A Chopard

A Kumar

C Kubrak

Jun-Feng Hu

M Bossola

MJ Tisdale

N Gullett

PK Paul

S Dodson

T Onodera

VE Baracos

WJ Evans

Xiao-Dong Xu

Yuan-Shui Sun

Zai-Yuan Ye

Zhen-Yuan Qian

Journal of Experimental & Clinical Cancer Research

Crossref

Sun et al. Journal of Experimental & Clinical Cancer Research 2012, 31:81http://www.jeccr.com/content/31/1/81RESEARCH Open AccessExpression of TRAF6 and ubiquitin mRNA inskeletal muscle of gastric cancer patientsYuan-Shui Sun*, Zai-Yuan Ye*, Zhen-Yuan Qian, Xiao-Dong Xu and Jun-Feng HuAbstractObjective: To investigate the prognostic significance of tumor necrosis factor receptor (TNFR),-associated factor 6(TRAF6),-and ubiquitin in gastric cancer patients.Methods: Biopsies of the rectus abdominis muscle were obtained intra operatively from 102 gastric cancer patientsand 29 subjects undergoing surgery for benign abdominal diseases, and muscle TRAF6 and ubiquitin mRNAexpression and proteasome proteolytic activities were assessed.Results: TRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles. TRAF6was upregulated in 67.65% (69/102) muscle of gastric cancer. Over expression of TRAF6 in muscles of gastric cancerwere associated with TNM stage, level of serum albumin and percent of weight loss. Ubiquitin was significantlyupregulated in muscle of gastric cancer compared with the control muscles. Ubiquitin was upregulated in 58.82%(60/102) muscles of gastric cancer. Over expression of ubiquitin in muscles of gastric cancer were associated withTNM (Tumor-Node-Metastasis) stage and weight loss. There was significant relation between TRAF6 and ubiquitinexpression.Conclusions: We found a positive correlation between TRAF6 and ubiquitin expression, suggesting that TRAF6 mayup regulates ubiquitin activity in cancer cachexia. While more investigations are required to understand itsmechanisms of TRAF6 and ubiquitin in skeletal muscle. Correct the catabolic-anabolic imbalance is essential for theeffective treatment of cancer cachexia.Keyword: Gastric cancer, TRAF6, Ubiquitin, Mrna, Skeletal muscle, CachexiaIntroductionCancer cachexia is a complex metabolic condition charac-terized by loss of skeletal muscle. Common clinical mani-festations include muscle wasting, anemia, reduced caloricintake, and altered immune function, which contribute toincreased disability, fatigue, diminished quality of life, andreduced survival [1-3]. Many patients with cancer presentwith weight loss at diagnosis, and much of this weight losscan be attributed to muscle wasting. Cancer cachexia hasbeen viewed as an end-of-life condition in patients withadvanced or incurable malignancies that was managed pri-marily through palliative approaches. However, cachexiaand associated skeletal muscle loss may be present early inthe progression of cancer, indicating the importance ofearlier diagnosis and treatment.* Correspondence: sunshui818@126.com; zaiyuanye@163.comDepartments of Gastrointestinal Surgery, Zhejiang Provincial People’sHospital, Hangzhou 310014, PR China© 2012 Sun et al.; licensee BioMed Central LtdCommons Attribution License (http://creativecreproduction in any medium, provided the orThe prevalence of cancer cachexia varies depending onthe type of malignancy, with the greatest frequency ofweight loss (50%–85% of patients) observed in gastrointes-tinal, pancreatic, lung, and colorectal cancers at diagnosisand before initiation of chemotherapy [4]. One commonmechanism associated with skeletal muscle proteindegradation in cancer cachexia is the activation of the ad-enosine triphosphate-dependent ubiquitin-proteasomeproteolytic path way [5,6]. This system plays a major rolein muscle wasting and, more specifically, in the breakdownof myofibrillar proteins. Certainly, the mechanisms ofmuscle wasting in cancer cachexia are complex. They in-volve multiple host and tumor factors, decreased levels oftestosterone and insulin-like growth factor-1 (IGF-1), anddecreased food intake, contributing to both antianabolicand procatabolic processes [7,8]. The study demonstratethat the expression level of tumor necrosis factor (α) re-ceptor adaptor protein 6 (TRAF6), a protein involved in. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.Table 1 Summary of characteristics of gastric cancer patients and controlControls (n = 29) Gastric cancer (n = 102) t/χ2 P ValueAge, y 61.88 ± 6.49 62.13 ± 6.54 0.053 0.959Sex (M:F) 21:8 72:30 0.037 0.848Weight loss 65.50 ± 4.84 57.38 ± 6.28 2.899 0.012BMI 24.13 ± 1.81 21.00 ± 1.31 3.96 0.001Serum albumin, g/L 41.38 ± 6.09 33.75 ± 3.11 3.15 0.007PNI 45.25 ± 3.62 37.18 ± 3.74 5.26 0.0001Table 2 The expression of TRAF6 in muscle of cancerpatientslow high χ2 P ValuePercent weight loss 12.9 0.001≥10 9 45<10 24 24Serum albumin,g/L 20.05 0.001≥35 24 18<35 9 51TNM stage 13.33 0.001I-II 21 18III-IV 12 51Sun et al. Journal of Experimental & Clinical Cancer Research 2012, 31:81 Page 2 of 5http://www.jeccr.com/content/31/1/81receptor-mediated activation of several signaling pathways,is enhanced in skeletal muscle during atrophy [9,10].Here, we analyzed the prognostic significance of tumornecrosis factor receptor (TNFR)-associated factor 6(TRAF6), and ubiquitin in gastric cancer patients. Thecurrent study were to examine the expression of TRAF6and ubiquitin in skeletal muscle specimens of patientswith gastric cancer, to explore the possible correlationamong TRAF6, ubiquitin mRNA expression and cachexia.MethodsPatients and tissue samplesSkeletal muscle tissues were collected from one hundredand two patients with gastric cancer (median age 61.0y,range 42–88y; 24 male, 10 female) from the Departmentof Surgery, Zhejiang Provincial People’s Hospital fromJanuary 2008 to January 2011. Patients’ characteristicsare showed in Table 1. Diagnosis of gastric cancer wasperformed by endoscopic biopsy. Twenty-nine patientsundergoing surgery for benign abdominal diseasesserved as a control group, there were 12 cholelithiasis, 9inguinal hernia, 8 hemangioma of liver. Gastric cancerpatients and controls were similar in terms of age andsex distribution. Nevertheless, gastric cancer patientsshowed a significantly lower body mass index, serum al-bumin levels and prognostic nutritional index. Exclusioncriteria for both groups were considered: acute orchronic renal failure, liver failure, diabetes, metabolicacidosis, sepsis, AIDS, inflammatory bowel disease, auto-immune disorders, chronic heart failure, and hyperthy-roidism. The study was approved by our hospital ethicscommittees. Written informed consent for the studyprocedures was obtained from the patients.Nutritional assessmentThe nutritional assessment included anthropometric[height, actual body weight, %WL, body mass index(BMI), usual body weight], immunological (totallymphocyte count), and biochemical (serum albumin)indexes. Routine blood test was determined usingcompletely automatic blood cell count analyzer (Beck-man-Coulter -MAXM, American). Liver function wasdetermined using Completely automatic biochemistryanalyzer (Beckman-Coulter SYNCHRON LX 20,American) (Table 1). The PNI(prognostic nutritionalindex) was calculated as follows: PNI = 10 × serum albu-min(g/100 ml) + 0.005 × total lymphocyte count/mm3 ofperipheral blood [11].Muscle biopsyA biopsy specimen was obtained from the rectus abdom-inis muscle during the initial phase of the operation. Theanterior sheet of the rectus abdominis muscle wasopened with scissors after skin incision and dissectionthrough the subcutaneous fat, and a muscle biopsy speci-men weighing about 1.0 g was obtained. The biopsy spe-cimen was divided into two portions that wereimmediately frozen in liquid nitrogen and then stored at−80°C until analysis. No complications occurred fromthe biopsy procedure.Real-time quantitative RT-PCRTotal RNA from rectus abdominis muscle was extractedby TRIzol reagent and cDNAs were reverse-transcribed byRevert Aid TM reverse transcriptase. Real-time PCR wascarried out using the ABI PRISM 7700 Sequence Detec-tion System (Applied Bio systems) at 50°C for 2 min, 95°Cfor 10 min, followed by 50 cycles at 95°C for 15 s, and at60°C for 1 min. The primers for GAPDH (224 bp) were 5'-TGAAGGTCGGAGTCAACGG-3' (sense) and 5'- CTGGAAGATGGTGATGGGATT-3' (antisense). The primersfor TRAF6 (134 bp) were 5'-GCCTGGGTGACAGAGTGC-3' (sense) and 5'-AATGACTACTTATGGCTCCTTTFigure 1 The level of TRAF6 protein in muscle of cancer patients and control.Table 3 The expression of ubiquitin in muscle of cancerpatientslow high χ2 P ValuePercent weight loss 11.78 0.001≥10 15 42<10 27 18Serum albumin,g/L 15.74 0.001≥35 27 15<35 15 45TNM stage 20.52 0.001I-II 27 12III-IV 15 48Sun et al. Journal of Experimental & Clinical Cancer Research 2012, 31:81 Page 3 of 5http://www.jeccr.com/content/31/1/81TC-3' (antisense). The primers for ubiquitin(165 bp) were5'-CCCTGGATGTGATGGTGTC-3' (sense) and 5'-CTCGTTGTCCCTGTTGCTG-3' (antisense). The expression ofGAPDH was used to normalize that of the target genes.Each assay was done in triplicate, the average was calcu-late, and the expression level of TRAF6 and ubiquitin wasexpressed as 2–ΔΔCt, ΔCt=Ct (Target)–Ct (GAPDH).ImmunoblottingCells were lysed in RIPA buffer (150 mM NaCl,10 mM Tris, pH 7.5, 1% NP40, 1% deoxycholate, 0.1%SDS, protease inhibitor cocktail (Roche)). Total pro-teins were fractionated using the NuPAGE 4–12% Bis-Tris gradient gel (Invitrogen) and transferred ontoPVDF membrane. Membranes were blocked with 5%non-fat milk in PBS/Tween-20, and incubated withantibodies against TRAF6 (Santa Cruz), ubiquitin(Santa Cruz), and β-actin (Abcam).Statistical analysisIn order to analyze the relationship among the expres-sion of TRAF6 and ubiquitin and nutritional status ofpatients (percent weight loss, serum albumin), accordingto the literature [12], they were divided into two groups(percent weight loss ≥ 10 and <10, serum albumin ≥ 35and <35). All statistical analyses were performed usingSPSS16.0 software. Measurement data were analyzedusing the Student’s t test, while categorical data werestudied using χ2 or Fisher exact tests. Statistical signifi-cance was set at P < 0.05.ResultsThe expression of TRAF6 in muscle of control and cancerpatientsTumor necrosis factor (α) receptor adaptor protein 6(TRAF6), a protein involved in receptor-mediated activa-tion of several signaling pathways, is enhanced in skel-etal muscle during atrophy. We assessed the expressionof TRAF6 in 29 control muscles and 102 patient mus-cles. TRAF6 was significantly upregulated in muscle ofgastric cancer compared with the control muscles(P < 0.05). TRAF6 was upregulated in 67.65% (69/102)muscle of gastric cancer. Overexpression of TRAF6 inmuscles of gastric cancer were associated with TNMstage, level of serum albumin and percent of weight loss(P > 0.05) (Table 2). We also analyze the expression ofTRAF6 in 8 muscles of control and cancer patients bywestern blotting, the results show the expression ofTRAF6 in muscle of cancer patients were higher thancontrol (Figure 1).The expression of ubiquitin in muscle of control andcancer patientsWe assessed the expression of ubiquitin in 29 controlmuscles and 102 patient muscles. Ubiquitin was signifi-cantly upregulated in muscle of gastric cancer comparedwith the control muscles (P< 0.05). Ubiquitin was upregu-lated in 58.82% (60/102) muscles of gastric cancer. Overexpression of ubiquitin in muscles of gastric cancer wereassociated with TNM stage and weight loss (P> 0.05)(Table 3). In order to analyze the expression of ubiquitinprotein with quantitation, 8 muscle of control and cancerpatients were detectec by western blotting, the study indi-cated the expression of ubiquitin in 5 muscle of cancerpatients were higher than control (Figure 2).Association between expression of TRAF6 and ubiquitinSeventeen cases of gastric cancer had high expression ofboth TRAF6 and ubiquitin, and eight cases of gastriccancer had low expression of both TRAF6 and ubiquitin.Figure 2 The level of ubiquitin protein in muscle of cancer patients and control.Sun et al. Journal of Experimental & Clinical Cancer Research 2012, 31:81 Page 4 of 5http://www.jeccr.com/content/31/1/81There was significant between TRAF6 and ubiquitin ex-pression (χ2 =6.68; P= 0.01) (Table 4, Figure 3).DiscussionIn healthy individuals, skeletal muscle metabolismrequires a balance of anabolic and catabolic processes,resulting in a continuous renewal of muscle proteinswithout a net change in overall muscle mass. However,in cancer cachexia and other chronic illnesses, themuscle wasting were associated with the reduced rate ofprotein synthesis, increased protein degradation, or acombination of both contributes [13]. One commonmechanism associated with skeletal muscle protein deg-radation in cancer cachexia is the activation of the ad-enosine triphosphate-dependent ubiquitin-proteasomeproteolytic path way, this system plays a major role inmuscle wasting [5,6]. The study showed muscle ubiqui-tin mRNA was hyper expressed in gastric cancerpatients compared to controls [14], the ubiquitin-proteasome proteolytic system play important role in thepathogenesis of muscle protein hyper catabolism in can-cer cachexia. To investigate the role of ubiquitin expres-sion in the skeletal muscle of gastric cancer patients. Weassessed the expression of ubiquitin in 29 control mus-cles and 102 patient muscles. Ubiquitin was significantlyupregulated in muscle of gastric cancer compared withthe control muscles. Over expression of ubiquitin inmuscle of gastric cancer were associated with TNMstage and weight loss.Skeletal muscle wasting is a major reason for morbid-ity and mortality in many chronic disease states, disuseconditions and aging. The ubiquitin-proteasome andautophagy-lysosomal systems are the two major proteo-lytic pathways involved in regulation of both physio-logical and pathological muscle wasting. The studyTable 4 Association between expression of TRAF6 andubiquitinClinicalparametersTRAF6high low χ2 Pubiquitin 20.05 0.001high 51(85.0%) 9(15.0%)low 18(42.9%) 24(57.1%)demonstrate that the expression level of tumor necrosisfactor (α) receptor adaptor protein 6 (TRAF6), a proteininvolved in receptor-mediated activation of several sig-naling pathways, is enhanced in skeletal muscle duringatrophy [9,10]. To explore the relation of TRAF6 expres-sion in the skeletal muscle of gastric cancer patients. Weassessed the expression of TRAF6 in 29 control musclesand 102 patient muscles. TRAF6 was significantly upre-gulated in muscle of gastric cancer compared with thecontrol muscles, Overexpression of TRAF6 in muscle ofgastric cancer were associated with TNM stage, the levelof serum albumin and percent of weight loss. The studyshowed overexpression of TRAF6 may play importantrole in gastric cancer cachexia.Paul’s study discover that TRAF6 possesses E3 ubiqui-tin ligase activity causing lysine-63-linked polyubiquiti-nation of target proteins. Muscle-wasting stimuli couldup regulate the expression of TRAF6 and auto-ubiquiti-nation. Muscle-specific depletion of TRAF6 preservesskeletal muscle mass in a mouse model of cancer cach-exia or denervation. Inhibition of TRAF6 also blocks theexpression of the components of the ubiquitin-proteasome system (UPS) and auto phagosome forma-tion in atrophying skeletal muscle [15]. We also exam-ined TRAF6 expression in skeletal muscle with gastricFigure 3 Association between expression of TRAF6 andubiquitin.Sun et al. Journal of Experimental & Clinical Cancer Research 2012, 31:81 Page 5 of 5http://www.jeccr.com/content/31/1/81cancer and its correlation with ubiquitin status. Wefound a positive correlation between TRAF6 and ubiqui-tin expression, suggesting that TRAF6 may up regulatesubiquitin activity in cancer cachexia. While more investi-gations are required to understand its mechanisms ofTRAF6 and ubiquitin in skeletal muscle. Correct thecatabolic-anabolic imbalance is essential for the effectivetreatment of cancer cachexia.Competing interestsThe authors declared that they have no competing interest.Authors' contributionsY-SS and Z-YY design the study, Z-YQ, X-DX, and J-FH carried out the Real-time quantitative RT-PCR and Immunoblotting, Y-SS drafted the manuscript.All authors read and approved the final manuscript.AcknowledgmentsWork was supported by Zhejiang Provincial Department of Science andTechnology Research Foundation (2011C33009).Received: 18 August 2012 Accepted: 24 September 2012Published: 26 September 2012References1. Gullett N, Rossi P, Kucuk O, Johnstone PA: Cancer-induced cachexia: aguide for the oncologist. J Soc Integr Oncol 2009, 7(4):155–169.2. Evans WJ: Skeletal muscle loss: cachexia, sarcopenia, and inactivity. Am JClin Nutr 2010, 91(4):1123S–1127S.3. Evans WJ, Morley JE, Argilés J, et al: Cachexia: a new definition. Clin Nutr2008, 27(6):793–799.4. Dodson S, Baracos VE, Jatoi A, et al: Muscle wasting in cancer cachexia:clinical implications, diagnosis, and emerging treatment strategies. AnnuRev Med 2011, 62:265–279.5. Baracos VE: Cancer-associated cachexia and underlying biologicalmechanisms. Annu Rev Nutr 2006, 26:435–461.6. Tisdale MJ: Mechanisms of cancer cachexia. Physiol Rev 2009, 89(2):381–410.7. Chopard A, Hillock S, Jasmin BJ: Molecular events and signalling pathwaysinvolved in skeletal muscle disuse-induced atrophy and the impact ofcounter measures. J Cell Mol Med 2009, 13(9B):3032–3050.8. Kubrak C, Olson K, Jha N, et al: Nutrition impact symptoms: keydeterminants of reduced dietary intake, weight loss, and reducedfunctional capacity of patients with head and neck cancer beforetreatment. Head Neck 2010, 32(3):290–300.9. Paul PK, Gupta SK, Bhatnagar S, et al: Targeted ablation of TRAF6 inhibitsskeletal muscle wasting in mice. J Cell Biol 2010, 191(7):1395–1411.10. Kumar A, Bhatnagar S, Paul PK: TWEAK and TRAF6 regulate skeletalmuscle atrophy. Curr Opin Clin Nutr Metab Care 2012, 15(3):233–239.11. Onodera T, Goseki N, Kosaki G: Prognostic nutritional index ingastrointestinal surgery of malnourished cancer patients. Nippon GekaGakkai Zasshi 1984, 85:1001.12. Bossola M, Muscaritoli M, Costelli P, Grieco G, Bonelli G, Pacelli F, RossiFanelli F, Doglietto GB, Baccino FM: Increased muscle proteasome activitycorrelates with disease severity in gastric cancer patients. Ann Surg 2003,237(3):384–389.13. Baracos VE: Management of muscle wasting in cancer-associatedcachexia: understanding gained from experimental studies. Cancer 2001,92(6 Suppl):1669–1677.14. Bossola M, Muscaritoli M, Costelli P, et al: Increased muscle proteasomeactivity correlates with disease severity in gastric cancer patients. AnnSurg 2003, 237(3):384–389.15. Paul PK, Kumar A: TRAF6 coordinates the activation of autophagy andubiquitin-proteasome systems in atrophying skeletal muscle. Autophagy2011, 7(5):555–556.doi:10.1186/1756-9966-31-81Cite this article as: Sun et al.: Expression of TRAF6 and ubiquitin mRNAin skeletal muscle of gastric cancer patients. Journal of Experimental &Clinical Cancer Research 2012 31:81.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color ﬁgure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submit

Expression of TRAF6 and ubiquitin mRNA in skeletal muscle of gastric cancer patients

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Expression of TRAF6 and ubiquitin mRNA in skeletal muscle of gastric cancer patients

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