Papillary thyroid cancer (PTC) typically has a favorable prognosis; however, patients with PTC carrying the BRAFV600E mutation show resistance to radioiodine treatment and have high rates of metastases and low survival rates. Inhibitors of BRAFV600E, including vemurafenib, have been tested recently as treatments for BRAFV600E-melanomas, with very promising results. However, development of secondary tumor resistance to these compounds has been reported. Another potential player in PTC aggressiveness is vascular endothelial growth factor receptor 2 (VEGFR2), a crucial regulator of angiogenesis. Sorafenib, which targets VEGFR2, was only partially effective against metastatic PTC, suggesting secondary resistance mechanisms to this drug as well. Our preliminary results show that metastatic primary human BRAFV600E positive PTC cells in vitro were resistant to high doses of either vemurafenib or sorafenib alone, but combined treatment with both drugs decreased cell viability by greater than 90%. BRAFV600E up-regulates VEGFR2 and pERK1/2 in PTC cells, triggering recruitment of endothelial cells and pericytes. We also observed upregulation of ACAC? (metabolic gene) suggesting that BRAFV600E harnesses a metabolic response that promotes PTC metastasis, a novel mechanism. Our preliminary data also indicate that VEGFR2 is associated with neck recurrence in BRAFV600E PTC. Our objective is to determine how BRAFV600E and VEGFR2 fit into the autocrine and paracrine signaling pathways that control PTC cell progression. Our central hypothesis is that BRAFV600E and VEGFR2 pathways synergize in PTC cells, increasing secretion of extracellular matrix (ECM) proteins and leading to increased PTC cell adhesion, migration/invasion, and angiogenesis. We propose a secondary pathway in which the BRAFV600E/ERK1/2 signaling cascade triggers hyper-expression of VEGFR2 in PTC cells. The alternate VEGFR2 pathway synergizes with residual BRAFV600E activity in the presence of vemurafenib, conferring secondary drug resistance in PTC cells. We will test this by manipulating VEGFR2 and BRAFV600E function and assessing tumorogenic properties in our novel models: a 3D co-culture system that recapitulates the PTC microenvironment, an orthotopic mouse with primary human PTC cells with heterozygous BRAFWT/V600E, and ChIp-seq analysis. Aim 1: To elucidate the autocrine and paracrine pathways by which BRAFV600E and VEGFR2 promote PTC aggressiveness. These studies will delineate VEGFR2-interacting ECM proteins that enhance ERK1/2 phosphorylation in BRAFV600E PTC and provide new insight into vemurafenib and sorafenib secondary drug resistance. Aim 2: To assess whether VEGFR2 protein expression is a long-term prognostic biomarker for PTC aggressiveness in BRAFV600E PTC. Aim 3: To assess efficacy of anti-BRAFV600E and anti-VEGFR2 combined therapy in a pre-clinical mouse model of human heterozygous BRAFWT/V600E PTC. These proposed studies are highly likely to provide evidence supporting clinical trials of combined targeted therapy for BRAFV600E-PTC and identify novel targets for drug development and biomarkers for aggressive PTC.