Summary for Project 2 led by Research Project Leader Sayem Miah, PhD The metastatic processes of breast cancer are complex and remain a key area of intense research. TGFβ/SMAD signaling has been identified as a critical pathway that can be targeted to impact breast cancer tumorigenesis and metastasis. We made the exciting discovery that breast tumor kinase (BRK)—a non-receptor tyrosine kinase (nRTK) that is overexpressed in most invasive ductal carcinomas, including the triple-negative subtype, and linked to proliferation, metastasis, and cancer development—catalyzes tyrosine phosphorylation of SMAD4, a transcription factor that is a major transducer of TGFβ signaling. Under normal cellular conditions, SMAD4 interacts with SMAD2/3 to form the SMAD complex and promote the transcription of genes involved in cellular homeostasis. However, we found that BRK-dependent phosphorylation of SMAD4 inhibits the formation of the SMAD complex and instead induces an interaction between SMAD4 and the nucleosome remodeling and deacetylase (NuRD) complex. NuRD is an epigenetic regulator that represses transcription and has been implicated in triple-negative breast cancer tumorigenesis. Based on our preliminary findings and previously published reports, we hypothesize that BRK-dependent phosphorylation of SMAD4 facilitates interactions with the multi-subunit NuRD complex that result in targeted chromatin modification and dysregulation of canonical TGFβ-stimulated transcription, which promotes tumorigenesis and metastasis. We will test our hypothesis in the following Specific Aims: (Aim 1) Identify macromolecular protein interactions between BRK-phosphorylated SMAD4 and the NuRD complex, (Aim 2) Determine how the BRK-phosphorylated SMAD4 with NuRD complex dysregulates canonical TGFβ-stimulated transcription, and (Aim 3) Determine whether BRK-mediated dysregulation of canonical TGFβ signaling promotes tumorigenesis and metastasis in vivo. The proposed work is significant because the oncogene BRK is highly expressed in >85% of human invasive ductal carcinomas, including the triple-negative subtype. In alignment with the scientific theme of the Center for Molecular Interactions in Cancer (CMIC), we will elucidate novel molecular features of BRK and SMAD4 that fuel pro- malignant signaling in breast cancer. COBRE support will help establish my independent research program through mentoring, career development, and access to Research Cores that are key to the success of the project.