Summary This application is being submitted in response to the Notice of Special Interest (NOSI) identified as “NOT-CA- 24-044”. The parent award R01CA is focused on high-grade serous ovarian carcinoma (HGSOC) and the development of in vitro model systems. In this supplement we will utilize the methods and insights from the parent award to analyze invasion and metastasis in uterine serous carcinoma (USC). USC is an aggressive subtype of uterine cancer that arises in elderly women and is responsible for 40% of uterine cancer deaths. USCs invade and metastasize early, and 40% of USC patients have advanced disease at diagnosis, receive adjuvant chemoradiation therapy, and have poor outcomes. In contrast, 60% of USC patients have apparently localized disease at diagnosis which is potentially amenable to surgical cure. However, USC still recurs in 25% of these patients. We previously implicated the mechanosensitive ion channel Piezo1 in regulating metastasis in HGSOC, a related gynecologic malignancy with similar histology, molecular pathogenesis, clinical behavior, and treatment paradigms compared to USC. In HGSOC, Piezo1 activation promotes detachment of tumor spheroids and increased metastatic tumor burden in disease-relevant animal models. However, the factors regulating Piezo1 expression remain poorly understood, and it’s unknown if Piezo1 may play a similar function in USC. In parallel to these studies, our co-investigator identified GATA Binding Protein 2 (GATA2), a zinc finger transcription factor normally expressed in healthy endometrium, as a critical regulator of USC invasion and as predictive of USC recurrence after surgery. GATA2 expression is aberrantly lost in 75% of USCs, and this event is associated with USC invasion, metastasis, disease recurrence, and poor survival, while GATA2 depletion increases the invasive capacity of patient-derived USC cell lines. Utilizing new RNAseq and genome-wide anti-GATA2 ChIPseq datasets that our co-investigator generated, we now identify multiple GATA2 binding sites within the first intron of Piezo1 and show that Piezo1 levels are increased following GATA2 depletion. This supports a model whereby localized USC tumors amenable to surgical resection lose expression GATA2, leading to increased Piezo1 levels which promotes invasion and metastasis. This collaborative project will dissect the mechanistic relationship between GATA2 and Piezo1 in USC, and test whether Piezo1 regulates USC invasion and metastasis. Aim 1 will test whether GATA2 binds GATA enhancer elements in Piezo1 to regulate Piezo1 expression levels. Aim 2 will measure the effects of Piezo1 expression on USC invasion and metastasis. The proposed studies will dissect the molecular events through which GATA2 and Piezo1 regulate USC behavior and provide a scientific foundation for planned prospective clinical trials in which GATA2 and/or Piezo1 immunohistochemistry will guide the option for therapy de-escalation in low-stage USC patients.