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HRG Switches TNFR1-Mediated Cell Survival to Apoptosis in Hepatocellular Carcinoma

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HRG Switches TNFR1-Mediated Cell Survival to Apoptosis in Hepatocellular Carcinoma 1 HRG switches TNFR1-mediated cell survival to apoptosis in hepatocellular carcinoma 2 Xuejing Zou1, 2*, Dongyan Zhang2, 3*, Yang Song 2, 3*, Shanshan Liu1, 2, Qian Long1, 2, Liheng 3 Yao 1, 2, Wenwen Li1, 2, Zhijiao Duan1, 2, Dehua Wu2, 3and Li Liu1, 4 4 1. Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Hepatology Unit and 5 Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, 6 Guangzhou 510515, China 7 2. State Key Laboratory of Organ Failure Research, Nanfang Hospital, Southern Medical 8 University, Guangzhou 510515, China 9 3. Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, 10 Guangzhou 510515, China 11 4.Department of Medical Quality Management, Nanfang Hospital, Southern Medical 12 University, Guangzhou 510515, China 13 These authors contributed equally: Xuejing Zou, Dongyan Zhang, Yang Song 14 Running title: HRG as a pro-apoptotic molecular switch in HCC 15 Corresponding authors: Prof. L. Liu, Guangdong Provincial Key Laboratory of Viral 16 Hepatitis Research, Hepatology Unit, Department of Infectious Diseases and Department of 17 Medical Quality Management, Nanfang Hospital, Southern Medical University, Guangzhou 18 510515, China. Phone: 86-18602062738. E-mail: [email protected]; and Prof. D. Wu, 19 Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, 20 Guangzhou 510515, China. Phone: 86-20-62787693; Fax: 86-20-62787631. E-mail: 21 [email protected]. 22 Conflict of interest: The authors have no conflicts of interest to declare. 1 23 Abstract 24 Background: Tumor necrosis factor receptor 1 (TNFR1) signaling plays a pleiotropic role in 25 the development of hepatocellular carcinoma (HCC). The formation of TNFR1-complex I 26 supports cell survival while TNFR1-complex Ⅱ leads to apoptosis, and the underlying 27 mechanisms of the transformation of these TNFR1 complexes in HCC remain poorly defined. 28 Methods: The interaction protein of TNFR1 was identified by GST pulldown assay, 29 immunoprecipitation and mass spectrometry. In vitro and in vivo assay were performed to 30 explore the biological features and mechanisms underlying the regulation of TNFR1 signals 31 by histidine-rich glycoprotein (HRG). Data from the public databases and HCC samples were 32 utilized to analyze the expression and clinical relevance of HRG. 33 Results: HRG directly interacted with TNFR1 and stabilized TNFR1 protein by decreasing 34 the Lys(K)-48 ubiquitination mediated-degradation. The formation of TNFR1-complex Ⅱ was 35 prompted by HRG overexpression via upregulating Lys(K)-63 ubiquitination of TNFR1. 36 Besides, overexpression of HRG suppressed expression of pro-survival genes by impairing 37 the activation of NF-κB signaling in the presence of TNFR1. Moreover, downregulation of 38 HRG was a result of feedback inhibition of NF-κB activation in HCC. In line with the pro- 39 apoptotic switch of TNFR1 signaling after HRG induction, overexpression of HRG inhibited 40 cell proliferation and increased apoptosis in HCC. 41 Conclusions: Our findings illustrate a crucial role for HRG in suppressing HCC via inclining 42 TNFR1 to a pro-apoptotic cellular phenotype. Restoring HRG expression in HCC tissues 43 might be a promising pharmacological approach to blocking tumor progression by shifting 44 cellular fate from cell survival to apoptosis. 2 45 46 Graphical abstract: In HCC cells, HRG expression is suppressed by NF-κB activation which 47 supports cell survival. Otherwise, forcing HRG expression increases cell death by enhancing 48 TNFR1-complex II formation and impairing NF-κB signaling. 49 50 Keywords: histidine-rich glycoprotein, tumor necrosis factor receptor 1, apoptosis 51 52 Background 53 TNFR1 is dysregulated in various cancers and is associated with malignant progression. 54 Study showed that TNFR1-mediated signaling cooperates with repressed keratinocyte NF-κB 55 in driving skin cancer development [1]. MUC13 interacts with TNFR1 and increases 56 clustering of the TNFR1 signaling complex, thereby amplifying the efficiency of TNF- 57 induced NF-κB activation, which supports colorectal cancer cells to survive under DNA- 58 damaging agents [2]. TNFR1 was also noted to be involved in regulating the tumorigenicity 59 of ovarian cancer [3]. In liver cancer, TNFR1 is reported to be vital for tumor promotion. 60 TNF-α/ TNFR1 signaling participates in the proliferation of oval cells during the 61 preneoplastic stage of liver carcinogenesis and loss of TNFR1 reduces the incidence of tumor 3 62 formation [4]. TNFR1 signaling contributes to obesity-induced carcinoma promotion as 63 depletion of TNFR1 abolished obesity-enhanced HCC development [5]. Based on such 64 tumor-promoting aspects of TNFR1, efforts have been made to target TNF-α or TNFR1 in 65 various malignancies with pharmaceuticals that include TNF-α inhibitors, TNFR1 66 monoclonal antibodies, and TNFR1-targeting nanomaterials. However, the implements of 67 TNF-α or TNFR1 therapeutic practices are unsatisfactory owing to the dual function and 68 complexity of TNFR1-mediated signaling during cancer development [6-8]. 69 TNFR1 governs either survival or death in cancer cells via formation of different 70 TNFR1-complexes [9,10]. Upon TNF-α stimulation, TNFR1 recruits receptor-interacting 71 protein 1 (RIP1) and TNFR1-associated death domain protein (TRADD) to form TNFR1- 72 complex I which increases the expression of a string of pro-survival genes by activating the 73 NF-κB signaling pathway [11,12]. Activation of TNFR1-complex I plays a vital role in 74 tumorigenesis as shown in previous studies that investigated the act of TNF-α in the initiation, 75 development, recurrence and therapy resistance of malignancies such as liver cancer, breast 76 cancer, bladder cancer, renal cell carcinoma and pancreatic cancer [13-17]. Besides, TNF-α 77 stimulation leads to TNFR1 internalization into the cytoplasm and formation of TNFR1- 78 complex Ⅱ, also known as death-inducing signaling complex (DISC). Next, TNFR1 recruits 79 fas-associated death domain protein (FADD) and caspase-8 to increase apoptosis, leading to 80 cell death [18]. Based on the cell death-promoting effect of TNFR1-complex Ⅱ, the potency 81 of human recombinant TNF-α as an anti-tumor therapy was tested in numerous clinical trials 82 but declined owing to the unclear role of the TNFR1-mediated signaling pathway in cancer 83 [6]. Suppressing NF-κB-dependent living signaling while inducing TNFR1-mediated cell 4 84 apoptosis is an unresolved conundrum for devising medicines based on TNFR1 modulation. 85 TNFR1 signaling is regulated by diverse processes that include ubiquitination, 86 glycosylation, endocytosis, lipid raft recruitment, and so on. Among these, ubiquitination is 87 one of the most central post-transcriptional modifications that controls the stability of TNFR1 88 protein and leads the pro-survival or pro-apoptotic cellular signaling transduction, which 89 determines cancer cell progression [19-22]. Given the limited understanding of governing 90 TNFR1-mediated signaling transduction in HCC, we performed this study to explore the 91 regulation of TNFR1 complex I/Ⅱ and the resulting influence on the cell fate. 92 Researches demonstrated that histidine-rich glycoprotein (HRG) involves both 93 inflammatory promoting effect in chronic liver disease and tumor suppression during the 94 development of HCC [23-25]. HRG was identified as a TNFR1 binding partner in our study. 95 Whether HRG influences the TNFR1 signaling mechanism within cancer cells from a pro- 96 survival to pro-death state has not been explored. A better understanding of the role of HRG 97 in TNFR1-mediated pleiotropic pathways ought to assist in formulating new anticancer 98 therapeutic strategies. 99 100 Materials and Methods 101 Patients and tissue samples 102 HCC samples and matching noncancerous tissues used in our study were collected from 103 HCC patients who underwent hepatectomy between January 2014 and December 2015 at 104 Nanfang Hospital (Guangzhou, China). Patients enrolled did not receive any anti-tumor 105 treatment before surgery. Prior patient consent and approval from the Institute Research 5 106 Ethics Committee were obtained for the use of the clinical materials for research. 107 All tissues were stored in a liquid nitrogen container prior to RNA or protein extraction. 108 Cell culture 109 HCC cell lines Huh7 and SMMC-7721 were purchased from the Cell Bank of the 110 Chinese Academy of Sciences (Chinese Academy of Sciences, Shanghai, China). Huh7 and 111 SMMC-7721 cells were incubated in DMEM and RPMI 1640 (Gibco, Grand Island, NY, 112 USA), respectively, with 10% fetal bovine serum (Gibco) at 37 ℃ in a humidified incubator 113 containing 5% CO2. Recombinant human TNF-alpha (PeproTech, Rocky Hill, NJ, USA), 114 FXR agonist GW4064 (Selleck Chemicals, USA), MG132 (Beyotime Biotechnology, China) 115 and cycloheximide (CHX, Sigma-Aldrich, USA) were used to treat cells for experiments. 116 Xenograft assays 117 BALB/c nude mice (4-6 weeks old, Central Laboratory of Animal Science, Southern 118 Medical University, Guangzhou, China) were used for in vivo studies. HRG-overexpressed 119 SMMC-7721 cells and the related controls were suspended at a density of 1×107 per 100 μL 120 and inoculated subcutaneously into the flanks of each mouse. After 30 days, the IVIS Lumina 121 II system (Caliper Life Sciences, Hopkinton, MA, USA) was used to measure the 122 fluorescence intensity of tumor before sacrifice. Xenograft tumors were then removed and 123 weighed. Hematoxylin and eosin (H&E), terminal deoxy-nucleotidyl transferase-mediated 124 dUTP nick-end labeling (TUNEL) assay (In Situ Cell Death Detection Kit, Roche, USA), and 125 immunohistochemical (IHC, Dako, Carpinteria, CA, USA) staining were performed to 126 evaluate the morphology, cell apoptosis, and cell proliferation in all tumor tissues, 127 respectively, according to the manufacturer’s guidelines. 6 128 Gene-expression datasets 129 TCGA cohort: A cohort containing 365 cases of HCC patients with HRG expression data 130 and follow-up information downloaded from The Cancer Genome Atlas Liver Hepatocellular 131 Carcinoma (TCGA-LIHC; https://tcga-data.nci.nih.gov/tcga/) dataset.
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