Etoposide-induced DNA damage is increased in p53 mutants: identification of ATR and other genes that influence effects of p53 mutations on Top2-induced cytotoxicity.

Daniel Menendez,Michael A Resnick,Nadia Slepushkina,Jiaqi Fu,Eugen Buehler,Carri C Murphy,John L Nitiss,Madhu Lal-Nag,Whitney J Bell,Jay R Anand,Scott E Martin

doi:10.18632/oncotarget.28195

Abstract

The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.

Highlights

Topoisomerases are ubiquitous enzymes involved in many aspects of DNA metabolism including DNA replication, transcription and recombination
We examined the consequences of p53 loss-offunction or the dominant-negative p53R273H mutation on levels of Top2 covalent complexes (Top2cc) induced by etoposide
We found that downregulation of DNA repair functions such as Rnf4 leads to elevated levels of Top2cc in the presence of etoposide [17], indicating that DNA repair defects can lead to enhanced levels of protein/DNA covalent complexes

Summary

Introduction

Topoisomerases are ubiquitous enzymes involved in many aspects of DNA metabolism including DNA replication, transcription and recombination. Topoisomerases mediate topological changes in DNA through these breaks that involve a protein/DNA covalent intermediate. Short-lived, various small molecules and other www.oncotarget.com conditions can trap covalent intermediates generating lesions with protein covalently bound to DNA at the DSB site, potentially resulting in genome instability or cytotoxicity. Because these small molecules lead to trapped topoisomerases on DNA, they are referred to as topoisomerase poisons. DNA damage response (DDR) pathways resulting in downstream events including γH2AX signaling appear to require processing of Top covalent complexes (Top2cc) to generate damage signals [10]

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Conclusion