PROJECT SUMMARY/ABSTRACT Patients with brain metastasis of lung cancer have extremely poor prognosis, high mortality rate, and frequent incidence of tumor recurrence. Understanding the pathological mechanism of brain metastasis is urgently needed to develop a novel and effective therapeutic strategy. Published data as well as the results of my own study for brain metastasis of lung cancer indicate that smoking and nicotine significantly increased the incidence and progression of brain metastasis, but the pathological mechanism by which the smoking promotes brain metastasis through modulating brain microenvironment is yet poorly understood. Our preliminary results showed that synaptic formation in brain metastasis region is strongly correlated with poor overall survival of patients with brain metastasis. However, the exact role of neurons in brain metastasis progression remains unclear. The goal of this proposal is to elucidate the mechanism of GABAnergic neuron activation in the brain metastasis in order to develop innovative strategies for the treatment of brain metastasis. I hypothesize that nicotine stimulates microglia to secrete exosomal miR-32-3p which promotes brain metastasis by augmenting GABAergic synaptic formation and hence releasing GABA that serves as metabolic substrate to fuel tumor cell growth. I also hypothesize that inhibiting the GABA transporter of tumor cell suppresses brain metastasis by blocking GABA shunt. In Aim 1, I will clarify the molecular pathway through which nicotine stimulates microglia to secrete exosomal miR32-3p and activates GABAergic neuron. In Aim 2, I will investigate the pathological mechanism by which the activated GABAergic neuron enhances brain metastasis by promoting GABA shunt of tumor cells. Aim 3 is to test the efficacy of inhibitors for GABA transporter on nicotine-stimulated brain metastasis. The K99 phase of the proposed research will be pursued at Wake Forest University (WFU), an interactive cancer biology community, and a wealth of intellectual and technical resources. The training plan, under the mentorship of Dr. Watabe, outlines a comprehensive strategy for acquiring the technical and the professional skills required to complete the proposed research and prepare me for an independent research career. Experienced six members of my mentor team will provide training in new techniques and analyses of data. By taking advantage of Wake Forest‘s exceptional resources for professional development, I will also improve my skills for communication, management, and leadership. The training I obtain at WFU will equip me to lead a laboratory that merges diverse approaches to investigate the mechanisms of initiation and progression to clinically significant brain metastasis and identify innovative strategies for prevention and treatment of brain metastasis.

PROJECT NARRATIVE The proposed research seeks to develop a functional and mechanistic understanding of how GABAergic neuron modulation by nicotine contributes to the brain metastasis of lung cancer. Here, we aim to define the newly discovered functional role of GABAergic neuron in supporting brain metastasis and provides a scientific basis for pursuing the inhibition of nicotine-activated GABA shunt as a novel therapeutic strategy that could significantly improve the treatment and prevention of brain metastasis in cancer patients with past and current smoking history. Information gleaned from this work may be used in the generation of future clinical trials focusing on therapeutic interventions for preventing brain metastasis.