Molecular Mechanisms of Small Cell Lung Cancer Brain Metastasis

PROJECT SUMMARY This proposal focuses on the molecular and cellular mechanisms of metastasis to the brain in small-cell lung cancer (SCLC), one of the most fatal forms of human cancer. Brain metastases are very frequent and a major cause of morbidity and mortality in SCLC patients. It has proven to be extremely challenging to investigate SCLC brain metastasis because surgery is very rarely performed to remove brain metastases in SCLC patients. There is also a dearth of pre-clinical models to study SCLC brain metastasis. In previous work using pre-clinical mouse models and intracranial injections of SCLC cells, we have found an upregulation of neuronal programs in SCLC cells during metastatic progression. Our preliminary data further indicate that this induction of neuronal programs endows SCLC cells with the ability to functionally interact with various cell types in the brain microenvironment, thereby promoting the ability of SCLC cells to grow in the brain. Mechanistically, this upregulation of neuronal programs is often due to the upregulation of the transcription factor NFIB, a known regulator of neuronal programs in the developing brain. My goal is to further investigate key cellular and molecular mechanisms of SCLC brain metastasis. I propose two Specific Aims. First, because ~15% of patients with SCLC have brain metastases at the time of their cancer diagnosis, it is important to investigate the mechanisms of SCLC growth in the brain. I will use intracranial injections of SCLC cells to model the growth of brain metastases in the brain of patients with SCLC. Using these cell models, I will perform in vivo CRISPR/Cas9 knockout screens focusing on candidates with known functions in neuronal biology during brain development. In parallel, I will perform similar screens in subcutaneous tumors to identify genes that are important for growth of SCLC specifically in the brain. A pilot screen has identified Leucine-rich repeat-containing 4B (LRRC4B, also known as Netrin-G ligand-3, NGL3), a synaptic adhesion molecule known for its involvement in excitatory synapse stability, to be important for SCLC growth in the brain. The role of LRRC4B will be investigated using gain- and loss-of-function studies in SCLC cell lines to understand its potential in facilitating interactions between SCLC cells and cells within the brain microenvironment. I will also use co-culture assays with murine neurons to investigate the role of LRRC4B in the functional interactions between SCLC cells and neurons. Second, because ~70% have brain metastases at the time of death from ~15% at the time of diagnosis, it is important to also investigate how new metastases are generated in the brain of patients with SCLC. I have identified human cell lines with brain tropism in mice. I will perform serial transplantation in the brain to enrich for brain metastasis phenotypes. I will also use these cell models to assess the importance of candidate genes, including NFIB, in the metastatic seeding of SCLC cells to the brain. The proposed experiments will uncover key genes and pathways that contribute to the striking ability of SCLC to metastasize to the brain and will identify mechanisms of SCLC brain metastasis that may serve as the basis to develop new strategies to treat SCLC patients with brain metastasis.