Abstract 2585: GABA shunt pathway promotes peri-synaptic brain metastasis by hijacking synaptic signaling

Abstract Metastatic brain tumors are the most common intracranial neoplasms in adults. Among all subtypes of breast cancer, triple-negative breast cancer (TNBC) has a significantly poorer prognosis compared to other subtypes. Pre-menopause and estrogen replacement therapy after menopause have been shown to increase the risk of brain metastases in TNBC patients. Estrogen blockade using tamoxifen demonstrates some efficacy in TNBC, suggesting that estrogen promotes brain metastases by modulating the tumor microenvironment in estrogen receptor (ER)-negative tumors. Neurons, as highly specialized cells responsible for cell-to-cell signal transmission, are among the most critical and abundant cell types in the brain. However, the role of tumor-associated neurons and their contributions to TNBC remain poorly understood. We hypothesize that estrogen promotes brain metastasis in hormone receptor-negative breast cancer by increasing the release of gamma-aminobutyric acid (GABA), which serves as a metabolic substrate to fuel tumor cell growth. We further hypothesize that blocking GABA transporter function will suppress brain metastases in hormone receptor-negative breast cancer. Our findings show that pre-menopausal TNBC patients exhibit higher synaptic formation and increased expression of GABA transporters in brain metastases, suggesting that neurons may contribute to tumor growth. To investigate the effect of estrogen on brain metastasis in ER-negative breast cancer aCnd to understand how estrogen depletion modulates metastatic growth in the brain, we performed bilateral ovariectomy in female mice and intracardially implanted luciferase-labeled human ER-negative brain metastatic breast cancer cells (231BrM). Our results demonstrate that estrogen increases synaptic formation and upregulates the expression of GABA and GABA transporter 1 (GAT1) in brain metastases more prominently than other neurotransmitter receptors. To examine the effect of GABA on tumor progression, 231BrM cells were treated with 0.5 mM GABA, mimicking physiological brain concentrations, or recombinant GABA. We found that GABA treatment upregulated GAT1 expression and enhanced colony formation and migration abilities, strongly suggesting that GABA promotes brain metastasis. Furthermore, treating 231BrM cells with conditioned medium (CM) obtained from GABAergic neurons exposed to estrogen led to increased tumor growth through activation of the GABA shunt pathway in tumor cells. Blocking GAT1 using CRISPR/Cas9 or a small-molecule GAT1 inhibitor effectively suppressed GABAergic neuron-induced tumor progression. Our results suggest that estrogen enhances GABA release from neurons, subsequently promoting activation of the GABA shunt pathway in tumor cells. This study highlights GAT1 inhibitors as promising therapeutic tools for treating TNBC patients with brain metastases. Citation Format: Shih-Ying Wu, Kerui Wu, Abhishek Tyagi, Ravindra Pramod Deshpande, Eleanor Cecile Smith, Jee Won Kim, Kounosuke Watabe. GABA shunt pathway promotes peri-synaptic brain metastasis by hijacking synaptic signaling [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 2585.