DESCRIPTION (provided by applicant) Triple negative breast cancer (TNBC) lacks estrogen and progesterone receptors (ER and PR), and amplification of the growth factor receptor HER2 such that this tumor type are unresponsive to endocrine or HER2-targeted therapies. Partially due to this lack of therapeutic options to treat tumors resistant to chemotherapy, but also because of particularly aggressive unique biological characteristics of this breast cancer subtype, TNBC patients have the poorest prognosis and the disease is often already metastatic at time of prognosis. The ability of carcinoma cells to resist programmed cell death following detachment from the basement membrane or detachment from the primary tumor is known as anoikis resistance, and is thought to be a critical step in the metastatic cascade. TNBC cells are more anoikis resistant than their less aggressive ER+ counterparts and ER+ disease usually takes longer to become metastatic and does so less often than TNBC. To begin to elucidate mechanisms by which TNBC cells survive in suspension culture, we performed global gene profiling comparing TNBC cells in forced-suspension or attached culture for 24 hours and made the novel discovery that multiple genes in the kynurenine pathway (KP) of tryptophan catabolism are upregulated in suspended, anoikis resistant TNBC cells. Acting as the primary pathway of tryptophan catabolism and de novo NAD+ synthesis, the KP and associated metabolites have been implicated in promoting tumorigenesis and immune suppression in other types of cancer. The intermediate tryptophan metabolite, kynurenine, binds to and activates the aryl hydrocarbon receptor (AhR). Importantly, both the AhR itself and AhR regulated genes were also upregulated in TNBC cells upon suspension. The goal of my proposal is to determine if and how intracrine/autocrine signaling through the kynurenine pathway and subsequent activation of AhR, is critical to TNBC survival in suspension, promoting TNBC metastasis. Specifically in Aim 1, I will define whether upregulation of TDO2 promotes survival in suspension and migration/invasion through Kyn mediated activation of AhR, in vitro. In Aim 2, I will define the role of the kynurenine pathway in survival in circulation and metastasis of TNBC cells, in vivo. This proposal emphasizes a molecular understanding of metabolic signaling through the kynurenine pathway that will provide the necessary framework to determine if therapeutic strategies targeting this pathway should will be viable and should target the rate limiting enzyme in this pathway, TDO2, in order to combat and/or prevent metastasis of TNBC. The completion of the proposed research will help the National Cancer Institute fulfill their mission to support cancer research leading to novel therapeutic targets and training future cancer biologists.

PUBLIC HEALTH RELEVANCE Patients with triple-negative breast cancer (TNBC) are unresponsive to current endocrine or HER2 targeted therapies and have poor overall survival, greatly attributed to the high rate of metastasis following diagnosis. My preliminary data demonstrate that key enzymes involved in the kynurenine pathway of tryptophan catabolism and the production of the intermediate metabolite kynurenine are upregulated in TNBC cells in forced-suspension culture and may contribute clinically to circulating tumor cell survival and invasive capacity. Therefore, we predit that targeting of key enzymes within the kynurenine pathway will inhibit an autocrine/intracrine survival signal through binding to the aryl hydrocarbon receptor necessary for TNBC metastatic progression and serve as a targeted therapeutic for this aggressive form of breast cancer.