The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) senses cytosolic DNA and activates an immune response. This signaling pathway is important for defending against viral infections and regulates cancer immunity. In order to study this signaling pathway, our goal was to develop an in vivo tool using the model organism Drosophila melanogaster to answer questions regarding the activity of STING signaling. To make this STING signaling reporter gene assay, we used molecular cloning to clone the promoter regions of three genes downstream of the STING pathway (Nazo, Srg1, Srg2). We used restriction enzymes to combine the promoter regions with a vector containing the reporter DsRed (sequence for red fluorescent protein). This DNA construct was injected into flies to create transgenic fly lines. When STING signaling is active, the reporter sequence is transcribed and translated along with the STING target genes. Thus, when the transgenic flies are dissected and stained for the reporter DsRed, the images show where and how much STING signaling is active in specific cells in tissue. We tested our reporter line with the antibiotic drug bleomycin which causes tissue and DNA damage that could activate STING signaling. We found that when flies from the reporter line were fed with sucrose containing bleomycin, images of their guts showed areas with high fluorescence that weren’t visible in flies fed with just sucrose. The fluorescent areas also aligned with areas appearing to have cells with broken nuclei, suggesting our reporter line was successful. This tool is useful for any project that needs to detect STING signaling, and is helpful for answering questions regarding the pathway. There are many unknowns regarding what regulates STING signaling and what STING signaling causes which can be further studied with this in vivo reporter.

Extracellular vesicles (EVs) are lipid-bilayer membrane-enclosed structures that cells produce and use for intercellular communication. Within the context of cancer, EVs have been shown to enhance cancer development by delivering cargo from malignant cells to recipient cells to promote survival, proliferation, and invasion. In a previous project, I conducted a chemical screen alongside my graudate mentor and other undergraduates to determine kinases that were important to EV biogenesis. One hit was the JNK pathway, which decreased EV production when inhibited. I studied the pathway in further detail utilizing a variety of experimental techniques to establish its importance for EV generation, and I was able to conclude that JNK regulates EV biogenesis. Another facet of cancer development is oxidative stress, caused by reactive oxygen species (ROS). When unregulated, these highly reactive free radicals and molecules derived from oxygen can damage DNA, facilitate metastasis, and aid in cancer progression. Given that surrounding literature revealed that JNK is activated by ROS, I hypothesized a connection between ROS and EV production. This project aims to more directly uncover the impact of ROS on EV generation by manipulating ROS-related genes in vivo. To do this, I knocked down ROS generator genes such as Dual Oxidase (Duox) in Drosophila melanogaster. I quantified ROS levels by staining the dissected tumor tissues with an ROS probe to ensure that the genes were functioning as expected. Then, I stained the tissues for phospho-JNK as a proxy for ROS quantification and to measure JNK activity. Finally, I conducted live imaging of the tumor tissues to quantify EV generation. I anticipate that impairing ROS generation will inhibit JNK activation, subsequently leading to a decrease in EV production. Understanding how factors involved in cancer development function in relation to each other is crucial for discovering novel cancer therapeutics.

Extracellular vesicles (EVs) are essential mediators in intercellular communication secreted by cells to transfer bioactive cargo that lead to biological effects. The crucial roles EVs have in maintaining biological homeostasis are similarly found within cancer cells in the tumor microenvironment, where they promote cell growth/survival, invasion, and metastasis. Investigating methods to reduce tumor-cell derived EVs could provide substantial remedies for cancer patients. One pathway of interest in cancer is the cellular response to reactive oxygen species (ROS)—highly reactive molecules which tumor cells use for oncogenic signaling, to damage macromolecules, and drive tumor progression. Modulation of ROS levels may yield anticancer effects, but research about the role of ROS in EV biogenesis has not been conducted. To assess their connection, I used MDA-MB-231 human breast cancer cells as an in vitro model for EV biogenesis. My interest in ROS and EVs began when I assisted my graduate mentor in an extensive chemical screen and found kinase inhibitors that altered EV production via an EV isolation protocol. From these hits, I identified ROS-activated pathways that promote cancer progression as important players in EV production. I then tested if chemicals known to directly affect ROS alter EV production by isolating and quantifying EVs and by imaging their production from MDA-MB-231 cells. To provide a comprehensive understanding of the pathway, I validated upstream interactions of EV biogenesis by measuring the production of ROS using a chemical marker that emits green fluorescence when oxidized. From this data, I can determine if there is a direct interaction between ROS and EV production. An understanding of EV biogenesis and its connection to ROS and cancer progression may unveil new opportunities for novel cancer therapeutics.

Approximately 500 million people worldwide live with osteoporosis, a disease of low bone mineral density (BMD) and bone fragility caused by a disequilibrium between osteoblasts, cells that build bone, and osteoclasts, cells that reabsorb bone. Existing osteoporosis treatments are single-action anti-resorptive or osteoanabolic (bone-promoting) drugs, which make them insufficient for individuals with severe disease or those at high imminent risk for fractures. RANK is a receptor on osteoclastic progenitor cells that, when activated by RANK ligand binding, induces osteoclast formation and spurs the translocation of a transcription factor, NFATc1, into the nucleus, where it initiates RANK transcription. The available literature on the topic has traditionally only acknowledged RANK to be present in osteoclasts. Contrary to this view, our lab recently identified Rank in osteoblasts. Thus, my project examines how Rank acts in osteoblasts to regulate bone formation. I hypothesize that in osteoblasts of developing bone, Rank signaling is regulated by Nfatc1 via a positive feedback loop, similar to what occurs in osteoclasts. Using in situ hybridization chain reaction, I found that nfatc1 is expressed strongly and specifically in the same developing skeletal structures as rank+ osteoblastic cells in 3, 5, 12, and 14 day post-fertilization zebrafish, supporting my hypothesis. My ongoing studies focus on identifying the Rank and Nfatc1 interactions that may promote osteoblast differentiation. I am achieving this by analyzing the skeletal phenotypes of a rank loss-of-function mutant I am crossing with a reporter transgene line that fluoresces when Nfatc1 signaling is activated, as well as analyzing fish chronically subjected to FK506—a pharmacological inhibitor of calcineurin, which is required for NFATc1 translocation. My preliminary data suggest that the proposed positive feedback loop between RANK and NFATc1 is conserved across osteoclasts and osteoblasts, revealing potential targets for dual-action (anti-resorptive and osteoanabolic) osteoporosis therapies.

Osteoporosis is the most common metabolic bone disease in the United States and worldwide. Genome-wide association studies (GWAS) have identified numerous loci associated with bone mineral density (BMD), however, the target genes at most of these loci remain unknown. Multiple GWAS have identified the TNFRSF11B-COLEC10 locus to be associated with BMD. TNFRSF11B, tumor necrosis factor receptor superfamily, member 11B, is a gene that encodes for osteoprotegerin (OPG), a key regulator of bone resorption. COLEC10, collectin subfamily member 10, encodes a C-lectin family protein involved in neural crest cell migration, endocrine function, and the nervous system, though its role in bone remains unknown. While TNFRSF11B is presumed to be the target gene at the TNFRSF11B-COLEC10 locus, we have obtained preliminary data that loss of COLEC10 in zebrafish results in altered bone morphology. However, these animals were mosaic for mutations in COLEC10, preventing a uniequivocal determination of its role in bone. The purpose of my study is to map genotype-to-phenotype relationships in COLEC10 and TNFRSF11B germline mutant zebrafish. Mutants for COLEC10 were generated by ENU mutagenesis as part of the Sanger Mutation Project. Mutants for TNFRSF11B were generated by our lab using CRISPR. I will genotype both mutants using Polymerase Chain Reaction (PCR) and gel electrophoresis. I will scan the adult fish (90 days post fertilization) using micro-computed tomography (microCT), and then utilize FishCuT for the segmentation and analysis of the vertebral column of each zebrafish. The primary outcomes will be the tissue mineral density (TMD), volume (Vol), thickness (Th), and length (Le), in the centrum, haemal arch, and neural arch of each vertebra. By determining whether COLEC10 is a gene of major effect compared to TNFRSF11B, my research will help to elucidate COLEC10’s skeletal function and its potential role as a casual gene underlying genetic risk for osteoporosis.