Project Summary: Uterine serous carcinomas (USC), uterine carcinosarcomas (UCS), and leiomyosarcomas represent the most aggressive subtypes of uterine cancer and account for a disproportionate number of uterine cancer related deaths in the United States. The lethality of these high-grade uterine cancers is largely due to the propensity of these cancers to disseminate and metastasize, leaving patients with a lack of effective treatment options. Despite this, the molecular drivers of the metastatic potential of these disease subtypes remain incompletely understood. Previous work by our group and others have identified a recurrent mutational hotspot within the gene encoding for the A scaffolding subunit of the protein phosphatase 2A (PP2A), PPP2R1A. These mutations, P179R and S256F, occur exclusively within these high-grade subtypes of uterine cancer and are not found in any other cancer type. Protein phosphatase 2A (PP2A), is a family of serine/threonine phosphatases, where the active heterotrimeric form is comprised of the catalytic (C) subunit, the scaffolding (A) subunit, and a substrate directing regulatory (B) subunit. In aggregate, PP2A is considered a tumor suppressor protein, but certain heterotrimers have tumor promoting roles. We have shown that these mutations contribute to uterine cancer tumorigenesis by modulating PP2A function in a manner that impairs the formation of tumor suppressive heterotrimers, while leaving tumor promoting heterotrimeric complexes intact. My preliminary data show that the PP2A P179R and S256F mutations enhance tumor initiating capacity, and the ability to form 3D tumor spheres in vitro. Importantly, uterine cancer cells expressing these mutations also form intraperitoneal nodules at an increased rate in vivo, suggesting that these mutations may enhance the establishment of new tumor formation in the peritoneal cavity. Conditioned media from the mutant expressing uterine cancer cells promoted tumor sphere formation of WT expressing uterine cancer cell growth, indicating that the mutant cells may create a more permissive tumor microenvironment for tumor formation. Preliminary data suggests that the mutant cells have increased -catenin phosphorylation and WNT ligand expression. The working hypothesis of this proposal is that the recurrent mutants, P179R and S256F, enhance uterine cancer metastasis through increased WNT signaling and secretion of WNT ligands. To test this hypothesis, we will develop PP2A A knock-in xenograft models to determine how these recurrent mutants contribute to enhanced metastasis in vivo (Specific Aim 1). Second, mechanistic studies will explore the secreted factors from the mutant uterine cancer cells, exploring WNT ligands, coupled with secretome analysis. These studies will also explore the link between loss of PP2A and WNT mediated EMT (Specific Aim 2). This proposal aims to decipher the functional role of recurrent PP2A A𝛼 mutations on uterine cancer metastasis, which may identif...