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Ubiquitination of the DNA-Damage Checkpoint Kinase CHK1 by TRAF4

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Ubiquitination of the DNA-Damage Checkpoint Kinase CHK1 by TRAF4 Yu et al. Journal of Hematology & Oncology (2020) 13:40 https://doi.org/10.1186/s13045-020-00869-3 RESEARCH Open Access Ubiquitination of the DNA-damage checkpoint kinase CHK1 by TRAF4 is required for CHK1 activation Xinfang Yu1,2†, Wei Li1,2,3†, Haidan Liu4, Qipan Deng1,2, Xu Wang1,2, Hui Hu1,2,5, Zijun Y. Xu-Monette6, Wei Xiong7, Zhongxin Lu5, Ken H. Young6, Wei Wang3* and Yong Li1,2* Abstract Background: Aberrant activation of DNA damage response (DDR) is a major cause of chemoresistance in colorectal cancer (CRC). CHK1 is upregulated in CRC and contributes to therapeutic resistance. We investigated the upstream signaling pathways governing CHK1 activation in CRC. Methods: We identified CHK1-binding proteins by mass spectrometry analysis. We analyzed the biologic consequences of knockout or overexpression of TRAF4 using immunoblotting, immunoprecipitation, and immunofluorescence. CHK1 and TRAF4 ubiquitination was studied in vitro and in vivo. We tested the functions of TRAF4 in CHK1 phosphorylation and CRC chemoresistance by measuring cell viability and proliferation, anchorage-dependent and -independent cell growth, and mouse xenograft tumorigenesis. We analyzed human CRC specimens by immunohistochemistry. Results: TRAF4catalyzedtheubiquitinationofCHK1inmultipleCRC cell lines. Following DNA damage, ubiquitination of CHK1 at K132 by TRAF4 is required for CHK1 phosphorylation and activation mediated by ATR. Notably, TRAF4 was highly expressed in chemotherapy-resistant CRC specimens and positively correlated with phosphorylated CHK1. Furthermore, depletion of TRAF4 impaired CHK1 activity and sensitized CRC cells to fluorouracil and other chemotherapeutic agents in vitro and in vivo. Conclusions: These data reveal two novel steps required for CHK1 activation in which TRAF4 serves as a critical intermediary and suggest that inhibition of the ATR–TRAF4–CHK1 signaling may overcome CRC chemoresistance. Keywords: Colorectal cancer, Checkpoint kinase 1, Tumor necrosis factor receptor-associated factor 4, Ubiquitination, Chemoresistance Introduction and its mechanism of action is direct or indirect in- Colorectal cancer (CRC) is the third most common duction of DNA damage response (DDR) pathways cancer and the second leading cause of cancer-related [2]. Although the majority of patients with advanced death worldwide [1]. Currently, fluorouracil (5-Fu)- CRC are initially responsive to the 5-Fu-based com- based chemotherapy is one of the most frequently bination chemotherapy, tumors frequently recur due used therapeutic strategies for advanced CRC patients, to drug resistance. Immune checkpoint inhibitors have recently shown promise in a limited subset of * Correspondence: [email protected]; [email protected] † patients as a treatment for CRC [3]. Therefore, fur- Xinfang Yu and Wei Li contributed equally to this work. ther elucidating the mechanisms of DDR and che- 3Department of Radiology, The Third Xiangya Hospital of Central South University, Changsha, Hunan, China moresistance in CRC and improving the efficacy of 1Department of Medicine, Baylor College of Medicine, Houston, TX, USA chemotherapy have important clinical significance. Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yu et al. Journal of Hematology & Oncology (2020) 13:40 Page 2 of 19 DNA-damage checkpoints are signaling cascades that routinely checked for mycoplasma and tested cytogeneti- either trigger cell cycle arrest, which allows time for cally to ensure their authentication. Chemical reagents, DNA repair or induce apoptotic cell death [4]. Trans- including Tris, NaCl, SDS, and dimethyl sulfoxide duction of the DDR signal, such as that induced by 5-Fu, (DMSO), were purchased from Sigma-Aldrich (St. Louis, is often carried out by a set of conserved checkpoint MO, USA). Oxaliplatin (cat. #S1224), 5-Fu (cat. #S1209), protein kinases, including ataxia telangiectasia mutated irinotecan (cat. #S2217), cisplatin (cat. #S1166), peme- (ATM), ATM- and Rad3-related (ATR) proteins, and trexed (cat. #S1135), gemcitabine (cat. #S1714), VE-821 the downstream checkpoint kinases CHK2 and CHK1. (cat. #S8007), AZ20 (cat. #S7050), and prexasertib were Phosphorylation of CHK1 at S317 and S345 by the ATR obtained from Selleck Chemicals (Houston, TX, USA). kinase is required for its activation in response to DNA Lipofectamine™ 2000 (cat. #11668019, Thermo Fisher damage [4]. CHK1 is upregulated in multiple human Scientific) transfection reagent was used for transient cancers, including CRC, and its overexpression and/or transfection experiments following the manufacturer’s hyperactivation is linked to chemo- and radiotherapy re- instruction. Ctrl siRNA (cat. #sc-93314), UbcH6 siRNA sistance [5–10]. CHK1-targeted therapy selectively elimi- (cat. #sc-61744), BTG3 siRNA (cat. #sc-43644), and nates replication-stressed, p53-deficient, and ATR siRNA (cat. #sc-29763) were purchased from Santa hyperdiploid colorectal cancer stem cells [11, 12]. Accu- Cruz Biotechnology (Dallas, TX, USA). Three independ- mulating evidence has shown that CHK1 ubiquitination ent experiments were completed for each cell line, treat- is critical for its stability, subcellular localization, and ment, and time point, as indicated. phosphorylation. Furthermore, K48-linked ubiquitina- tion downregulates CHK1 expression, whereas K63- Immunoblotting (IB) and immunoprecipitation (IP) linked ubiquitination upregulates CHK1 activity [13–15]. Whole-cell lysates were extracted with RIPA buffer (cat. However, how the DDR triggers the ubiquitination- #89900, Thermo Fisher Scientific) supplemented with dependent activation of CHK1 to promote CRC cell sur- phosphatase inhibitors (cat. #78428, Thermo Fisher Sci- vival remains elusive. entific). The lysates were sonicated and centrifuged at Tumor necrosis factor receptor-associated factor 4 12000×g for 15 min at 4 °C. The BCA Assay Reagent (TRAF4, also known as RING finger protein 83 or (cat. #23228, Thermo Fisher Scientific) was used to deter- RNF83) is emerging as a critical regulator for cancer cell mine protein concentration. For co-immunoprecipitation proliferation, survival, and metastasis [16–18]. TRAF4 (co-IP) assays, cells were lysed with IP Lysis Buffer (cat. promotes the invasion of CRC cells by regulating Wnt/ #87787, Thermo Fisher Scientific). IB and co-IP were per- β-catenin signaling [19, 20]. Recent reports suggest the formed as previously described [16]. All antibodies for IB N-terminal RING finger domain of TRAF4 has E3 ligase analysis were diluted in phosphate-buffered saline (PBS) activity that ubiquitinates AKT, SMURF2, and TrkA to buffer with 5% non-fat milk. Antibodies against Bax (cat. promote tumor growth and metastasis [16–18]. Due to #5023; IB, 1:1000), Bik (cat. #4592; IB, 1:1000), Bim (cat. the critical role of TRAF4 in tumorigenesis and its drug- #2933; IB, 1:1000), Bid (cat. #2002; IB, 1:1000), Bak (cat. gable enzymatic activity, identification of novel TRAF4 #12105; IB, 1:1000), survivin (cat. #2808; IB, 1:1000), Bcl-2 substrates will be helpful for developing new cancer (cat. #4223; IB, 1:1000), Bcl-xL (cat. #2764; IB, 1:1000), treatment strategies. Mcl-1 (cat. #5453; IB, 1:1000), γ-H2AX (cat. #9718; IB, 1: Here, we identified TRAF4 as an E3 ligase that ubiqui- 4000), α-tubulin (cat. #2144; IB, 1:10000), ubiquitin (cat. tinates CHK1. During DDR, TRAF4 ubiquitinates #3936; IB, 1:1000), cleaved-caspase 3 (cat. #9664; IB, 1: CHK1, which is a prerequisite for CHK1 chromatin as- 2000), cleaved-PARP (cat. #5625; IB, 1:2000), p-(Ser/Thr) sociation and subsequent phosphorylation and activation ATM/ATR substrate (cat. #2851; IB, 1:1000), p-ATR by ATR in CRC cells. The absence of TRAF4 sensitizes (S428) (cat. # 2853; IB, 1:1000), p-ATR (Thr1989) (cat. CRC cells to chemotherapeutic agents in a CHK1 ubi- #30632; IB, 1:1000), ATR (cat. # 13934; IB, 1:1000), p- quitination- and activation-dependent manner, suggest- CHK1 (S317) (cat. #12302; IB, 1:1000), p-CHK1 (S345) ing that CHK1 ubiquitination by TRAF4 is a potential (cat. #2348; IB, 1:1000), CHK1 (cat. #2360; IB, 1:1000; IP, therapeutic target to overcome CRC chemoresistance. 1:200), p-CDC25C (Ser216) (cat. #4901; IB, 1:1000), CDC25C (cat. #4688; IB, 1:1000), GST tag (cat. #2624; IB, Materials And methods 1:5000; IP, 1:200), K63-linkage-specific polyubiquitin (cat. Cell culture and reagents #12930; IB, 1:1000), rabbit IgG HRP (cat. #7074; IB, 1: HT29, SW620, HCT-8, FHC, HeLa, U2OS, A549, 10000), and mouse IgG HRP
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