Project Summary This proposal will investigate the molecular drivers that contribute to the progression of one of the most aggressive and metastatic types of breast cancer. Triple Negative Breast Cancer (TNBC) is characterized by the lack of hormone receptors (estrogen and progesterone) and HER2 expression and accounts for approximately 20% of all breast cancers. Due to its heterogeneity and dearth of defined specific molecular targets, TNBC treatment remains challenging. Accordingly, this study seeks to elucidate the molecular role of secretory carrier- associated membrane protein 3 (SCAMP3) on TNBC pathogenesis, with the goal of developing new therapies that will improve the survival rate of patients. Recently, SCAMP3 has been found overexpressed and associated with poor prognosis in hepatocellular carcinoma, melanoma, glioma, and breast cancer, suggesting that SCAMP3 has a key role in oncogenesis. However, the molecular mechanisms of how SCAMP3 promotes TNBC progression are poorly understood. SCAMP3 is a regulator of epidermal growth factor receptor (EGFR) trafficking within endosomal membranes promoting receptor recycling. EGFR mediated endocytosis is a mechanism of transport of cancer-associated signaling proteins, such as signal transducer and activator of transcription 3 (STAT3). EGFR and STAT3 have been associated with TNBC proliferation, invasion, metastasis, and cancer stem cell (CSC) maintenance. Our objectives are to determine the mechanisms by which SCAMP3 promotes TNBC progression and determine the interplay between SCAMP3/EGFR and STAT3 signaling pathways. We hypothesize that: 1) SCAMP3/EGFR mediated endocytosis regulates the activation of STAT3. 2) SCAMP3 signaling pathway promotes and maintains TNBC stemness via the modulation of EGFR/STAT3, and 3) SCAMP3 contributes to TNBC tumor formation and metastasis. To test these hypotheses, we propose the following aims: 1) To elucidate the mechanism of action of SCAMP3 on STAT3 activation; 2) Define the role of SCAMP3 in TNBC and CSCs regulation via STAT3 modulation: 2a) To elucidate the molecular mechanisms of SCAMP3 in CSCs maintenance in vitro; 2b) To investigate the effect of SCAMP3 in tumorigenesis and metastasis. We propose experiments that will monitor EGFR and STAT3 internalization, STAT3 DNA-binding activity, and transcriptional activity. We will identify the relevant driver genes that maintain cancer stemness in SCAMP3 overexpressing cells using RNAseq, which will be confirmed in patient samples. The studies have the potential to be rapidly translated to the clinic and significantly reduce the incidence and number of deaths related to TNBC.