ABSTRACT PAI-1-mediated early-onset endometrial cancer Over the last 15 years, an increase in obesity-associated endometrial cancer has been observed, coinciding with the globesity epidemic in the US. These patients are 15-30 years younger than the typical patient demographics, and thus experience clinical burden linked to fertility preservation and disease recurrence. To understand the contribution of obesity to early-onset endometrial cancer, we recently found increased infiltration of adipose stromal cells (ASCs) in endometrial microenvironments of obese patients. Preliminary studies implicate that ASCs directly influence expression changes of loci associated with intercellular permeability and polarity (IPP) in endometrial epithelial cells (EECs). Single-cell transcriptomic profiling has further identified that plasminogen activator inhibitor-1 (PAI-1), an abundant ASC-secreted adipokine, plays a key role in deregulating IPP functions. Therefore, we hypothesize that aberrant PAI-1 signaling interferes with IPP transcription, disrupting intercellular communication homeostasis to promote neoplastic EECs. In Aim 1, the contribution of ASC-secreted PAI-1 to transcription reprogramming of IPP will be determined in EEC exposure models. When PAI-1 is tethered to LDL receptor-related protein 1 (LRP1) on the cell surface, the internalized signaling engenders E3 ubiquitin-mediated degradation of SMAD4 that attenuates TGFβ tumor- suppressive transcription program. Single-cell proteomic profiling will confirm whether IPP repression preferentially occurs in SMAD4-underexpressed cell subpopulations of primary tumors in young obese patients. In Aim 2, phenotypic influences of PAI-1 on cellular transformation will be assessed in EEC exposure models with IPP knockdowns or knockins. Dye-transfer assay and atomic force microscopy will be used to probe intercellular properties of permeability and polarity in EECs and to determine whether these altered biophysical features represent a neoplastic phenotype of EECs. When validated in a tissue microarray panel of 230 tumors and 30 uninvolved normal samples (sample size justified), decreased expression of candidate loci is expected to correlate with the young age of patients with high body mass indices (BMIs). In Aim 3, PAI-1- mediated recruitment of DNA methyltransferases will be examined in susceptible IPP loci. Persistent exposure of EECs to PAI-1 will facilitate methylation propagation within IPP promoters, leaving permanent epigenetic footprints in the neoplastic progeny. When confirmed in an endometrial cancer cohort, increased DNA methylation of these candidate loci is frequently present in primary tumors of young obese patients. The proposed study not only gives insights into a novel role of PAI-1 in early-onset endometrial cancer, but also identifies epigenetic biomarkers for cell-free DNA monitoring of young patients at risk of developing future recurrence.