• Deciphering the Role of Sumoylation During EBV Replication

      Jenkins, Jessica L; School of Medicine
      Epstein Barr Virus, a gamma herpes virus, is the known causative agent in infectious mononucleosis and is highly ubiquitous in nature. Although primary infection typically yields no long term issues, viral latency is associated with lymphomas and epithelial cell carcinomas. We documented that the presence of LMP1, the principal EBV oncogene, dysregulates cellular sumoylation processes in lymphoma tissues, modulates innate immune response, and maintains viral latency. Sumoylation is a dynamic process were target proteins are modified with free small ubiquitin like modifier (SUMO) proteins. The SUMO modification is vital for cellular processes including: immune response, DNA damage repair sensing, cell cycle progression, resistance to apoptosis, and metastasis. Several cancers display dysregulation of the sumoylation process, making the SUMO machinery a sufficient target for anti-cancer therapies. Known sumoylation inhibitors include natural extracts and antibiotics. However, many of these agents are nonspecific and/or demonstrate adverse effects like allergic reactions with botanical extracts. This piqued our interest in investigating synthetically engineered compounds along with a well-known natural extract inhibitor, Ginkgolic Acid (GA). ML-792, 2-D08, and TAK-981 are synthetically derived small molecule inhibitors that were identified as selective SUMO-inhibitors, interfering at different stages of the sumoylation process. We hypothesize that the SUMO-inhibitors will have therapeutic effects for the treatment of EBV-associated malignancies by modulating the EBV life-cycle. Results showed that each of the tested inhibitors decreased global levels of sumoylated proteins, though ML-792 and TAK-981 showed greater inhibition when compared to GA and 2-D08. Additionally, the SUMO-inhibitors induced low levels of spontaneous reactivation in latently infected B cells. We also confirm that sumoylation is important for maintaining EBV latency and lytic replication in B cells. Lastly, we note anti-viral potential for each tested inhibitor, particularly GA and 2-D08 have a better affect than ML-792 and TAK-981 in this regard. Of the tested sumoylation inhibitors, we now propose 2-D08 as the best potential therapeutic drug to aid the treatment of EBV-associated malignancies due to its ability to significantly reduce viral DNA levels following induced reactivation and decrease the ability of produced virus to infect additional cells.
    • The Influence of Hyaluronic Acid Metabolism on the Development of Chemoresistance in 3D Breast Cancer Cell Models

      Iyahen, Violet; School of Medicine
      Breast cancer is the most diagnosed malignancy in the world. Of the various subtypes of breast cancer cells, the ductal carcinoma is responsible for 70% to 80% of worldwide breast cancer diagnoses. Current treatments for breast cancer include hormonal therapy, surgery, radiation therapy, and chemotherapy. Unfortunately, many breast cancer tumors become resistant to these therapies overtime prompting the need for new targeted therapies. It is becoming increasingly clear that the tumor microenvironment plays a critical role in tumor cell survival and development of resistance. In particular, the CD44 receptor and its ligand hyaluronic acid (HA) have been implicated in a number of processes related to tumor progression and survival. HA is a major component of the extracellular matrix (ECM) in both normal and abnormal tissues. In this current study we focused on examining the role of HA metabolism on the resistance of breast cancers to the commonly used chemotherapeutic agent doxorubicin (dox). Specifically, we examined the influence of HA in the ductal carcinoma cell line, MCF-7 using both a 2D and 3D tumor model. The results from this study supported previous findings that 3D MCF-7 cultured cells are resistant to doxorubicin treatment when compared to 2D models and further established the novel finding that cells grown in 3D models have increased gene expression of hyaluronic acid synthase. Consequently, 4-methylumbelliferone (4MU) was chosen due to its inhibiting mechanism during HA synthesis. For this reason, 4MU’s effect was vital to our hypothesis regarding modifications of HA content to augment the strength of low concentrations of dox on tumor cells. MCF-7 cells were grown and cultured two-dimensionally and three-dimensionally using unique methods and specialized plates. Assays were used to quantify and contrast the HA content in each culture along with the specific enzymes responsible for HA’s anabolic and catabolic processes. Then, overall cellular proliferation was measured after administering dox and 4MU separately followed by combination treatments of both. Results revealed an increase in HA synthase enzymes in the 3D cultures; however, overall HA concentration was lower when compared to the 2D cultures. Cellular proliferation was repeatedly measured, and on average, 3D cultures were more resistant to individual treatments of dox and 4MU. Similar results were seen when combination treatments were administered, and cellular proliferation did not decrease in the 3D groups.