Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

Kaisa R Luoto,Amanda R Wasylishen,Linda Z Penn,Robert G Bristow,Alice X Meng,Carla L Coackley,Helen Zhao

doi:10.1158/0008-5472.can-10-0944

Abstract

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.

Highlights

MYC is a basic helix-loop-helix leucine zipper transcription factor that dimerizes with its binding partner MAX and associates with gene promoters containing the E-box motifs CACGTG or CACATG to induce gene transcription [1]
We show that MYC occupies most double-strand break (DSB) repair gene promoters and regulates RAD51 expression
Previous Chromatin immunoprecipitations (ChIP) studies completed by our group showed that MYC can bind the Rad51 gene promoter [9]

Summary

Introduction

MYC is a basic helix-loop-helix leucine zipper transcription factor that dimerizes with its binding partner MAX and associates with gene promoters containing the E-box motifs CACGTG or CACATG to induce gene transcription [1]. MYC controls a broad spectrum of functions including proliferation and cell cycle, differentiation, sensitization to apoptotic stimuli, and genetic instability [1]. These functions are deregulated in most human cancers by a variety of mechanisms including gene amplification, insertional mutations, or chromosomal translocation of the myc gene. This central role in oncogenesis makes MYC a promising target for stand-alone molecular cancer therapies in cells undergoing oncogene addiction [2]. Anti-MYC agents such as antisense oligonucleotides, small interfering RNA (siRNA), or phosphorodiamidate morpholino oligomers (PMO) have been developed to induce tumor cell growth arrest, differentiation, or apoptosis [4,5,6,7]

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Conclusion