Project Summary: Breast cancer is the second leading cause of cancer death in women in the U.S. In 2020, about 276,000 new cases of invasive breast cancer are expected to be diagnosed in women in the United States. Steroid hormone receptors, estrogen receptor (ER) and progesterone receptor (PR), are critical for normal breast tissue development as well as breast tumor development. The majority of breast cancers (~70%) express those hormone receptors. Function of ER in breast cancer has been extensively studied and ER-targeting therapy such as tamoxifen is routinely used for ER-positive breast cancers. However, acquisition of resistance to anti-ER therapies in breast cancers constitutes a major obstacle to successful treatments. Therefore, there is an urgent need to develop a new therapeutic strategy. Surprisingly, in contrast to ER, PR function in breast cancer is not well-studied. Progesterone (a ligand of PR) is known to inhibit ER and PR positive breast cancer growth in vitro and in vivo. Importantly, progesterone is also known to facilitate breast cancer cell cycle and increase breast cancer risk Although progesterone treatment has been considered as a promising breast cancer treatment, because of this bipolar function of PR in breast cancer and our lack of knowledge for the molecular mechanism of PR action, the clinical significance of progesterone treatment is still obscure. Particularly, the mechanism of PR-mediated cell cycle regulation remains elusive. In this proposal, we aim to identify the mechanism of progesterone-induced cell cycle arrest in breast cancer cells. Our central hypothesis is that PR regulate cell cycle regulatory miRNAs by forming chromatin loops (3D interaction) between PR bound enhancers and miRNA- coding loci. The central hypothesis will be tested by pursuing three specific aims: (1) Identify cell cycle gene targeting miRNAs activated by PR; (2) Define the role of chromatin loop formation induced by PR; (3) Determine the impact of PR reactivation in PR deficient breast cancer cells. We will utilize the innovative combination of techniques - an advanced genomic technique (HiChIP), CRISPR-mediated knockout, and our PR impaired breast cancer cell line to identify progesterone-induced miRNAs that regulate breast cancer cell proliferation. The proposed research is significant because it is expected to contribute a missing and fundamental element to our understanding of the role of PR in breast cancer growth. Ultimately, such knowledge has the potential to offer development of new therapeutic strategies of breast cancer treatment. Characterized miRNAs in this study also have potential to be used as biomarkers for progesterone-treatable breast cancers.