SUMMARY Lymphangioleiomyomatosis (LAM) is a progressive neoplastic lung disease associated with inactivation of tuberous sclerosis complex 1/2 (TSC1/2) genes and upregulation of mTOR signaling. LAM is characterized by proliferative lung nodules that destroy surrounding parenchyma and aberrant lymphangiogenesis causing chylous effusions. LAM lesions express high levels of urokinase plasminogen activator (uPA), an enzyme implicated in the proliferation and spread of cancers, but not its primary inhibitor (PAI-1). We demonstrate that genetic ablation of TSC2 leads directly to upregulation of uPA but not PAI-1. Deletion of uPA attenuates the growth of TSC2-null lung tumors and inhibits aberrant lymphangiogenesis in vivo. Surprisingly, inhibition of mTORC1 by rapamycin, currently used to treat LAM, further up- regulates uPA and dramatically upregulates its receptor (uPAR), but not PAI-1, in TSC2- compromised cells, consistent with its failure to eradicate the disease. In this proposal we will examine the mechanism by which loss of TSC leads to upregulation of uPA expression and activity and the consequences of uPA upregulation on the proliferation of LAM cells and the development of lymphatics in vitro and in vivo. In Aim 1, we will delineate the pathway by which loss of TSC induces uPA and examine how this is amplified by inhibition of mTORC1. In Aim 2, we will examine how uPA and VEGF-D secreted by TSC2-null LAM cells promotes uPA overexpression and subsequent growth, migration, lymphangiongesis and growth of LAM lung lesions. We will delineate the contribution of specific uPA domains that mediate tissue proteolysis, intracellular signaling and nuclear translocation/gene transcription. In Aim 3, will determine whether blocking upregulation of uPA will act in concert with inhibition of mTORC1 to control the growth TSC2-null tumors and aberrant lymphangiongesis in a murine model of LAM. The results of this study will provide new insights into the pathogenesis of LAM, advance our understanding of the mechanism by which uPA promotes neoplasia, and potentially identify a novel biomarker and auxiliary therapeutic target for the diagnosis and management of LAM.