PROJECT SUMMARY/ABSTRACT Therapy failure remains an overarching clinical challenge for patients with aggressive Triple Negative Breast Cancer (TNBC). Two important factors that regulate TNBC metastasis and recurrence are: (i) the differentiation status of the cancer cells, and (ii) the functionality of anti-tumor immunity, both of which are highly influenced by the tumor microenvironment (TME). We find that the TME cytokines Transforming Growth Factor β (TGFβ) and Interferon β (IFNβ) oppose one another in regulating both cancer cell differentiation and immune system function in TNBC. TGFβ and its receptors are frequently upregulated in aggressive, therapy-resistant cancers, such as TNBC. Mechanistically, we find that TGFβ effector SMAD3 cooperates with STAT3, an important effector of the IL-6 family of cytokines, to induce stem-like/mesenchymal reprogramming, which enhances TNBC invasiveness, tumor-initiating capacity, and therapeutic resistance. At the same time, TGFβ strongly suppresses IFNβ production. Since IFNβ promotes a less aggressive cancer cell state and activates anti-tumor immunity, the TGFβ-mediated suppression of IFNβ and its downstream effectors, STAT1 and STAT2, is an important contributor to TNBC progression and immune system impairment. Importantly, restoration of IFNβ signaling results in the reversal of TGFβ-mediated stem-like/mesenchymal program and re-engages anti-tumor immunity. We hypothesize that the relative amounts of TGFβ and IFNβ cytokine activity in both cancer and immune cells dictate TNBC aggressiveness and ultimately, patient outcomes. The purpose of our study is to define the molecular mechanisms by which TGFβ and IFNβ antagonize one another in regulating stem-like/mesenchymal reprogramming and anti-tumor immunity. Our Project has strong connections with Project 1, which will assess the regulation of STAT2 by a novel threonine phosphorylation, and with Project 3 which will examine how TGFβ synergizes with another pro-tumor cytokine, IL-17, to amplify the signals critical for repressing IFNβ production. The unifying studies proposed in our Program will (i) identify common molecular mechanisms that promote the development of resistance to chemo- and immune-therapies and (ii) assess novel therapeutic combinations aimed at shifting the equilibrium of IFNβ and TGFβ/IL-17 signaling in the TME. By shifting this balance, we propose to induce the differentiation of cancer cells and enhance anti-tumor immunity to increase the sensitivity of hard-to-treat cancers to chemo- and immuno-therapy.