Degree Type

Thesis

Date of Award

2020

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

Major

Microbiology

First Advisor

Cathy L Miller

Abstract

The International Agency for Research on Cancer estimates that 9.6 millionpeople died of cancer in 2018. Prostate cancer (PCa) is the fourth most commonlydiagnosed cancer worldwide, and is the most common in men within the United States.Development of new cancer therapies has helped many PCa patients experienceremission, but far too many die due to tumor resistance toward therapy. Tumors areheterogeneous collections of aberrant cells growing uncontrollably, and as they progress,they evolve becoming more genetically and metabolically diverse throughout the tissue.Within the tumor, microenvironments form that differ dramatically from one region toanother. One tumor microenvironment of particular interest is tumor hypoxia, an area oflow oxygen that is increasingly resistant to cancer therapy. Within these hypoxic regions,the hypoxia-inducible factor 1 alpha subunit (HIF-1α) accumulates, enters the nucleus,and enhances transcription of genes involved in growth, survival, angiogenesis, andmetastasis, resulting in increased tumor aggressiveness. When patients have highamounts of HIF-1α accumulation within the blood serum, they typically have muchpoorer prognosis as compared to those without elevated HIF-1α. Novel HIF-1α inhibitorsare being investigated for safety and efficacy to inhibit these effects, as well as tosensitize hypoxic tumors to current cancer therapies. While viruses are not often thoughtof as therapies, mammalian orthoreovirus (MRV) has been shown to inhibit HIF-1α invitro and in vivo. MRV is a cancer killing, oncolytic, virus that specifically targets,infects, replicates, and lyses cancerous tissue while leaving healthy tissue undamaged.The virus has undergone various Phase I-III clinical trials and has been shown to be safefor the treatment of humans, but it has only been shown to be effective in some patients while others receive little to no benefit. Therefore, research is needed to investigate theviral impact on the cell to better understand which tumor types or tumor environments aremost amenable to MRV therapy.This work began investigating the basic biology of MRV factories utilizing amodified virus so that infection could be observed in live-cell microscopy. By adding atetracysteine (TC) tag within the viral µNS protein, the viral factories could be observedusing the TC labeling FlAsH-EDT2 reagent. This study provided data about factorydynamics and presented a potential tool for further investigation of MRV oncolytics. Atthe same time systematic investigation into the stage of viral replication necessary toinhibit HIF-1α was investigated. Using a transcriptional deficient virus along withendosomal breakdown inhibitors, a step between viral capsid cleavage and transcriptionwas determined to be sufficient for HIF-1α inhibition. Subsequent work looked into therole of viral components, protein or dsRNA, and cellular stress granule (SG) formationcaused by virus infection to further elucidate the mechanism of MRV-induced HIF-1αinhibition. Evidence suggests that the viral capsid proteins and dsRNA are not required todecrease HIF-1α accumulation, but early during infection SG formation is. Altogetherthis work has produced valuable information into the mechanism of MRV-inducedinhibition of HIF-1α and provided a new technique for further study of MRV replicationand oncolysis.

DOI

https://doi.org/10.31274/etd-20200624-34

Copyright Owner

Luke Daniel Bussiere

Language

en

File Format

application/pdf

File Size

143 pages

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