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TRIM22 functions as an oncogene in gliomas through regulating the Wnt/b-catenin signaling Cite this: RSC Adv.,2018,8, 30894 pathway

Shi-lei Tang,†a Yuan-lin Gao†b and Wen-zhong Hu *a

The tripartite motif-containing (TRIM) family is a group of proteins that are implicated in a plethora of pathological conditions. TRIM22 has been found to be involved in various cancers; however, the role of TRIM22 in gliomas has not been reported. The present study aimed to evaluate the expression pattern of TRIM22 and its function in gliomas. TRIM22 expressions in glioma tissues and cell lines were measured by RT-PCR and western blot analysis. To knockdown TRIM22 by small hairpin RNAs (shTRIM22), the U118 cells were transfected with pLKO.1-shTRIM22 plasmid or pLKO.1 plasmid. Cell proliferation was measured using CCK-8 assay. Transwell assays were performed to evaluate the migration and invasion. The epithelial–mesenchymal transition (EMT) was assessed by detecting the expressions of E-cadherin,

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. N-cadherin and vimentin with western blot analysis. A xenograft mouse model was established to evaluate the effect of TRIM22 silencing on tumor growth in vivo. The expressions of b-catenin, cyclin D1, and c-Myc were analyzed by western blot analysis. TRIM22 was significantly overexpressed in glioma tissues and cell lines. In vitro studies demonstrated that TRIM22 knockdown inhibited cell proliferation, migration, and invasion. Additionally, TRIM22 silencing increased the expressions of E-cadherin, and decreased the expressions of N-cadherin and vimentin. Nude mouse xenograft assay showed that TRIM22 silencing inhibited tumor growth in vivo. Furthermore, silencing of TRIM22 inhibited the Received 3rd July 2018 activation of the Wnt/b-catenin pathway. Treatment with LiCl, an activator of the Wnt/b-catenin Accepted 20th August 2018 pathway, attenuated the effects of shTRIM22 on U118 cells. Silencing of TRIM22 inhibited proliferation, This article is licensed under a DOI: 10.1039/c8ra05684f migration and invasion, as well as repressing the EMT process in glioma cells. The Wnt/b-catenin rsc.li/rsc-advances pathway was involved in the effect of TRIM22.

Open Access Article. Published on 03 September 2018. Downloaded 9/29/2021 1:08:09 AM. 1. Introduction The tripartite motif-containing (TRIM) family is a group of proteins that are characterized by a conserved structure with Glioma is a type of tumor originating in the glial cells in the a RING domain, one or two B-box domains, a predicted coiled brain.1 It accounts for about 33 percent of all brain tumors, and coil, and usually a C-terminal domain.5 Several lines of evidence 80 percent of all malignant brain tumors.2 It represents a rela- suggest that TRIM proteins have a broad range of functions in tively serious health burden.1 On the basis of their histopa- regulation of many cellular processes such as cell diﬀerentia- thology, gliomas can be subdivided into grades I–IV according tion, proliferation/cycle, and apoptosis.6 Consistently, the to the World Health Organisation (WHO) classication of alterations of these proteins are implicated in a plethora of tumors in the central nervous system.3 The treatment of gliomas pathological conditions.6,7 Some members are thought to be is a combined therapy with surgery, radiation therapy and important regulators of carcinogenesis.8,9 Among these chemotherapy, which depends on the location and grade of members, TRIM22 has been proved to be involved in various malignancy.4 However, the conventional treatment has an cancers, such as endometrial cancer,10 leukemia,11 non-small alarmingly dismal prognosis. Gliomas generally recur even aer cell lung cancer,12 and breast cancer.13 However, the role of optimal initial treatment. Therefore, novel approaches in the TRIM22 in glioma has not been investigated. The present study treatment of this disease are still needed. aimed to evaluate expression pattern of TRIM22 and its function in glioma.

a RETRACTED Department of Neurosurgery, Huaihe Hospital of Henan University, No. 8 Baobei Road, Kaifeng 475000, Henan Province, China. E-mail: [email protected]; Fax: 2. Materials and methods +86-0371-23906516; Tel: +86-0371-23906516 2.1 Patient samples bDepartment of Neurology, Kaifeng Central Hospital, Kaifeng 475000, Henan Province, China The clinical samples used in this study are consisted of 21 † These authors contributed equally to this work. glioma tissues and matched normal brain tissues were collected

30894 | RSC Adv.,2018,8,30894–30901 This journal is © The Royal Society of Chemistry 2018 View Article Online Paper RSC Advances

from patients with a mean age of 44 5 years. The patients 2.5 Sh-TRIM22 transfection underwent surgical treatment at Huaihe Hospital of Henan Small hairpin RNAs (shRNA) targeting TRIM22 (sh-TRIM22) and University between May 2014 and September 2017. This study its negative control (sh-control) were purchased from Shanghai was performed in strict accordance with the NIH guidelines for Genechem Co., Ltd. (Shanghai, China). The shTRIM22 was the care and use of laboratory animals (NIH Publication No. 85- constructed into pLKO.1 plasmids (Sigma). The pLKO.1-sh- 23 Rev. 1985) and was approved by the Institutional Animal TRIM22 plasmid or pLKO.1-sh-control was transfected into Care and Use Committee of Huaihe Hospital of Henan U87 cells. Then the transfected cells were subjected to 14 days of University (Kaifeng, China). All patients were informed and selection in DMEM medium containing 1.0 mgmL 1 puromycin have signed the informed consent. (Sigma). Finally, the individual puromycin-resistant cells were isolated and collected for further experiments as stable 2.2 Cell culture expressing cells. Normal microglia cell line HEB, and glioma cell lines including A172, LN229, U251, and U87 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells 2.6 Cell proliferation assay  were grown in Dulbecco's modi ed Eagle's medium (DMEM; Cell counting-kit-8 (CCK-8) assay was used for the determina- Hyclone, Logan, UT, USA) containing 10% fetal bovine serum tion of cell proliferation. U87 cells were seeded in 96-well plates (FBS; Gibco Laboratories, Grand Island, NY, USA) and 1% at a density of 1 104 cells per well. Then 10 ml CCK-8 solution penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at (Dojindo, Kumamoto, Japan) was added to each well at 0 h, 24 h, 37 C in an atmosphere with 5% CO2. 48 h, and 72 h, and incubated for 1 h at 37 C. Subsequently, the absorbance values were determined at 450 nm using a micro- 2.3 Quantitative real-time polymerase chain reaction (qRT- plate reader (Bio-Rad). PCR) analysis Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Total RNA was extracted from the tissues and cells by Trizol reagent (Invitrogen-Thermo Fisher Scientic, Waltham, MA, 2.7 Cell migration and invasion assays USA). Then the obtained RNA was used to synthesize cDNA Cell invasive and migratory abilities were assayed using using a cDNA Reverse Transcription Kit (Fermentas-Thermo transwell assays with 8 mm transwell plates (Corning Inc., Fisher Scientic, USA) following the manufacturer's protocol. Corning, NY, USA). Cells (100 ml) suspended in DMEM at The mRNA levels of TRIM22 were quantied using the SYBR® adensityof1 105 cells per ml were added to the upper Green PCR Master Mix (Thermo) on an ABI-7300 sequence chambers coated with matrigel (for invasion assay) or without detection system (Applied Biosystems, Foster, CA, USA). The matrigel (for migration assay). And DMEM containing 10% 0

This article is licensed under a primers used are as follows: TRIM22, forward 5 -GCAC GCTC FBS (600 ml) was added to the lower chambers. Aer incuba- 0 0 ATCT CAGA TCTC C-3 and reverse 5 -TTTT GGCT TTTC AATG tion for 24 h, the inserts were removed and xed with 4% 0 0 0 TCCA G-3 ; GAPDH, forward 5 -AATC CCAT CACC ATCT TC-3 methanol for 15 min, followed by staining with 0.1% crystal 0 0 and reverse 5 -AGGC TGTT GTCA TACT TC-3 . GAPDH was used violet for 15 min. Images of ve random elds were captured Open Access Article. Published on 03 September 2018. Downloaded 9/29/2021 1:08:09 AM. as a reference gene. The relative mRNA levels of TRIM22 to under a microscope (Olympus Corporation, Tokyo, Japan). The DDC GAPDH were calculated by the 2 t method. numbers of the cells were counted, and the average numbers were calculated. 2.4 Western blot analysis Total proteins of tissues and cells were prepared using RIPA  buﬀer (Invitrogen). Protein concentrations were determined 2.8 Nude mouse xenogra model using the BCA protein assay kit (Thermo). The proteins (30 mg) The animal experiments were approved by the Institutional were separated by 10% SDS-PAGE and then transferred to Animal Care and Use Committee of Huaihe Hospital of Henan nitrocellulose membranes (Millipore, Billerica, MA, USA) using University (China). The experiments were performed according semi-dry method. Subsequently, the membranes were blocked to the National Institute of Health guidelines. Twelve female 6 for 1 h with 5% non-fat milk at room temperature. Aer that, week-old athymic nude mice (HFKBio, Peking, China) were the membranes were subjected to incubation with primary randomly separated into two groups: control group (n ¼ 6) and antibodies against TRIM22 (Sigma; 1: 1000); E-cadherin, N- TRIM22 silencing group (n ¼ 6). The pLKO.1-shTRIM22 cadherin, vimentin (Thermo; 1:1000); b-catenin, cyclin D1, c- plasmid or pLKO.1 plasmid transfected cells (5 106 cells per Myc or b-actin (Abcam, Cambridge, MA, USA; 1: 800) over- mouse) were subcutaneously inoculated into le right of the night at 4 C. The membranes were then incubated with dorsal midline. The xenogras were allowed to grow for 4 respective horseradishRETRACTED peroxidase (HPR)-conjugated secondary weeks. The tumor sizes (mm) were measured every seven days antibodies (Sigma; 1: 3000) at 37 C for 1 h. Finally, the signals using digital caliper. The tumor volumes (v,mm3) were calcu- were detected using enhanced chemiluminescence (ECL) lated using the following formula: length (mm) width (mm) reagents (Thermo). The intensity of bands was analyzed using width (mm)/2. Aer 28 days, all mice were sacriced with Quantity One Soware (Bio-Rad Laboratories, Hercules, CA, general anesthesia, and the tumors were removed and USA). weighted.

This journal is © The Royal Society of Chemistry 2018 RSC Adv.,2018,8,30894–30901 | 30895 View Article Online RSC Advances Paper Creative Commons Attribution-NonCommercial 3.0 Unported Licence. This article is licensed under a Open Access Article. Published on 03 September 2018. Downloaded 9/29/2021 1:08:09 AM.

Fig. 1 TRIM22 was highly expressed in glioma tissues and cell lines. (A and B) The protein and mRNA levels of TRIM22 in glioma tissues and normal brain tissues. *p < 0.05 vs. normal brain tissues. (C and D) The protein and mRNA levels of TRIM22 in normal microglia cell line (HEB) and glioma cell lines (A172, LN229, U118, U251, and U87). n ¼ 3. *p < 0.05 vs. HEB cells.

2.9 Statistical analysis 3. Results All data were expressed as the means SD, and analyzed by 3.1 TRIM22 was highly expressed in glioma tissues and cell SPSS 21.0 (SPSS Inc.,RETRACTED Chicago, IL, USA). The diﬀerences among lines groups were analyzed by ANOVA or unpaired Student's t-test. Diﬀerences were considered statistically signicant when p Compared with the TRIM22 expression levels observed in value < 0.05. normal brain tissues, the protein and mRNA levels of TRIM22 were found to be signicant abundant in the glioma tissues

30896 | RSC Adv.,2018,8,30894–30901 This journal is © The Royal Society of Chemistry 2018 View Article Online Paper RSC Advances Creative Commons Attribution-NonCommercial 3.0 Unported Licence. This article is licensed under a Open Access Article. Published on 03 September 2018. Downloaded 9/29/2021 1:08:09 AM.

Fig. 3 Silencing of TRIM22 inhibited the migration and invasion of U87 cells. Transwell assays were performed to evaluate the migration (A) and invasion (B) of U118 cells with 8 mm transwell plates. The migration and invasion were reﬂected by the numbers of transferred cells. n ¼ 3. *p < 0.05 vs. pLKO.1 plasmid transfected cells.

Fig. 2 Silencing of TRIM22 inhibited the cell proliferation of U87 cells. Because the highest expression level of TRIM22 was detected in U118 cells were transfected with pLKO.1-shTRIM22 plasmid or pLKO.1 U87 cell line, thus, the U87 cells were applied in the following plasmid. The expressions of TRIM22 in the transfected cells were assessed by RT-PCR (A) and western blotting (B). (C) The cell prolif- studies. eration was measured using CCK-8 assay. n ¼ 3. *p < 0.05 vs. pLKO.1 plasmid transfected cells. RETRACTED3.2 Silencing of TRIM22 inhibited the proliferation of (Fig. 1A and B). Furthermore, we detected the TRIM22 expres- glioma cells sion levels in normal microglia cell line HEB, and glioma cell The expressions of TRIM22 in the transfected cells were lines including A172, LN229, U251, and U87. As shown in assessed. The RT-PCR and western blotting showed that Fig. 1C and D, the protein and mRNA levels of TRIM22 were TRIM22 expression was signicantly reduced in the cells markedly higher in glioma cell lines than that of in HEB cells. transfected with pLKO.1-shTRIM22 plasmid in both mRNA

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Fig. 4 Silencing of TRIM22 inhibited the EMT process in U87 cells. (A) The biomarkers of EMT including E-cadherin, N-cadherin, vimentin

This article is licensed under a were measured by western blot to evaluate the process of EMT. (B) Quantiﬁcation analysis was performed using Gel-Pro Analyzer version 4.0 software. n ¼ 3. *p < 0.05 vs. pLKO.1 plasmid transfected cells.

Open Access Article. Published on 03 September 2018. Downloaded 9/29/2021 1:08:09 AM. and protein levels, as comparedtothecellstransfectedwith pLKO.1 plasmid (Fig. 2A and B). Next, we examined the cell proliferation using CCK-8 assay. A trend towards a signicant decrease in cell proliferation was detected in cells transfected with pLKO.1-shTRIM22 plasmid when compared with the cells Fig. 5 Silencing of TRIM22 attenuated xenograft tumor growth in transfected with pLKO.1 plasmid (Fig. 2C). nude mice. Xenograft model was established in nude mice to evaluate the eﬀect of TRIM22 silencing on tumor growth in vivo. The tumor 3.3 Silencing of TRIM22 inhibited the migration and sizes (mm) were measured every 7 days from the tenth day post injection, and the tumor volumes (v,mm3) were calculated using the invasion of glioma cells following formula: length (mm) width (mm) width (mm)/2. The Transwell assays were performed to evaluate the migration and tumors were removed and weighted at the last period of the experiments. (A) Tumor volume in each group. (B) Growth rate of xenograft invasion of glioma cells. Fig. 3A showed that the migration was n ¼ *p  tumors in each group. (C) Tumor weight in each group. 6. < markedly decreased a er pLKO.1-shTRIM22 transfection. 0.05 vs. control group (mice injected with pLKO.1 plasmid transfected Fig. 3B showed that pLKO.1-shTRIM22 transfection also led to cells). a reduction in invasion ability of U87 cells. The results indicated that silencing of TRIM22 dramatically inhibited migration and invasion of U87 cells. biomarkers of EMT including E-cadherin, N-cadherin and RETRACTEDvimentin were measured by western blot. As shown in Fig. 4, there were obvious higher expressions of E-cadherin as well as 3.4 Silencing of TRIM22 inhibited the epithelial– lower expressions of N-cadherin and vimentin in pLKO.1- mesenchymal transition (EMT) in glioma cells shTRIM22 transfected cells than those in pLKO.1 transfected EMT is a critical process that occurs in the initiation of cells. metastasis and progression of malignant tumors.14 The

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Fig. 6 Silencing of TRIM22 inhibited the activation of Wnt/b-catenin pathway in U87 cells. (A) The expressions of b-catenin, cyclin D1, and c-Myc were analyzed by western blot. To further investigate the role of Wnt/b-catenin pathway in the effect of TRIM22, the cells were treated with LiCl, which is an activator of Wnt/b-catenin pathway. (B) Quantification analysis of b-catenin, cyclin D1 and c-Myc. (C) Effect of LiCl on cell proliferation. (D) Effect of LiCl on cell migration. (E) Effect of LiCl on cell invasion. n ¼ 3. *p < 0.05 vs. cells transfected with pLKO.1, #p < 0.05 vs. cells transfected with pLKO.1-shTRIM22.

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3.5 Silencing of TRIM22 attenuated xenogra tumor growth associated with proliferation and differentiation of leukemic U- in nude mice 937 cells, indicating that TRIM22 may function as a tumor suppressor.22 However, Li et al.11 reported that TRIM22 knock- Next, we established xenogra model to evaluate the effect of down inhibits cell proliferation, and induces cell cycle arrest TRIM22 silencing on tumor growth in vivo. As illustrated in and cell apoptosis of chronic myeloid leukemia through regu- Fig. 5A, the subcutaneous tumors in control group were lation of PI3K/Akt/mTOR pathway. In addition, TRIM22 was dramatically larger than that in TRIM22 silencing group. Fig. 5B found to be up-regulated in 55.6% cases of non-small cell lung revealed that the growth rate of xenogra tumors was obviously 12 reduced in the TRIM22 silencing group compared with control cancer (NSCLC) tissues. TRIM22 overexpression is correlated group. Furthermore, compared with control group, a decrease with advanced TNM stage, positive nodal metastasis and poor prognosis in patients with NSCLC, and induces proliferation, in tumor weight was observed in TRIM22 silencing group colony formation, invasion and EMT in A549 cells.12 These (Fig. 5C). ndings suggest that TRIM22 serves as an oncogene in NSCLC. Consequently, the role of TRIM22 in cancer is controversial. 3.6 Silencing of TRIM22 inhibited the activation of Wnt/b- These dual roles of TRIM22 could attribute to organ-specic catenin pathway in glioma cells actions and different cellular contexts. Consistent with the Wnt/b-catenin pathway has been demonstrated to play an data of TRIM22 in NSCLC, the present study showed that important role in caner development.15 To investigate whether TRIM22 was up-regulated in glioma tissues and cell lines. the Wnt/b-catenin pathway was involved in the effect of TRIM22 TRIM22 knockdown inhibited cell proliferation, migration, in glioma cells, the expressions of b-catenin, cyclin D1, and c- invasion, and EMT in U118 cells, as well as attenuated tumor Myc were analyzed by western blot. As indicated in Fig. 6A, growth in vivo. Our results suggested that TRIM22 might func- shTRIM22 resulted in signicant decrease in expressions of b- tion as an oncogene in glioma. Therefore, it is important to catenin, cyclin D1, and c-Myc in U87 cells. Additionally, we developing inhibitory agents of TRIM22 for their use as thera- applied the LiCl for the activation of Wnt/b-catenin pathway.

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. peutic tools for glioma treatment. Fig. 6C–E showed that LiCl attenuated the inhibitory eﬀects of Wnt signaling is an important intracellular signal trans- shTRIM22 on cell proliferation, migration, and invasion in U87 duction pathway that is implicated in various cellular functions.24 cells. Wnt signaling is classied as canonical (b-catenin dependent) and the non-canonical (b-catenin independent) pathway.24 In 4. Discussion canonical pathway, Wnt signaling can be activated by binding of Wnt proteins to surface receptors, which leads to the inhibition Despite signicant advances are achieved in diagnostics and of phosphorylation and degradation of b-catenin. The stabilized therapeutics of glioma over the past decades, the therapies for b-catenin then translocates into the nucleus and regulates the

This article is licensed under a patients with high-grade glioma are inadequate. In recent transcription of target genes such as c-Myc and cyclin D1.24 It has decades, targeted therapy, a form of molecular medicine, has recently been proven that Wnt/b-catenin pathway exerts onco- attracted many attentions for the treatment of cancers with genic activities in regulating proliferation, apoptosis, invasion more effective and less harmful properties.16 TRIM proteins are and EMT in glioma cells.25 Therefore, inhibition of the Wnt/b- Open Access Article. Published on 03 September 2018. Downloaded 9/29/2021 1:08:09 AM. a large family that consists of more than 80 members, and most catenin pathway activation might be a new therapeutic approach of the members have E3 ubiquitin ligase activity.17 Since the for glioma. In the present study, our results showed that silencing ubiquitin system is responsible for degradation of target of TRIM22 inhibited the activation of Wnt/b-catenin pathway. In protein and plays crucial roles in multiple cellular processes, addition, LiCl treatment attenuated the effects of TRIM22 on U87 therefore, TRIM proteins have various functions.18 Accumu- cells. These ndings suggested that TRIM22 executed its effects, lating studies have shown that TRIM proteins have unique and at least in part, through regulating Wnt/b-catenin pathway. important roles in several dysregulation diseases such as Several other pathways are known to contribute to glioma cell immunological diseases, developmental disorders, and invasiveness and motility, such as PI3K/AKT, JAK/STAT3 and NF- cancers.19 Recently, pathological analyses prove that aberrant kB signaling pathway,26–28 thus, more studies are needed to clarify expressions of TRIM proteins are intensely correlated with the the role of TRIM22 in these pathways. malignancy of cancers and prognosis.19 To date, many TRIM proteins have been shown to play critical roles in EMT, trans- 5. Conclusion formation, and metastasis, which are considered as the most important factors in the cancers development.6,9 In summary, the present study demonstrated that TRIM22 was TRIM22, also known as Staf50, has been identied as an signicantly up-regulated in glioma tissues and cell lines. In interferon (IFN)-inducible protein.20 It was reported that vitro studies demonstrated that TRIM22 knockdown inhibited TRIM22 could suppressRETRACTED hepatitis C virus replication in Con1b cell proliferation, migration, invasion, and EMT in U118 cells. and Huh7.5.1 cells.21 TRIM22 has been found to be a direct Nude mouse xenogra assay denoted that TRIM22 silencing target gene p53, which is a tumor suppressor gene that medi- inhibited tumor growth in vivo. Furthermore, TRIM22 executed ates cell cycle arrest and apoptosis.22,23 TRIM22 is up-regulated its effects through regulating the Wnt/b-catenin pathway. Our in response to p53, and inhibits clonogenic growth of results suggested that TRIM22 might function as an oncogene leukemic U-937 cells.22 TRIM22 has been proven to be in glioma.

30900 | RSC Adv.,2018,8,30894–30901 This journal is © The Royal Society of Chemistry 2018 View Article Online Paper RSC Advances

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