Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer.

S Stolzenburg,A G Rivenbark,T Swift-Scanlan,A S Beltran,P Blancafort,R Rashwan

doi:10.1038/onc.2014.470

Abstract

With the recent comprehensive mapping of cancer genomes, there is now a need for functional approaches to edit the aberrant epigenetic state of key cancer drivers to reprogram the epi-pathology of the disease. In this study we utilized a programmable DNA-binding methyltransferase to induce targeted incorporation of DNA methylation (DNAme) in the SOX2 oncogene in breast cancer through a six zinc finger (ZF) protein linked to DNA methyltransferase 3A (ZF-DNMT3A). We demonstrated long-lasting oncogenic repression, which was maintained even after suppression of ZF-DNMT3A expression in tumor cells. The de novo DNAme was faithfully propagated and maintained through cell generations even after the suppression of the expression of the chimeric methyltransferase in the tumor cells. Xenograft studies in NUDE mice demonstrated stable SOX2 repression and long-term breast tumor growth inhibition, which lasted for >100 days post implantation of the tumor cells in mice. This was accompanied with a faithful maintenance of DNAme in the breast cancer implants. In contrast, downregulation of SOX2 by ZF domains engineered with the Krueppel-associated box repressor domain resulted in a transient and reversible suppression of oncogenic gene expression. Our results indicated that targeted de novo DNAme of the SOX2 oncogenic promoter was sufficient to induce long-lasting epigenetic silencing, which was not only maintained during cell division but also significantly delayed the tumorigenic phenotype of cancer cells in vivo, even in the absence of treatment. Here, we outline a genome-based targeting approach to long-lasting tumor growth inhibition with potential applicability to many other oncogenic drivers that are currently refractory to drug design.

Highlights

Cytosine methylation in mammalian DNA is regarded as a key epigenetic modification controlling essential processes such as imprinting, silencing of retrotransposons and cell differentiation.[1]
The 6ZF constructs were delivered into MCF7 cells using inducible retroviral vectors[17] and stable clones were isolated in which the temporal expression of the ZF598-DNA methyltransferase 3A (DNMT3A) and ZF598-SKD fusions was controlled by doxycycline (Dox) treatment
When Dox was removed for up to 8 days (~10 cell generations) from the culture media (R, Figures 1b and c) cells transduced with ZF598-SKD restored SOX2 expression to levels that were similar to those of uninduced cells, whereas cells expressing the ZF598-DNMT3A showed a persistent decrease of SOX2 expression (85% relative to uninduced cells) upon Dox removal (Figure 1b)

Summary

Introduction

Cytosine methylation in mammalian DNA is regarded as a key epigenetic modification controlling essential processes such as imprinting, silencing of retrotransposons and cell differentiation.[1]. Aberrant incorporation or removal of DNAme in cancer cells leads to genome-wide alteration of gene expression, including inactivation of tumor suppressor genes and reactivation of oncogenes.[4] Notably, changes in DNAme patterns are characteristic hallmarks of the distinct intrinsic subtypes of breast cancer. Highly proliferative basal-like breast cancers associated with stem/progenitor celllike features are significantly hypomethylated relative to the other breast cancer subtypes. These tumors are driven by aberrant activation of multiple developmental transcription factors (TFs), which fuel the tumor with sustained proliferation, drug resistance and metastatic capacity.[5,6,7]

Methods

Results

Conclusion