Kathleen O'Neill - US grants

Uterine natural killer cells (uNK) are an innate lymphoid population that are critical determinants of pregnancy success. Despite extensive research, fundamental knowledge gaps remain regarding the origin and composition of uNK cells in the endometrium and the molecular mechanisms driving uNK differentiation. These gaps result in part from our inability to address these essential questions in a human model. We have combined cutting-edge next generation sequencing methodologies with access to human uterus transplant recipients to answer questions about uNK trafficking, function and differentiation that will advance our understanding of pregnancy and its complicatons. The central premise of this proposal is that placentation, and therefore pregnancy fate, is pre-determined in part by the differentiation of uNK cells which populate the maternal-fetal interface prior to embryo implantation. Herein, we build upon this premise and test the following hypotheses: 1) uNKs which derive from peripheral and tissue-resident progenitor populations possess distinct transcriptional signatures, and 2) the Nuclear Factor of Activated T cells (NFAT) is a key transcription factor that promotes the differentiation of immature endometrial NK cells into mature decidual NK cells. Uterus transplant recipients provide an opportunity to answer foundational questions about uNK cell biology in human beings and will yield new insights into the pathogenesis of pregnancy complications.

Project Summary Triple Negative Breast Cancer (TNBC) is an aggressive subtype representing 15-20% of newly diagnosed breast cancer cases. Due to a lack of known molecular targets, treatment options for TNBC are limited, and 30-50% of patients acquire resistance to standard care. I aim to better understand the metabolic dependencies of TNBC cells to reveal novel targets that could be exploited therapeutically. TNBC cells tend to exist in a mesenchymal-like state, having undergone epithelial-to-mesenchymal transition (EMT) during which cells acquire a more motile and invasive phenotype. Because EMT is associated with poor breast cancer prognosis, understanding the molecular drivers and adaptations during this process will identify targetable dependencies of metastatic cancer cells. microRNA-200c (miR-200c) is well-established as a suppressor of EMT that tends to be lost in TNBC. Using miR-200c as a tool to push TNBC cells into a more epithelial-like phenotype, I have identified novel changes to cholesterol metabolism occurring during EMT. This project delineates how EMT regulates cholesterol metabolism and the functional consequences of these changes. In the F99 portion of this grant, I establish how reversal of EMT by miR-200c alters cholesterol metabolism proteins including the low-density-lipoprotein (LDL) receptor (LDLR) and Niemann-Pick-Type-C1 (NPC1). LDLR is responsible for endocytosing LDL particles, which are the primary source of cholesterol for breast tumor cells. After fusion of endocytic vesicles with the lysosome, NPC1 is specifically required for the processing and transport of cholesterol from lysosomes to other cellular components. These changes suggest an altered demand for cholesterol mesenchymal-like versus epithelial-like TNBC, the consequences of which are evaluated in Aim 1B of this project. My preliminary data also demonstrates that NPC1 activity is critical to survival of TNBC cells in vitro. In Aim 1B, I outline experiments to understand overall cholesterol uptake and intracellular cholesterol levels during EMT and delineate the critical role of NPC1 on TNBC cell viability. This work is the first to establish altered dependency and expression of LDLR and NPC1 in EMT and breast cancer progression. In the K00 phase of the proposed project, I will expand on my interest in the metabolic requirements of the metastatic cascade. I will move into investigating how extracellular mechanical cues driven by the extracellular matrix (ECM) drive metabolic adaptations to promote invasion and migration during metastasis. Further, I will evaluate whether the EMT phenotype of cells influences these adaptations. Gaining a better understanding of how cancer cell metabolism supports changing ECM and how mechanical demands encountered during metastasis will provide insights into development of therapeutic targets within tumor metabolism.

The female reproductive system, the uterus, fallopian tubes and the ovaries, is a complex interrelated set of organs that is physiologically dynamic and not only important for fertility but critically interrelated with general health. Single cell studies of the human female reproductive system and related tissues have been previously studied, but, as of yet, a comprehensive program, aligned with the goals of the HuBMAP, to define a molecular map of the entire system, integrating multi-modal assays, spatial diversity, and individual variations has not been established. The Penn Department of Obstetrics and Gynecology performs approximately 3,500 surgical procedures annually, of which many procedures allow sampling of multiple organs and locations from the same subject under normal conditions. Here, we propose to leverage the sampling opportunities afforded by the Penn ObGyn group and the single cell biology expertise of Penn investigators to establish a Penn Center for Multi-scale Molecular Map of the Female Reproductive System. We will obtain a comprehensive molecular characterization of the female reproductive system using six different molecular assays for at least ~700 tissue samples in anatomically indexed samples, creating a key resource for both basic science and women's health. The molecular assays include single cell RNAseq, clampFISH spatial transcriptomics, simultaneous single cell open chromatin and RNA assays, and spatial open chromatin assay, among others. We will also generate a 3D anatomical model to provide spatial coordinate for our molecular characterization. All assay data will be registered to our 3D anatomical map that will be integrated with the HIVE Common Coordinate Framework. All metadata from subject records, clinical procedures, molecular procedures, and informatics pipelines will be collected, curated, and deposited as structured data. All data, including an extensive set of metadata, will be made available as a public resource. The completion of this resource will impact reproductive medicine for women's health and also inform basic biology of human cell communities.