Degree Type

Dissertation

Date of Award

2014

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

Major

Genetics

First Advisor

Cathy L. Miller

Abstract

Mammalian Orthoreovirus (MRV), a double-stranded RNA virus, is a potent oncolytic virus, which specifically replicates in tumor cells and consequently kills them. Hypoxia, a condition of low oxygen, is a characteristic feature of most solid tumors and develops due to tumor cells outgrowing their blood supply. Hypoxic cell survival is driven by the transcriptional activity of hypoxia inducible factor-1 (HIF-1). We examined the ability of MRV to infect hypoxic prostate tumor cells and found that not only did it successfully translate proteins and replicate in hypoxic prostate tumor cells, it also induced apoptosis. In addition, MRV induced downregulation of the HIF-1α protein, through a ubiquitin-dependent proteasome-mediated pathway, required the PAS domain of HIF-1α and was rescued by knockdown of the Receptor for Activated C Kinase 1 (RACK1).

We investigated the direct involvement of any MRV proteins in the downregulation of HIF-1α and found that proteins μ1 and μ2 were able to downregulate HIF-1α independently. Strain specific difference (specifically the aa at 208th position of the protein) was found to affect the ability of μ2 to induce downregulation of HIF-1α; the T1L and T3DC causing more downregulation than the T3DF MRV strains. The region of μ2 containing aa 188-380 and the φdomain of μ1 were determined to be needed for HIF-1α downregulation.

The dependence of Prostate Cancer (PCa) on androgens for growth and survival is exploited by the hormone therapy, where androgen deprivation is used as a viable therapy. Although initially successfully, this androgen dependent PCa progresses to androgen independent PCa, in which hypoxia has been proven to play a vital role. We examined the effect of MRV infection on hypoxic androgen dependent LNCaP cells and found that MRV induced apoptosis in hypoxic and normoxic LNCaP cells and also inhibited Akt activity and downregulated Prostate Specific Antigen (PSA). MRV infection also caused the downregulation of Androgen receptor (AR) activity and protein, primarily by translation inhibition.

Overall, our work highlights the potential use of MRV at various stages of PCa and lays important groundwork for the design of clinical trials based on MRV targeting hypoxic PCa cells at all stages.

Copyright Owner

Pooja Gupta

Language

en

File Format

application/pdf

File Size

167 pages

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