Project Summary/Abstract Breast cancer (BC) is the second leading cause of cancer-related deaths among women in the United States. A subset of BC called triple-negative breast cancer (TNBC) has the worst prognosis among BC subtypes, due to both the frequency and location of the resulting metastases. In TNBC, the underlying mechanisms that mediate metastatic outgrowth after disseminated tumor cells implant into distal sites is unknown. However, our laboratory recently identified the long noncoding RNA, BORG (BMP/OP-Response Gene), as a driver of TNBC metastasis. Indeed, BORG expression enhances neoplastic proliferation and chemoresistance, which are important cellular functions associated with metastatic phenotypes. Mechanistically, our group showed that BORG complexes with the E3 SUMO ligase TRIM28 in order to enhance neoplastic proliferation by repressing the expression of the growth arrest genes, p21 and gadd45a. Herein, I will show that the proliferative and cell survival pathways activated by BORG reflect breast cancer stem cell (BCSC) phenotypes. Importantly, I demonstrate that BORG enhances BCSC mammosphere formation and tumor growth both in vitro and in vivo, and that this phenotype relies in part on the formation of BORG:TRIM28 complexes. However, the mechanisms through which BORG and TRIM28 enhance BCSC plasticity are not yet understood. To this end, I find that BORG expression causes significant transcriptomic reprogramming, suggesting that BORG enhances cellular plasticity by mediating transcriptomic changes (e.g., alternative splicing) which broadly influences BCSC-like phenotypes. Accordingly, I showed that BORG expression plays a role in alternative splicing by upregulating 61 alternative splicing events (ASEs) and downregulating 83 ASEs, including a number of ASEs whose genes are involved in key metastatic pathways. Moreover, I determined that BORG downregulates the expression of 38 splicing factors, and up- regulates the expression of 6 splicing factors. Interestingly, the downregulation of 16 of these splicing factors relies upon BORG:TRIM28 complexes. Based on these preliminary data, I hypothesize that BORG:TRIM28 complexes drive BCSC plasticity and metastasis by regulating the epigenome and ASEs involved in metastatic phenotypes. To elucidate how BORG influences alternative splicing, I will (i) determine the mechanism(s) by which BORG:TRIM28 complexes regulate the expression and activity of splicing factors to drive variations in alternative splicing; and (ii) elucidate the consequences of BORG-induced ASEs on tumor cell plasticity and BCSC phenotypes. To our knowledge, my innovative analyses are the first to investigate the interaction between BORG and alternative splicing in promoting the metastatic features of TNBCs. By elucidating molecular mechanisms whereby BORG drives alternative splicing, we can better understand how lncRNAs elicit such drastic phenotypic changes to drive metastatic behaviors in TNBCs. Importantly, BORG...