Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer (BC), evades hormonal treatment modalities, and patients with TNBC experience high rates of metastasis and have a poor prognosis. Therefore, there is a critical need to find better approaches to treat TNBC. Sirtuin 1 (SIRT1) represents an interesting target in this regard, as SIRT1 has been shown to be implicated in cancer as a tumor suppressor, and in fact, SIRT1 mRNA and protein are significantly downregulated in TNBC. Previous work has shown that decreasing SIRT1 in human TNBC cells promotes the generation of a secretome containing soluble hydrolases and a large number of exosomes, a specific class of extracellular vesicles, with unique cargo. Furthermore, the hydrolases and exosomes produced by TNBC cells depleted of SIRT1 were shown to promote the aggressive phenotype of TNBC cells by promoting cell survival, invasive activity, and metastasis. Thus, it is important to determine whether decreasing SIRT1 expression or activity in vivo in mouse models of breast cancer promotes tumorigenesis and metastasis. Additionally, the mechanism for decreased expression of SIRT1 in TNBC is largely unknown, and a better understanding of SIRT1 regulation will uncover changes in cancer-promoting pathways that make TNBC so aggressive. In this proposal, the effects of decreased SIRT1 expression or activity on tumorigenesis and metastasis will be elucidated (Aim 1), and the transcriptional and post-transcriptional regulatory factors that decrease SIRT1 expression in TNBC will be identified (Aim 2). In Aim 1, three distinct, yet complementary, mouse models of breast cancer will be used to determine the effects of altered SIRT1 expression or pharmacologic modulation of SIRT1 activity on cancer progression in the tumor microenvironment, including tumor growth, invasiveness, and angiogenesis, as well as rate of metastasis. In Aim 2, precision nuclear run-on RNA sequencing (PRO-Seq) and small RNA sequencing will be performed to elucidate transcriptional and post-transcriptional regulators of SIRT1, respectively. For this aim, a system of three human BC cell lines with varying levels of SIRT1 expression will be used. Changes in transcriptional regulation and microRNA expression with PRO-Seq and small RNA-Seq, respectively, will be determined by comparing across these three cell lines. Due to the highly aggressive nature of and difficulty to treat TNBC, it is imperative to develop new therapeutic strategies that slow tumorigenesis and metastasis. Through the outlined six-year training plan in Drs. Richard Cerione and Robert Weiss’s labs, which have expertise in elucidating underlying molecular mechanisms and their effects in vivo through mouse models, by determining the effects of low SIRT1 levels in mouse models of BC and the regulatory factors that mediate this downregulation, this proposal holds promise for clarifying the role of SIRT1 as a tumor suppressor in TNBC.