Alternative Splicing Rewiring by Neurofibromin Isoforms Through ER-alpha and RAS Dependent Mechanisms - PROJECT SUMMARY Alternative splicing (AS) of transcripts is an essential post-transcriptional process that is instrumental for numerous cell functions including proliferation, differentiation, and survival. The process of AS is regulated by RNA binding proteins (RBPs) and their proper nuclear localization and interactions with spliceosome components which form visibly dense compartments called nuclear specks (NS). The ability for cancer to hijack and utilize AS by targeting genes involved in EMT, invasion, cell cycle regulation, and transcriptional control has been described to influence metastatic potential and therapeutic resistance. More recently, it has been shown that 50% of breast cancers contain overexpression or amplification of at least one RBP which consequently targets gene transcripts for AS to induce invasive characteristics. Furthermore, a novel function of estrogen receptor alpha (ERα) recently revealed non-canonical RNA binding of ERα to influence post- transcriptional regulation and promote cell fitness in response to environmental stressors. Our laboratory, along with other, recently established a critical link between NF1 and ERα in regulation of ERα signaling. NF1 is a tumor suppressor gene, encoding for the protein neurofibromin, that when mutated drives of a subset of breast cancers with poor prognosis. These studies have sparked motivation to investigate mechanisms in which NF1-deficient breast cancers contribute to metastasis and endocrine resistance. The interaction between neurofibromin and ERα describes neurofibromins ability to act as a co- repressor of ERα’s transcriptional activity. However, it still remains unknown whether this neurofibromin-ERα interaction regulates ERα’s post-transcriptional regulation or the importance of neurofibromin localization for this regulation. The overall goal of my F99 training phase (Aim 1) is to determine the role neurofibromin plays in ERα post-transcriptional regulation. Specifically, the Aims are 1.1) to define neurofibromin’s impact on ERα dependent post-transcriptional regulation in NF1 mutant ER+ breast cancer models and 1.2) determine the efficacy of selective estrogen receptor modulators (SERMs) to impact ERα post-transcriptional regulation in NF1-deficient cells. The training objectives in the F99 phase will expand my molecular techniques, sequencing, and data analysis in cancer biology. During my K00 phase (Aim 2) I will define the impact nuclear neurofibromin has on post-transcriptional regulation through nuclear speck formation and function. NF1- deficient breast cancer models will be used to determine nuclear speck formation, composition, and post- transcriptional and gene regulation. The training and data resulting from this fellowship award will establish the foundation of scientific and professional skills for my career as an independent researcher.
