Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Gordon Ginder

Abstract

Methyl-CpG Binding Proteins (MCBPs) are thought to function as the interpreters of epigenetic information encoded in cytosine methylation. Their ability to translate DNA methylation into local transcriptional repression has sparked interest in the role of Methyl-Binding Domain Proteins (MBDs) in cancer, where repatterning of CpG methylation is common. In this dissertation I summarize and discuss observations made in the Ginder Lab linking MCBPs to the progression of neoplastic disease. It is clear from our work that the Methyl Binding Domain Protein 2 (MBD2) is necessary for the persistent repression of critical tumor suppressor genes in breast cancer. We show that stable knockdown of MBD2 also leads to growth suppression in cultured human mammary epithelial cancer lines (MCF-7, 49% suppression; MDA-MB-231, 77%; MDA-MB-435, 94%; SK-BR-3, 92%) with the peak cytotoxicity and anti-proliferative effect occurring as late as 2-3 weeks after knockdown. MBD2 knockdown also led to a decrease in viable tumor cells at equivalent doses of the histone deacetylase inhibitor, SAHA (Vorinostat™), and chemotherapeutic agents Doxorubicin, and Paclitaxel. Stable MBD2 knockdown in MCF7 cells led to an increased proportion of normal epithelial structures in 3D culture (70%, [CI=0.55-0.83]) when compared to untransfected (46%, [CI=0.39-0.53], p≤0.038) or scrambled shRNA transfected (37%, [CI=0.29-0.45], p≤0.012) controls. In vivo xenograft studies show tumor growth in BALB/C nu/nu mice was significantly impaired when mice were implanted with human breast cancer cells harboring MBD2 targeted shRNA. Following MBD2 knockdown, tumor suppressor promoter methylation remained unchanged despite sustained increases in gene expression, arguing against the convention that passive demethylation occurs with increased transcription. Our data suggest that uncoupling CpG methylation from histone modifications or other repressor functions by removing MBD2 is sufficient to initiate and maintain anti-tumor gene transcription in the absence of secondary changes in DNA methylation. In this dissertation I present evidence for the pathologic role of MBD2 in breast cancer and provide mechanistic support for the prospect of targeting MBDs in neoplastic disease..

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

2-2-2010

Share

COinS