Elsevier

Free Radical Biology and Medicine

Volume 160, 20 November 2020, Pages 403-417
Free Radical Biology and Medicine

Original article
UNG2 deacetylation confers cancer cell resistance to hydrogen peroxide-induced cytotoxicity

https://doi.org/10.1016/j.freeradbiomed.2020.06.010Get rights and content

Highlights

  • UNG2 confers cell resistance to H2O2, which can be targeted for cancer treatment.

  • UNG2 is deacetylated and stabilized in response to H2O2 treatment.

  • E3 ligase UHRF1 interacts with and ubiquitinates acetylated UNG2 for proteasomal degradation.

  • Acetylation at K78 of UNG2 promotes UNG2-UHRF1 interaction and UNG2 degradation.

  • HDACi promotes acetylated-UNG2 degradation and enhances cancer toxicity of ROS reagents.

Abstract

Cancer therapeutics produce reactive oxygen species (ROS) that damage the cancer genome and lead to cell death. However, cancer cells can resist ROS-induced cytotoxicity and survive. We show that nuclear-localized uracil-DNA N-glycosylase isoform 2 (UNG2) has a critical role in preventing ROS-induced DNA damage and enabling cancer-cell resistance. Under physiological conditions, UNG2 is targeted for rapid degradation via an interaction with the E3 ligase UHRF1. In response to ROS, however, UNG2 protein in cancer cells exhibits a remarkably extended half-life. Upon ROS exposure, UNG2 is deacetylated at lysine 78 by histone deacetylases, which prevents the UNG2–UHRF1 interaction. Accumulated UNG2 protein can thus excise the base damaged by ROS and enable the cell to survive these otherwise toxic conditions. Consequently, combining HDAC inhibitors (to permit UNG2 degradation) with genotoxic agents (to produce cytotoxic cellular levels of ROS) leads to a robust synergistic killing effect in cancer cells in vitro. Altogether, these data support the application of a novel approach to cancer treatment based on promoting UNG2 degradation by altering its acetylation status using an HDAC inhibitor.

Introduction

Clinical ionizing irradiation and numerous chemotherapeutic agents kill cancer cells by producing reactive oxygen species (ROS) [[1], [2], [3]]. Unfortunately, many cancer cells are resistant to ROS, and thus survive [4]. Many molecular mechanisms have been described that might underlie cancer-cell resistance to ROS [[5], [6], [7], [8]], targeting these ROS-resistance mechanisms in cancer cells might serve as a promising strategy to re-sensitize cancer cells to ROS-generating agents.

ROS-induced DNA damage is a threat to cell survival; therefore, the timely repair of this damage might counteract ROS-induced cytotoxicity in cancer cells [4,8]. Uracil-DNA N-glycosylase (UNG) is one such enzyme that might help repair DNA. This highly conserved glycosylase excises oxidized cytosine/uracil bases damaged by ROS, creating apyrimidinic/apurinic (AP) sites for base excision repair [9,10]. The UNG gene encodes two isoforms that have identical C-terminal enzymatic sequences but different N-terminal sequences and different subcellular localizations [11,12]. UNG2 is transported into nucleus and maintains its full-length protein, whereas UNG1 is transported to the mitochondrion where its N-terminus is processed [12]. While UNG1 mainly functions in the mitochondrion, UNG2 interacts with replication protein A2 (RPA2), proliferating cell nuclear antigen (PCNA) and single strand–double strand DNA boundaries that are localized at replication sites [[13], [14], [15]]. In addition, nuclear UNG2 is overexpressed during S phase and has a specialized function in post-replicative base repair [[16], [17], [18]]. These data suggest that UNG2 has important roles in preserving DNA fidelity during DNA replication. Moreover, UNG depletion in cancer cells has been associated with increased genomic DNA damage and cell death in the presence of ROS and other genotoxic substances [[19], [20], [21]]. This finding suggests that nuclear UNG2 might facilitate cancer-cell resistance to ROS-induced cytotoxicity.

The cellular responses to ROS stimulation are closely related to protein stabilization [22,23]. Because genomic inhibition of UNG2 can sensitize cancer cells to ROS, targeting UNG2 protein turnover might also serve as a promising strategy in cancer treatment. To date, only UNG2 phosphorylation has been shown to affect its turnover [24]. UNG2 stabilization is modulated by the ubiquitin–proteasome system [24], but the precise mechanism is unclear. Ubiquitin-like with PHD and ring-finger-domains 1 (UHRF1) is one of only a few E3 ligases that shows specificity for glycosylases but its relationship with UNG2 is unknown [25]. Interestingly, UHRF1 closely regulates ROS responses by degrading Keap1 and releasing NRF-2 [26]. In addition, UHRF1 participates in the repair of DNA intercross linking [27], which is frequently induced during oxidative stress [28]. These data implicate UHRF1 in the cellular response to ROS. Whether this response occurs by modulating glycosylases, and specifically UNG2, is unknown.

Proteins are usually post-translationally modified before degradation. So far, UNG2 phosphorylation alone has been reported to involve in its turnover [24]. Other modifications of UNG2 in degradation or protein stability have not been investigated. Actually, beside protein phosphorylation, acetylation or deacetylation is also well associated with protein degradation [[29], [30], [31]]. Histone deacetylases (HDACs) catalyze deacetylations of histones and other proteins, and are closely related to cancer malignancies and overexpressed in cancer [32]. Therefore HDAC inhibitors are developed for cancer treatment. Evidences show that HDAC inhibitors can increase the anti-neoplastic effects of many therapeutic agents to synergistically kill cancer cells, which encourages the combined clinical uses of HDAC inhibitors with other cancer therapies [[33], [34], [35]]. However, the mechanism underlying these anti-neoplastic synergies remain unclear.

Here we aimed to determine the detailed mechanism for UNG2-dependent resistance towards ROS-induced cytotoxicity which could be potentially targeted for ROS-generating cancer therapy. We addressed these aims by monitoring the alterations of UNG2 protein in response to ROS stimulation, and identified the E3 ligase that interact with and degrade UNG2. We ultimately delineate a novel acetylation-based regulatory mechanism that underlies UNG2 protein stabilization and provide data to support that UNG2 might be a promising target to enhance the anti-tumor effects of ROS-generating treatments.

Section snippets

Cell lines and culture

Human colon cancer cell line HCT116 and human lung cancer cell line A549 cancer cell lines were purchased from the American Type Culture Collection (ATCC) and tested for mycoplasma contamination. The cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (PAN-Biotech) and cultured in a 37 °C, 5% CO2 conditioned incubator.

Reagents and antibodies

The following chemical reagents were used: Trichostatin A, Nicotinamide and Depsipeptide (Sigma-Aldrich), H2O2, PL and Doxorubicin (Sigma-Aldrich). The

UNG2 prevents ROS-induced DNA damage

A previous study showed that UNG is responsible for promoting cellular resistance to ROS-induced cytotoxicity [19], but the underlying mechanism was not delineated. To confirm the involvement of UNG in cancer-cell resistance to ROS, we knocked down (KD) UNG in human colon cancer HCT116 cells and human lung cancer A549 cells using a shRNA targeting both UNG1 and UNG2 mRNA (Supplementary Fig. S1A); we then exposed the cells to hydrogen peroxide (H2O2). We analyzed the cells using an UNG-modified

Discussion

This study has found that UNG2 protein is involved in mediating cellular resistance to ROS. To the best of our knowledge, ours is the first demonstration that UNG2 acetylation at K78 modulates its protein turnover by UHRF1-mediated ubiquitin-proteasome degradation. In response to ROS treatment, UNG2 is deacetylated by HDACs, preventing an UNG2–UHRF1 interaction from forming and thus blocking UHRF1-mediated UNG2 degradation. Overall, this effect leads to cancer cell resistance towards ROS.

Author contributions

Y.B. and W.-G. Z. designed the study; Y.B., L.T., B.S. and G.L. performed experiments; Q.Z., X.L., J.Z., Y.-F. L., H.W. and Y.T. analyzed data; Y.B., Y.S. and W.-G. Z. wrote the manuscript; W.-G. Z. approved the final version for submission.

Funding

This work was supported by National Key R&D Program of China [2017YFA0503900]; National Natural Science Foundation of China [81720108027, 81530074]; Science and Technology Program of Guangdong Province in China [2017B030301016]; Shenzhen Municipal Commission of Science and Technology Innovation [JCYJ20170818092450901]; Discipline Construction Funding of Shenzhen [(2016)1452]; Shenzhen Bay Laboratory [SZBL2019062801011]; China Postdoctoral Science Foundation [2017M622785].

Declaration of competing interest

The authors declare no conflicts of interest in this work.

Acknowledgements

We thank Dr. Jessica Tamanini of ETediting, Shenzhen University for language editing.

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