APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells.

Md Akram Hossain,Yusaku Nakabeppu,Jia Li,Garrett Driscoll,Shan Yan,Yunfeng Lin,Daisuke Tsuchimoto,Joshua Matos,Jianjun Zhao,Anne Mcmahon,Haichao Zhao

doi:10.3389/fcell.2021.738502

Abstract

The maintenance of genome integrity and fidelity is vital for the proper function and survival of all organisms. Recent studies have revealed that APE2 is required to activate an ATR-Chk1 DNA damage response (DDR) pathway in response to oxidative stress and a defined DNA single-strand break (SSB) in Xenopus laevis egg extracts. However, it remains unclear whether APE2 is a general regulator of the DDR pathway in mammalian cells. Here, we provide evidence using human pancreatic cancer cells that APE2 is essential for ATR DDR pathway activation in response to different stressful conditions including oxidative stress, DNA replication stress, and DNA double-strand breaks. Fluorescence microscopy analysis shows that APE2-knockdown (KD) leads to enhanced γH2AX foci and increased micronuclei formation. In addition, we identified a small molecule compound Celastrol as an APE2 inhibitor that specifically compromises the binding of APE2 but not RPA to ssDNA and 3′-5′ exonuclease activity of APE2 but not APE1. The impairment of ATR-Chk1 DDR pathway by Celastrol in Xenopus egg extracts and human pancreatic cancer cells highlights the physiological significance of Celastrol in the regulation of APE2 functionalities in genome integrity. Notably, cell viability assays demonstrate that APE2-KD or Celastrol sensitizes pancreatic cancer cells to chemotherapy drugs. Overall, we propose APE2 as a general regulator for the DDR pathway in genome integrity maintenance.

Highlights

Cells undergo continuous bombardments of exogenous and endogenous factors that can lead to genomic instability
To validate the phenotype of oxidative stress-induced ATR DNA damage response (DDR) pathway is due to APE2 reduction, we performed complementation assays by transfecting recombinant plasmid of full-length Xenopus APE2 tagged with YFP (YFP-xAPE2) or control plasmid of YFP in APE2-KD PANC1 cells (Lane 5–8, Figure 1B)
Due to the sequence difference between Xenopus APE2 and human APE2 in the four targeting regions of APE2-siRNA, YFP-xAPE2 cannot be targeted for protein reduction by APE2-siRNA

Summary

Introduction

Cells undergo continuous bombardments of exogenous and endogenous factors that can lead to genomic instability. It is critical for a cell to maintain genome integrity and fidelity for proper cellular function and survival in stress conditions. This task is daunting due to constant insults on the DNA by genotoxic agents, nucleotide mis-incorporation or deprivation during DNA replication, and the intrinsic biochemical instability of the DNA itself (Lindahl, 1993). Regulation of DDR by APE2 exogenous and endogenous sources can result in DNA replication stress and/or DNA lesions that include DNA double-strand breaks (DSB), DNA single-strand breaks (SSBs), and oxidative DNA damage (Ciccia and Elledge, 2010; Yan et al, 2014; Tubbs and Nussenzweig, 2017). The ATR DDR pathway is critical for duplicating DNA under stressful conditions (Saldivar et al, 2017), and ATR inhibitors as either monotherapy or combination therapy have been in different phases of clinical trials of cancer patients (Karnitz and Zou, 2015; Bradbury et al, 2020)

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