Expression of 4E-BP1 and phospho-4E-BP1 correlates with the prognosis of patients with clear cell renal carcinoma

Cancer Management and Research, Jun 2018

Expression of 4E-BP1 and phospho-4E-BP1 correlates with the prognosis of patients with clear cell renal carcinoma Feng Li,1,2,* Qingshui Wang,3,* Xiaoxue Xiong,3 Chenyi Wang,3 Xiaohua Liu,4 Ziqiang Liao,3 Ke Li,3 Bifeng Xie,5 Yao Lin3 1Department of Pathology, Provincial Clinical Medical College, Fujian Medical University, Fuzhou, Fujian Province, People’s Republic of China; 2Department of Pathology, Fujian Provincial Hospital, Fuzhou, Fujian Province, People’s Republic of China; 3Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian Province, People’s Republic of China; 4Department of Obstetrics, Anxi County Hospital, Anxi, Fujian Province, People’s Republic of China; 5College of Life Sciences, Fujian Normal University, Fuzhou, Fujian Province, People’s Republic of China *These authors contributed equally to this work Background: Eukaryotic translation initiation factor 4E (eIF4E) is a key regulator of protein synthesis. Changes in eIF4E activity disproportionally affect the translation of a subset of oncogenic mRNAs in some cancers. Materials and methods: We have assessed the expression levels of vascular endothelial growth factor C (VEGFC), eIF4E, eIF4E-binding proteins (4E-BPs) and phospho-4E-BP1 in clear cell renal carcinoma (ccRCC; n=101) using immunohistochemistry and analyzed the relevant mRNA levels and survival using online databases. Results: The protein levels of VEGFC, an eIF4E-regulated gene, were upregulated in ccRCC tissues compared with adjacent normal renal tissues, indicating an enhanced eIF4E activity in ccRCC. The expression of eIF4E had no significant changes in ccRCC tissues. However, 4E-BP1 and phospho-4E-BP1 were found to be overexpressed in ccRCC tissues (P<0.05), and the high mRNA and protein levels of 4E-BP1 and phospho-4E-BP1 correlated with an unfavorable clinical outcome in ccRCC patients. Meanwhile, the mRNA expression of PIK3CD and PIK3CG were enhanced in ccRCC. Conclusion: From these results, we could infer that the increase in eIF4E activity may be caused by the increased phospho-4E-BP1 level, which was probably due to the activation of phosphoinositide 3-kinase (PI3K) pathway. Keywords: eIF4E, 4E-BP1, phospho-4E-BP1, VEGFC, PI3K, ccRCC

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Expression of 4E-BP1 and phospho-4E-BP1 correlates with the prognosis of patients with clear cell renal carcinoma

Cancer Management and Research Expression of 4E-BP1 and phospho-4E-BP1 correlates with the prognosis of patients with clear cell renal carcinoma Feng Li 1 2 Qingshui Wang 0 Xiaoxue Xiong 0 Chenyi Wang 0 Xiaohua Liu 4 Ziqiang Liao 0 Ke Li 0 Bifeng Xie 3 Yao Lin 0 0 Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Sciences, Fujian Normal University , Fuzhou, Fujian Province , People's Republic of China 1 Department of Pathology, Fujian Provincial Hospital , Fuzhou, Fujian Province , People's Republic of China 2 Department of Pathology, Provincial Clinical Medical College, Fujian Medical University , Fuzhou, Fujian Province , People's Republic of China 3 College of Life Sciences, Fujian Normal University , Fuzhou, Fujian Province , People's Republic of China 4 Department of Obstetrics, Anxi County Hospital , Anxi, Fujian Province , People's Republic of China 8 1 0 2 - l u J - 2 1 n o 5 0 2 . 7 . 8 6 . 1 5 y b / m o c . s s e r p e v .do l.y ww no //w se : tsp lau th on s rom rpe fded roF PowerdbyTCPDF(ww.tcpdf.org) *These authors contributed equally to this work Background: Eukaryotic translation initiation factor 4E (eIF4E) is a key regulator of protein synthesis. Changes in eIF4E activity disproportionally affect the translation of a subset of oncogenic mRNAs in some cancers. Materials and methods: We have assessed the expression levels of vascular endothelial growth factor C (VEGFC), eIF4E, eIF4E-binding proteins (4E-BPs) and phospho-4E-BP1 in clear cell renal carcinoma (ccRCC; n=101) using immunohistochemistry and analyzed the relevant mRNA levels and survival using online databases. Results: The protein levels of VEGFC, an eIF4E-regulated gene, were upregulated in ccRCC tissues compared with adjacent normal renal tissues, indicating an enhanced eIF4E activity in ccRCC. The expression of eIF4E had no significant changes in ccRCC tissues. However, 4E-BP1 and phospho-4E-BP1 were found to be overexpressed in ccRCC tissues (P<0.05), and the high mRNA and protein levels of 4E-BP1 and phospho-4E-BP1 correlated with an unfavorable clinical outcome in ccRCC patients. Meanwhile, the mRNA expression of PIK3CD and Conclusion: From these results, we could infer that the increase in eIF4E activity may be caused by the increased phospho-4E-BP1 level, which was probably due to the activation of phosphoinositide 3-kinase (PI3K) pathway. eIF4E; 4E-BP1; phospho-4E-BP1; VEGFC; PI3K; ccRCC - open access to scientific and medical research PIK3CG were enhanced in ccRCC. Introduction Kidney cancer is one of the most common tumors in the urinary system. In the past 2 decades, the amount of kidney cancer has been increasing. Kidney cancer consists of multiple types, including transitional cell carcinoma of kidney, renal cell carcinoma (RCC), inverted papilloma and kidney lymphoma. Among the four types of kidney cancer, RCC accounts for the largest proportion. RCC contains multiple pathological categories, including chromophobe RCC, clear cell renal carcinoma (ccRCC) and papillary RCC. Among them, ccRCC accounts for the highest proportion with ~ 75% of cases.1 The translations of mRNAs need to be tightly controlled in cells as the imbalance of translation leads to cancer occurrence and development.2–4 At the transcription elongation step, nuclear-transcribed mRNAs have 5′-cap added. Translation in eukaryotic cells begins with binding between 5′-cap and the eukaryotic translation initiation factor 4F (eIF4F).5,6 eIF4F complex includes eIF4A, eukaryotic translation initiation factor 4E (eIF4E) and eIF4G. eIF4E is able to recognize and bind to 5′-cap of mRNA, allowing the translation initiation.7,8 Changes in eIF4E activity only have a tiny influence on the mRNA translation of house-keeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).9 Most house-keeping mRNAs have short and low complexity 5′-untranslated regions (UTRs). Unlike them, some 5′-cap-dependent mRNAs own long 5′-UTRs with a high complexity, whose protein translation is tightly controlled by eIF4E including the mRNAs of cyclin D3, 2108 phosphoribosyl pyrophosphate synthetase 2 (PRPS2) and l-Ju vascular endothelial growth factor (VEGF).3,10–13 21 Besides the amplification of eIF4E, eIF4E activity is also no5 controlled by eIF4E-binding proteins (4E-BPs) such as 4E-BP1, .270 4E-BP2 and 4E-BP3.14 4E-BP1 is the most common isoform . .86 of 4E-BPs. 4E-BPs and eIF4G compete with eIF4E for the y51 same binding site.15–17 The phosphoinositide 3-kinase (PI3K) /bm pathway can phosphorylate 4E-BPs, causing the separation of .sco 4E-BPs and eIF4E.6 Therefore, the phosphorylated 4E-BP1 is rsep able to promote protein translation. PI3K can phosphorylate ve phosphatidylinositol 3,4 (PIP2) and activate the AKT.18,19 The .do l.y ww no activity of AKT can be inhibited by phosphatase and tensin //w se homolog (PTEN).6,20,21 PI3Ks belong to three distinct categories : ttsph lauon called Class I, II and III.22 PI3K class I consists of multiple rom rpe genes including PIK3CA, PIK3CB, PIK3CD and PIK3CG. s The expression level of eIF4E or 4E-BP1 is deregulated in many cancers such as bladder, ovary and prostate.23–33 In the present study, we focused on the expression and active levels of 4E-BP1 and eIF4E to confirm the prognostic values of these factors in ccRCC. Materials and methods Expression and clinical data obtained from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas (TCPA) databases In the research, ccRCC datasets were extracted from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). Four datasets with >400 specimens, GSE40435,34 GSE46699,35 GSE1564136 and GSE66272,37 were obtained. The probe ID was converted into a gene symbol first. When a gene was mapped to different probes, the genic expression value was calculated by the average expression value. Next, the data were translated into log2 logarithms, and the median normalization was performed using the robust multichip averaging method.38 We compared the normal control vs cancer datasets using the Student’s t-test. A P value of <0.05 was considered as statistically significant. The clinical data and mRNA expression data of eIF4E and 4E-BP1 in ccRCC 1554 were extracted from the TCGA database.39,40 The survival analysis of eIF4E and 4E-BP1 protein expression was directly obtained from the TCPA database (http://www.tcpaportal.org/ tcpa/index.html).41–44 According to the median value of gene or protein expression, samples were divided into low- and high-expression groups. Patients and specimens The research consisted of 202 samples from 101 patients with ccRCC. All the patients had a renal resection at the Fujian Provincial Hospital between January 2016 and June 2017. The standard requirements for patients included in the study were 1) histologically confirmed ccRCC; 2) no history of other malignancy, and 3) no prior neoadjuvant chemotherapy. The study was performed with the approval of the ethics committee of Fujian Provincial Hospital. Written informed consent was given by the patients for their information, and specimens were stored in the hospital database and used for research. Immunohistochemical staining Paraffin blocks that contained sufficient formalin-fixed tumor specimens were serial sectioned at 3 mμ and mounted on silane-coated slides for immunohistochemical staining analysis. Dimethylbenzene rehydrated through 100% ethanol, 100% ethanol, 95% ethanol, 85% ethanol and 75% ethanol were applied to deparaffinize. In all, 0.01 mol/L of sodium citrate buffer (pH 6.0) was used to the progress of antigen retrieval treatment (autoclaved at 121°C, 2 min). Then, 3% H2O2 was applied to block endogenous peroxidase at room temperature for 10 min. The sections were washed in PBS solution subsequently and blocked with 10% goat serum (ZhongShan Biotechnology, Beijing, China) for 30 min and incubated with anti-eIF4E (ab33766, 1:100 dilution, monoclonal; Abcam, Cambridge, MA, USA) or anti-eIF4E-BP1 (ab32024, 1:100 dilution, monoclonal; Abcam) antibody or anti-vascular endothelial growth factor C (VEGFC; ab83905, 1:100 dilution, polyclonal; Abcam) or anti-phospho-4E-BP1 (Thr37/46) (236B4) (2855, 1:200 dilution, monoclonal; Cell Signaling Technology, Boston, MA, USA) at 4°C for 12 h. The sections were washed in PBS solution three times and incubated with HRP-conjugated secondary antibody for 30 min at room temperature. All slides were counterstained with diaminobenzidine (DAB) solution and 20% hematoxylin and dehydrated. The primary antibody diluent was regarded as negative control. Evaluation of immunostaining intensity Immunohistochemical staining tissue sections were reviewed and scored by two independent pathologists. The score was 8 1 0 2 l u J 2 1 n o 5 0 2 . 7 . 8 6 . 1 5 y b / m o c . s s e r p e v .do l.y ww no //w se : tsp lau th on s rom rpe fded roF a o l n w o d h c r a e s e R d n a t n e m e g a n a M r e c n a C calculated according to the proportion of stained tumor cells and intensity of cellular staining. The intensity of cellular staining was scored between 0 and 3: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The proportion of stained tumor cells was scored between 1 and 4: 1, 0–25%; 2, 26–50%; 3, 51–75% and 4, 75–100%. The multiplication of these two variables was calculated as the final score. The staining was divided into five grades according to the final score as follows: 0 score, 0; 1 score, 1–2; 2 score, 3–4; 3 score, 6–8 and 4 score, 9–12. Statistical analyses In the research, the student’s t-test was used to calculate the mRNA expression level in ccRCC tissues and adjacent normal renal tissues by using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Log-rank test was used to calculate the survival analysis by using IBM SPSS version 19.0 (IBM Corporation, Armonk, NY, USA). Multivariate survival analysis was performed by using stepwise Cox proportional hazards regression model. P value of <0.05 was considered as statistically significant. Results Expression levels of eIF4E were not changed in ccRCC In order to explore the expression of eIF4E in ccRCC patients, a total of four related publications were used. Based on these datasets (GSE40435, GSE46699, GSE15641, GSE66272; Table S1), no change in eIF4E expression was observed in the tissues of ccRCC compared with adjacent normal renal tissues (Figure 1A–D). These results suggested that the mRNA of eIF4E expression did not change in ccRCC. Moreover, the expression of eIF4E protein in 101 tissues of ccRCC and their adjacent normal renal tissues was detected by immunohistochemical staining. Representative immunohistochemical-stained tissue sections and the frequency distributions of the immunohistochemical scores are presented (Figure 1E–G). The mean scores of eIF4E proteins in ccRCC and adjacent normal renal tissues were 2.11 and 2.29, respectively (Figure 1H), consistent with the conclusion from mRNA analysis that the level of eIF4E did not change in ccRCC. Protein expression level of VEGFC was upregulated in ccRCC VEGFC, an important downstream target of eIF4E, is regulated by eIF4E activity. In order to further investigate the activity of eIF4E in ccRCC, we set out to evaluate the expression of VEGFC protein in ccRCC. The expression of VEGFC protein in 101 tissues of ccRCC and their adjacent normal renal tissues was detected by immunohistochemical staining. Representative immunohistochemical-stained tissue sections and the frequency distributions of the immunohistochemical scores are presented (Figure 2A–C).The mean scores of GSE40435 ccRCC and adjacent specimens (n=202) NS submit your manuscript | www.dovepress.com Dovepress 1555 C G E 7 4 F I e – lve 5 e li ssnoe r xp 5 vaee ilt eR 4 80 cy 60 n e rque 40 F 20 0 D H 7.5 E I4F7.0 e – lilvsseee66..50 n o r xp litveeeR55..05 a 3 E 4 F I fe 2 o csoe r C H Irge 1 vea A 0 8 1 0 2 l u J 2 1 n o 5 0 2 . 7 . 8 6 . 1 5 y b / m o c . s s e r p e v .do l.y ww no //w se : tsp lau th on s rom rpe fded roF a o l n w o d h c r a e s e R d n a t n e m e g a n a M r e c n a C ccRCC Normal P<0.0001 *** P<0.001 0 1 3 4 2 Score VEGFC proteins in ccRCC and adjacent normal renal tissues were 2.09 and 0.97, respectively(Figure 2D), suggesting that although eIF4E expression levels were unchanged, the activity of eIF4E in ccRCC tissues was enhanced. Expression levels of phospho-4E-BP1 and 4E-BP1 were enhanced in ccRCC In order to investigate how the activity of eIF4E was enhanced in ccRCC, we next investigated the expression and activity of 4E-BP1. To our surprise, the mRNA and protein expression of 4E-BP1 were overexpressed in ccRCC tissues based on four datasets (GSE40435, GSE46699, GSE15641, GSE66272; Figure 3A–D and Table S1) and immunohistochemical staining of 4E-BP1 in 101 ccRCC tissues and their adjacent normal renal tissues. Representative immunohistochemicalstained tissue sections of 4E-BP1 and the frequency distributions of these scores are presented (Figure 3E–G). The mean scores of 4E-BP1 proteins in ccRCC and adjacent normal renal tissues were 3.10 and 0.19, respectively (Figure 3H). 1556 Then, we detected the protein expression of phospho-4E-BP1 in the 101 sample cohort. Representative immunohistochemical-stained tissue sections of phospho-4E-BP1 and the frequency distributions of these scores are presented (Figure 4A–C). The mean scores of phospho-4E-BP1 in ccRCC and adjacent normal renal tissues were 2.52 and 0.18, respectively (Figure 4D), suggesting that both 4E-BP1 and phospho-4E-BP1 levels were much higher in ccRCC tissues. Expression levels of PIK3CD and PIK3CG were upregulated in ccRCC 4EBPs could be phosphorylated due to the activation of PI3K pathway. Phosphorylated 4EBPs enable the assembly of eIF4F by dislodging 4EBPs from eIF4E. In order to further investigate the mechanisms underlying eIF4E activation in ccRCC, the expression levels of PIK3CA, PIK3CB, PIK3CD, PIK3CG and PTEN were examined (Table S2). The mRNAs of PIK3CD and PIK3CG were upregulated in ccRCC tissues based on the TCGA database (Figure 5). GSE40435 ccRCC and normal specimens (n=202) GSE46699 ccRCC and normal specimens (n=130) 1 4 P B E I4F 3 e f o re 2 o c s C IH 1 e g a r ve 0 A *** P<0.001 A 1P8.5 B E I4F8.0 e – llvee 7.5 n o isse 7.0 r xp litvee 6.5 a eR6.0 110 P B E I4F 9 e – lvee 8 l n o isse 7 r xp litveeeR 56 a Normal ccRCC P<0.001 C 110 P B E I4F 9 e – lvee 8 l n o isse 7 r xp iltveeeR 56 a C 100 80 y cen 60 u q reF 40 20 0 B D 3 f o e 1 r P2 sco -EB IHC -o4 h e p a o1 g s rve ph A 0 fded roF a o l n w o d h c r a e s e R d n a t n e m e g a n a M r e c n a C submit your manuscript | www.dovepress.com Dovepress 1557 fded roF a o l n w o d h c r a e s e R d n a t n e m e g a n a M r e c n a C P<0.01 P<0.01 E 40 N E T P le30 v e l n o is20 s e r p x ee10 v it a l e R 0 NS P<0.05 Normal specimens (n=72) ccRCC specimens (n=538) Notes: The mRNA expression levels of PIK3CA (A), PIK3CB (B), PIK3CD (C), PIK3CG (D) and PTEN (E) in ccRCC were analyzed using the TCGA database. **P<0.01; ***P<0.001. Expression levels of 4E-BP1 and phospho4E-BP1 were strongly associated with ccRCC survival In the TCGA ccRCC cohort, we observed that patients with advanced stage and grade were at significantly increased risk of death. Patients with age >60 years, laterality=left, pharmaceutical therapy and radiation therapy also have a high risk of death in ccRCC (Table 1). 1558 associated with the clinical outcomes of ccRCC patients (Table S3), after adjusting for tumor location, grade, stage and patients’ age, gender and race (Table 2). The survival analyses of 4E-BP1, eIF4E and phospho-4E-BP1 protein were obtained from the TCPA database. With the cutoff value Variables set at the median, we found that the protein expression of 4E-BP1 (Figure 6C) and phospho-4E-BP1 (Figure 6E), but not eIF4E (Figure 6D), was also associated with the clinical outcomes of 445 ccRCC patients. Discussion In this study, the expression and prognostic relevance of eIF4E, 4E-BP1 and phospho-4E-BP1 in ccRCC were examined. Our analysis included four datasets with >400 specimens from the GEO database, one web tool for interactively exploring survival correlations performed by the TCPA database and one ccRCC cohort (n=101) with immunohistochemistry (IHC) analyses. eIF4E is highly elevated and deregulated in many cancers such as lung and breast.45,46 Surprisingly, no association was found between eIF4E expression and survival (Figure 6B and D), whereas the protein level of VEGFC (Figure 2) was upregulated in ccRCC tissues compared with adjacent normal renal tissues. The protein translation of VEGFC mRNA was regulated by eIF4E. Our results indicated that eIF4E activity is upregulated in ccRCC. In a simple model to calculate the regulatory effect toward eIF4E activity, one phospho-4E-BP1 protein could eliminate the inhibitory effect of two 4E-BP1 proteins on eIF4E activity,47,48 suggesting that phospho-4E-BP1 plays a greater role than unphosphorylated 4E-BP1 in the regulation of eIF4E activity. In our study, high mRNA and protein expression level of 4E-BP1 were observed in ccRCC tissues compared with adjacent normal renal tissues (3.10 vs 0.19, tumor vs normal; Figure 3). At the same time, a strong phosphorylation status of the 4E-BP1 in ccRCC was also observed (2.52 vs 0.18, tumor vs normal; Figure 4). Therefore, the increased eIF4E activity observed in our study may be due to an enhanced proportion of phospho4E-BP1 in ccRCC compared with adjacent normal renal tissues (Figure 7). As we know, eIF4E activity can be controlled by the phospho-4E-BP1 and the expression levels of eIF4E, and the increased eIF4E activity drives cancer progression. Therefore, phosphorylated 4E-BP1 and eIF4E overexpression synchronously drive disease progression in ccRCC.49 The simultaneous increase in 4E-BP1 protein expression and phosphorylation may be due to the malfunction of the feedback loop between 4E-BP1 and eIF4E. When eIF4E is overactivated, the enhanced 4E-BP1 is supposed to suppress its activity. However, due to the upregulation of the PI3K pathway, i.e., overexpression of PIK3CD and PIK3CG mRNA (Figure 5), most of newly synthesized 4E-BP1 proteins were phosphorylated and lost the ability to suppress eIF4E activity. Thus, the protein expression of 4E-BP1 cannot be restored and remained high in ccRCC; albeit most of the 4E-BP1 protein were phosphorylated and inactive. Collectively, our data displayed enhanced activity of eIF4E in ccRCC, which is probably due to the increased ratio of phospho-4E-BP1 against 4E-BP1 (Figure 7). In addition, there was a significantly unfavorable influence of 4E-BP1 and phospho-4E-BP1 expression on the survival (Figure 6). These results suggested that 4E-BP1, eIF4E and phospho4E-BP1 are important determinants of diagnosis and disease progression in ccRCC. fded roF a o l n w o d h c r a e s e R d n a t n e m e g a n a M r e c n a C PowerdbyTCPDF(w w.tcpdf.org) A 8 1 0 2 l u J 2 1 n o 5 0 2 . 7 . 8 6 . 1 5 y b / m o c . s s e r p e v .do l.y ww no fded roF a o l n w o d h c r a e s e R d n a t n e m e g a n a M r e c n a C PowerdbyTCPDF(w w.tcpdf.org) Covariates Gender (female vs male) Age (≤60 vs >60 years) Race (white vs other) Location (left vs right) Tumor stage (I/II vs III/IV) Histologic grade (G1/G2 vs G3/G4) eIF4E expression (low vs high) 4E-BP1 expression (low vs high) Coefficient Standard error HR Acknowledgment Author contributions This work was funded by the Youth Scientific Research Feng Li and Qingshui Wang performed the experiments, Project of Fujian Provincial Heath Department, China (2015wrote the paper and prepared figures and/or tables. 1-2); the International S&T Cooperation Program of China (2016YFE0121900) and the Scientific Research Innovation Team Construction Program of Fujian Normal University (IRTL1702). Xiaoxue Xiong and Chenyi Wang performed the experiments and analyzed the data. Xiaohua Liu, Ziqiang Liao, and Bifeng Xie contributed to reagents/analysis tools. submit your manuscript | www.dovepress.com Dovepress 1561 8 1 0 2 l u J 2 1 n o 5 0 2 . 7 . 8 6 . 1 5 y b / m o c . s s e r p e v .do l.y ww no //w se : tsp lau th on s rom rpe fded roF a o l n w o d h c r a e s e R d n a t n e m e g a n a M r e c n a C PowerdbyTCPDF(w w.tcpdf.org) Yao Lin conceived and designed the experiments, wrote the paper and prepared figures and/or tables. All authors contributed toward data analysis, drafting and revising the paper and agree to be accountable for all aspects of the work. Disclosure The authors report no conflicts of interest in this work. submit your manuscript | www.dovepress.com Dovepress 8 1 0 2 l u J 2 1 n o 5 0 2 . 7 . 8 6 . 1 5 y b / m o c . s s e r p e v .do l.y ww no //w se : tsp lau th on s rom rpe a o l n w o d h c r a e s e R d n a t n e m e g a n a M r e c n a C Publish your work in this journal Cancer Management and Research is an international, peer-reviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes, enhanced survival and quality of life for the cancer patient. The manuscript management system is completely online and includes 1. Nickerson ML , Jaeger E , Shi Y , et al. Improved identification of von Hippel-Lindau gene alterations in clear cell renal tumors . Clin Cancer Res . 2008 ; 14 ( 15 ): 4726 - 4734 . 2. Ruggero D. Translational control in cancer etiology . Cold Spring Harb Perspect Biol . 2013 ; 5 ( 2 ): 152 - 158 . 3. 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Feng Li, Qingshui Wang, Xiaoxue Xiong, Chenyi Wang, Xiaohua Liu, Ziqiang Liao, Ke Li, Bifeng Xie, Yao Lin. Expression of 4E-BP1 and phospho-4E-BP1 correlates with the prognosis of patients with clear cell renal carcinoma, Cancer Management and Research, 2018, 1553-1563, DOI: 10.2147/CMAR.S158547