Defense Date

2011

Document Type

Thesis

Degree Name

Master of Science

Department

Biochemistry

First Advisor

Dr. Jessica K. Bell

Abstract

DETERMINING THE EFFECT OF DOUBLE-STRANDED RNA TREATMENT IN OVARIAN CANCER By Charlotte Faye Roberts, B.A. A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science in Biochemistry at Virginia Commonwealth University.Virginia Commonwealth University, 2011Major Director: Jessica K. Bell, Ph.D.Assistant Professor, Department of Biochemistry & Molecular Biology. Epithelial ovarian cancer is a lethal gynecological malignancy. Due to its asymptomatic nature it is typically detected in the latter metastatic stages. Standard treatment protocol involves surgical cytoreduction, followed by a combination of taxane and platinum-based chemotherapeutics. Initially this treatment is successful however, most patients face recurring tumors that over time become resistant to current drug regimens. Thus, novel chemotherapeutic development is necessary. Cancer cells express receptors of the innate immune system, pattern recognition receptors (PRRs) that function to alert the host of invading pathogens. PRRs such as toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and dsRNA-dependent protein kinase receptor (PKR) recognize double-stranded RNA (dsRNA), a viral replication intermediate, and trigger apoptosis. Numerous studies have been conducted on the four dsRNA receptors in cancer. The findings have shown that stimulation of individual or a group of these receptors have led to a multitude of responses such as activation of apoptosis, inhibition of tumorigenic growth, and inhibition of metastasis in several cancer types (prostate, breast, nasopharyngeal, and melanoma cancer). Previous work in the Bell lab has shown that within a panel of ovarian cancer cell lines, one subset upregulates dsRNA receptors upon stimulation with polyinosinic-polyuridylic acid (pI:pC) and leads to apoptosis. A second subset of ovarian cancer cells do not upregulate dsRNA receptors and their survival is not affected by dsRNA treatment. We hypothesize that all or a subset of dsRNA receptors are required to elicit a dsRNA-induced apoptotic response. To test this hypothesis we examined the dsRNA-induced apoptotic response the responding cell lines (CAOV3 and OVCAR3) via three methods: selective ligand assays, transient knockdowns with siRNA, and stable lentiviral knockdowns with shRNA. Then we examined the dsRNA-induced apoptotic response in the non-responders (DOV13 and SKOV3). The first objective was to determine if all or a subset of these four dsRNA receptors were required for the dsRNA-induced apoptotic response. The second objective of this thesis was to examine if dsRNA receptor expression in cell lines resistant to dsRNA-induced apoptosis could restore dsRNA responsiveness. To execute the first objective, we first examined receptor contribution to the dsRNA-induced apoptotic response via a selective antagonist (2- aminopurine) to PKR and a selective agonist (polyadenylic-polyuridylic acid, pA:pU) to TLR3. Inhibition of PKR did not blunt the apoptosis levels in the responders and was determined to be inessential for the dsRNA-induced apoptosis. Selective ligation of TLR3 with pA:pU showed an increase in apoptosis, but not to levels seen with pI:pC. Objective one was also carried out via transient knockdown using siRNA. Knockdowns via this method were less than 70% and the lipid vehicle of one of the transfection reagents was found to be sensitizing to the cells. Stable lentiviral knockdowns with shRNA were utilized to conduct the knockdown assays. By qPCR, lentiviral knockdown of TLR3 showed an 85% decrease and showed a great decrease in the dsRNA-induced apoptotic response in the cell death assay. The lentiviral knockdown of RIG-I showed a 54% decrease via qPCR and did not alter dsRNA-induced apoptotic responses. The lentiviral knockdown of MDA5 could only be assessed via the TLR3/MDA5 double knockdown, and it showed a 53% decrease via qPCR analysis. The cell death assay of the TLR3/MDA5 double knockdown showed a great decrease in the dsRNA-induced response. The work presented in this thesis is the first to address the contribution of all four dsRNA receptors to the dsRNA-induced apoptotic response in one study. In this work, we have found that PKR is not needed for the dsRNA-induced apoptosis. Loss of TLR3 in the responders reduces death, but not back to basal levels. This may be due to the delivery method of pI:pC such that it goes directly to the endosome. Forced expression of the dsRNA receptors (TLR3, MDA5, and RIG-I) can all induce apoptosis to similar levels indicating redundancy. The importance of this work reveals that any of the three dsRNA receptors, TLR3, MDA5, and RIG-I, could be possible targets for individualized chemotherapeutic regimens for women with ovarian cancer expressing these receptors.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2011

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