and metastasis, but will also engage students in hands-on research in both neuroscience and cancer labs.
