Published OnlineFirst September 25, 2018; DOI: 10.1158/0008-5472.CAN-18-2063 Cancer Molecular Cell Biology Research An ATM/TRIM37/NEMO Axis Counteracts Genotoxicity by Activating Nuclear-to- Cytoplasmic NF-kB Signaling Geyan Wu1,2, Libing Song3, Jinrong Zhu1,2, Yameng Hu1,2, Lixue Cao1,2, Zhanyao Tan1,2, Shuxia Zhang1,4, Ziwen Li1,2, and Jun Li1,2 Abstract Blocking genotoxic stress-induced NF-kB activation damaging anticancer drug cisplatin in vitro and in vivo would substantially enhance the anticancer efficiency of through activation of the NF-kB pathway. Genotoxic genotoxic chemotherapy. Unlike the well-established classical stress-activated ATM kinase directly interacted with and NF-kB pathway, the genotoxic agents-induced "nuclear-to- phosphorylated TRIM37 in the cytoplasm, which induced cytoplasmic" NF-kB pathway is initiated from the nucleus translocation of TRIM37 into the nucleus, where it formed a and transferred to the cytoplasm. However, the mechanism complex with NEMO and TRAF6 via a TRAF6-binding motif linking nuclear DNA damage signaling to cytoplasmic IKK (TBM). Importantly, blocking the ATM/TRIM37/NEMO axis activation remains unclear. Here, we report that TRIM37, a via cell-penetrating TAT-TBM peptide abrogated genotoxic novel E3 ligase, plays a vital role in genotoxic activation of agent-induced NEMO monoubiquitination and NF-kB NF-kB via monoubiquitination of NEMO at K309 in the activity, resulting in hypersensitivity of cancer cells to gen- nucleus, consequently resulting in nuclear export of NEMO otoxic drugs. Collectively, our results unveil a pivotal role and IKK/NF-kB activation. Clinically, TRIM37 levels correlated for TRIM37 in genotoxic stress and shed light on mechan- positively with levels of activated NF-kB and expression of isms of inducible chemotherapy resistance in cancer. Bcl-xl and XIAP in esophageal cancer specimens, which also associated positively with clinical stage and tumor-node- Significance: In response to genotoxic stress, TRIM37 acti- metastasis classification and associated inversely with overall vates NF-kB signaling via monoubiquitination of NEMO, and relapse-free survival in patients with esophageal cancer. which subsequently promotes cisplatin chemoresistance and Overexpression of TRIM37 conferred resistance to the DNA- tumor relapse in cancer. Cancer Res; 78(22); 6399–412. Ó2018 AACR. Introduction Meanwhile, cells have evolved a precisely controlled network of DNA damage response (DDR) to respond to genotoxic stresses, Chromosomal integrity of all living organisms is endlessly includes sensing of damaged DNA, activation of cell cycle check- jeopardized by genotoxic stress generated from endogenous met- points, assembly of DNA repair machineries, and transactivation abolic sources, such as reactive oxygen species (ROS), and envi- of DNA damage-responsive gene expression. For instance, ATM ronmental resources, such as ionizing radiation and genotoxic kinase and PARP-1 act as sensor proteins to detect DNA lesions chemicals (1–4). Unrepaired or inappropriately repaired DNA and modify a variety of proteins, which initiate DNA repair and damage consequently attribute to genetic variation, apoptosis, cell-cycle checkpoint control (6, 7). aging, degenerative diseases, inflammation, and cancer (2–5). On the other hand, the NF-kB signaling pathway was emerged as a vital mediator for cellular responses to genotoxic threats via inducing survival genes that allow cells to repair damaged DNA 1Key Laboratory of Liver Disease of Guangdong Province, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, P.R. China. 2Department of bio- and promote survival (8, 9). However, unlike the well-established chemistry, Zhongshan school of medicine, Sun Yat-sen University, Guangzhou, classical NF-kB pathway in which signals are initiated from P.R. China. 3State Key Laboratory of Oncology in South China, Collaborative cell surface receptors and transduced from the cytoplasm to Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, the nucleus (10, 11), genotoxic agents triggered the "nuclear-to- 4 Guangzhou, P.R. China. Key Laboratory of Protein Modification and Degrada- cytoplasmic" NF-kB pathway that is initiated from the nucleus fi tion, School of Basic Medical Sciences, Af liated Cancer Hospital &Institute of and transferred to the cytoplasm (8, 9). In response to DNA Guangzhou Medical University, Guangzhou Medical University, Guangzhou, P.R. China. damage signals, PARP-1 is rapidly recruited to DNA damage sites and induces auto-poly(ADP-ribosyl)ation (PARylation), which Note: Supplementary data for this article are available at Cancer Research assembles NEMO, PIASy, ATM, PIDD, and LRP16 into a nucle- Online (http://cancerres.aacrjournals.org/). oplasmic signalosome (12–16). Furthermore, signalosome for- G. Wu, L. Song, and J. Zhu contributed equally to this article. mation induces sumoylation of NEMO at K277/K309 in a PIDD/ Corresponding Author: Jun Li, Sun Yat-sen University, 74 Zhongshan Road II, PARP1/PIASy-dependent manner and ATM-dependent phos- Guangzhou, Guangdong 510080, P.R. China; Phone: 86-20-87335828; phorylation of NEMO at S85 (12–17). Then phosphorylated- Fax: 86-20-87335828; E-mail: [email protected] NEMO is monoubiquitinated and exported from the nucleus doi: 10.1158/0008-5472.CAN-18-2063 together with ATM and ELKS, which form a complex with IKK Ó2018 American Association for Cancer Research. catalytic subunits in the cytoplasm, consequently resulting in www.aacrjournals.org 6399 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst September 25, 2018; DOI: 10.1158/0008-5472.CAN-18-2063 Wu et al. activation of IKK/NF-kB signaling (18). Therefore, nuclear mono- Plasmids, virus constructs, and retroviral infection of target ubiquitin of NEMO, which is essential for its nuclear export, is the cells key step in genotoxic agent-triggered "nuclear-to-cytoplasmic" Human TRIM37, NEMO, and TRAF6 were amplified by PCR NF-kB signaling. However, the E3 ligase responsible for NEMO andclonedintothepSin-EF2vector.Fragmentsofthehuman nuclear monoubiquitination remains unknown. TRIM37 and TRAF6-coding sequence were amplified by PCR Tripartite motif containing 37 (TRIM37) is a newly identified andclonedintothepSin-EF2vector.Theindicatedmutants E3 ubiquitin ligase that comprises a tripartite motif (TRIM, were created using primers and a Stratagene Mutagenesis Kit RING-B-box-coiled-coil) domain, TRAF domain (TD), and according to the protocol recommended by the manufacturer. polyacidic domain (19–21). It has been recently reported that pNF-kB-luc and control plasmids (Clontech) was used to TRIM37 plays vital roles in various biological processes quantitatively examine NF-kB activity. Transfection of siRNAs depending on TRIM domain-dependent E3 ligase activity, such or plasmids was performed using the Lipofectamine 3000 as promotion of peroxisomal matrix protein import via direct reagent (Invitrogen) according to the manufacturer's instruc- monoubiquitination of PEX5 at K464 and silencing of gene tion. Retroviral production and infection were performed as expression through monoubiquitination of histone H2A (22, described previously (25). Stable cell lines expressing indicated 23). In this study, we unveiled a novel function of TRIM37 in genes were selected for 10 days with 0.5 mg/mL puromycin regulating nuclear-to-cytoplasmic NF-kB signaling. We found 48 hours after infection. The primers used were listed in that, upon genotoxic stimulation, TRIM37 rapidly translocated Supplementary Table S4. into the nucleus where it interacted directly with TRAF6 to catalyze monoubiquitination of NEMO at K309. Blocking Immunohistochemistry TRIM37/TRAF6 interaction using a cell-penetrating TAT-TD IHC analysis was performed to study altered protein expression peptide abrogated NEMO monoubiquitination-dependent in 441 human esophageal cancer tissues according previous report NF-kB signaling, resulting in hypersensitivity of esophageal (26). Paraffin-embedded tissues were analyzed using IHC with cancer cells to DNA-damaging chemotherapeutics. Hence our anti-TRIM37 antibody (Abcam; 1:200), anti-NF-kB p65 antibody study reveals a crucial role of TRIM37 in genotoxic stress- (Abcam; 1:200), anti-Bcl-XL antibody (Cell Signaling; 1:100), induced NF-kB activation and sheds light on mechanisms of anti-XIAP (1007-1008) antibody (Proteintech; 1:100). All the inducible chemotherapy resistance in esophageal cancer. antibodies used in this study has been listed in Supplementary Table S5. The degree of immunostaining of formalin-fixed, par- fi Materials and Methods af n-embedded sections were reviewed and scored separately by two independent pathologists uninformed of the histopathologic Ethics statement features and patient data of the samples. The scores were deter- Informed consent was signed by all patients, and the investi- mined by combining the proportion of positively-stained tumor gation has been conducted in accordance with the ethical stan- cells and the intensity of staining. The scores given by the two dards according to the Declaration of Helsinki, national and independent pathologists were combined into a mean score for international guidelines, which has also been approved by the further comparative evaluation. Tumor cell proportions were authors' Institutional Review Board. scored as follows: 0, no positive tumor cells; 1, <10% positive tumor cells; 2, 10% to 35% positive tumor cells; 3, 35% to 75% Tissue