Date of Award

2-14-2008

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Edward Chu

Abstract

The therapeutic potential of a novel multi-targeted small-interfering RNA (siRNA) was investigated in human breast cancer cells. Previous studies had identified an siRNA that specifically and potently inhibited expression of thymidylate synthase (TS) by directly targeting human TS mRNA. TS is a folate-dependent enzyme that catalyzes the key reaction involved in synthesizing nucleotide precursors for DNA biosynthesis, and as such, it plays a critical role in maintaining cell growth. The goal of this thesis was to design and develop a novel siRNA molecule that targeted TS mRNA as well as a cellular mRNA that encodes a different cellular protein involved in cancer cell growth and proliferation, such as a member of the ErbB family. Gene sequence analysis was performed and identified an overlapping sequence between TS and ErbB2 mRNAs. An siRNA duplex was then designed to simultaneously target human TS and ErbB2 mRNA. Transfection of the multi-targeted siRNA (TS1M17) revealed that both ErbB2 and TS proteins were significantly suppressed in a time and dose-dependent manner in ErbB2-overexpressing human breast cancer SKBR3 cells. The corresponding mRNA levels, as determined by RT-PCR, were also decreased. Protein levels of other ErbB family members, including ErbB1 and ErbB3, remained unchanged with siRNA treatment. An ErbB2-specific siRNA (B2450) inhibited ErbB2, but had no effect on TS expression demonstrating the specificity of the multi-targeted siRNA against both TS and ErbB2. Mismatched (TS1-Mismatch) and control (GL2) siRNAs had no inhibitory effects on expression of the two target proteins. Suppression of activated ErbB2, as determined by expression of phosphorylated ErbB2 protein, was observed with transfection of TS1M17 siRNA. In addition, the expression of downstream signaling proteins, such as phosphorylated mitogen activated protein kinase (p-MAPK), p27Kip1, p21Cip1, cyclin D1, and survivin were significantly changed. In contrast, control siRNAs did not exert any inhibitory effects on downstream signaling. Taken together, these findings suggest that TS1M17 siRNA inhibits signaling of the ErbB2 pathway. The effect of TS1M17 siRNA on cytotoxicity was analyzed by WST-1 assay. Upon transfection into SKBR3 cells, the TS1M17 siRNA significantly suppressed cell proliferation with an IC50 value of 0.65 nM, which is 154-fold more potent than ErbB2- and TS-specific siRNAs. This study suggests that targeting expression of ErbB2 and TS, two key proteins involved in distinct and critical pathways for cancer growth and proliferation, with a single siRNA molecule may provide a novel approach for cancer chemotherapy.

Comments

This is an Open Access Thesis.

Download
Open Access

This Article is Open Access

Share

COinS