Expression of TRAF6 and ubiquitin mRNA in skeletal muscle of gastric cancer patients
© Sun et al.; licensee BioMed Central Ltd. 2012
Received: 18 August 2012
Accepted: 24 September 2012
Published: 26 September 2012
To investigate the prognostic significance of tumor necrosis factor receptor (TNFR),-associated factor 6 (TRAF6),-and ubiquitin in gastric cancer patients.
Biopsies of the rectus abdominis muscle were obtained intra operatively from 102 gastric cancer patients and 29 subjects undergoing surgery for benign abdominal diseases, and muscle TRAF6 and ubiquitin mRNA expression and proteasome proteolytic activities were assessed.
TRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles. TRAF6 was upregulated in 67.65% (69/102) muscle of gastric cancer. Over expression of TRAF6 in muscles of gastric cancer were associated with TNM stage, level of serum albumin and percent of weight loss. Ubiquitin was significantly upregulated 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 with TNM (Tumor-Node-Metastasis) stage and weight loss. There was significant relation between TRAF6 and ubiquitin expression.
We found a positive correlation between TRAF6 and ubiquitin expression, suggesting that TRAF6 may up regulates ubiquitin activity in cancer cachexia. While more investigations are required to understand its mechanisms of TRAF6 and ubiquitin in skeletal muscle. Correct the catabolic-anabolic imbalance is essential for the effective treatment of cancer cachexia.
KeywordsGastric cancer TRAF6 Ubiquitin Mrna Skeletal muscle Cachexia
Cancer cachexia is a complex metabolic condition characterized by loss of skeletal muscle. Common clinical manifestations include muscle wasting, anemia, reduced caloric intake, and altered immune function, which contribute to increased disability, fatigue, diminished quality of life, and reduced survival[1–3]. Many patients with cancer present with weight loss at diagnosis, and much of this weight loss can be attributed to muscle wasting. Cancer cachexia has been viewed as an end-of-life condition in patients with advanced or incurable malignancies that was managed primarily through palliative approaches. However, cachexia and associated skeletal muscle loss may be present early in the progression of cancer, indicating the importance of earlier diagnosis and treatment.
The prevalence of cancer cachexia varies depending on the type of malignancy, with the greatest frequency of weight loss (50%–85% of patients) observed in gastrointestinal, pancreatic, lung, and colorectal cancers at diagnosis and before initiation of chemotherapy. One common mechanism associated with skeletal muscle protein degradation in cancer cachexia is the activation of the adenosine triphosphate-dependent ubiquitin-proteasome proteolytic path way[5, 6]. This system plays a major role in muscle wasting and, more specifically, in the breakdown of myofibrillar proteins. Certainly, the mechanisms of muscle wasting in cancer cachexia are complex. They involve multiple host and tumor factors, decreased levels of testosterone and insulin-like growth factor-1 (IGF-1), and decreased food intake, contributing to both antianabolic and procatabolic processes[7, 8]. The study demonstrate that the expression level of tumor necrosis factor (α) receptor adaptor protein 6 (TRAF6), a protein involved in receptor-mediated activation of several signaling pathways, is enhanced in skeletal muscle during atrophy[9, 10].
Here, we analyzed the prognostic significance of tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), and ubiquitin in gastric cancer patients. The current study were to examine the expression of TRAF6 and ubiquitin in skeletal muscle specimens of patients with gastric cancer, to explore the possible correlation among TRAF6, ubiquitin mRNA expression and cachexia.
Patients and tissue samples
Summary of characteristics of gastric cancer patients and control
Controls (n = 29)
Gastric cancer (n = 102)
61.88 ± 6.49
62.13 ± 6.54
65.50 ± 4.84
57.38 ± 6.28
24.13 ± 1.81
21.00 ± 1.31
Serum albumin, g/L
41.38 ± 6.09
33.75 ± 3.11
45.25 ± 3.62
37.18 ± 3.74
The nutritional assessment included anthropometric [height, actual body weight, %WL, body mass index (BMI), usual body weight], immunological (total lymphocyte count), and biochemical (serum albumin) indexes. Routine blood test was determined using completely automatic blood cell count analyzer (Beckman-Coulter -MAXM, American). Liver function was determined using Completely automatic biochemistry analyzer (Beckman-Coulter SYNCHRON LX 20, American) (Table1). The PNI(prognostic nutritional index) was calculated as follows: PNI = 10 × serum albumin(g/100 ml) + 0.005 × total lymphocyte count/mm3 of peripheral blood.
A biopsy specimen was obtained from the rectus abdominis muscle during the initial phase of the operation. The anterior sheet of the rectus abdominis muscle was opened with scissors after skin incision and dissection through the subcutaneous fat, and a muscle biopsy specimen weighing about 1.0 g was obtained. The biopsy specimen was divided into two portions that were immediately frozen in liquid nitrogen and then stored at −80°C until analysis. No complications occurred from the biopsy procedure.
Real-time quantitative RT-PCR
Total RNA from rectus abdominis muscle was extracted by TRIzol reagent and cDNAs were reverse-transcribed by Revert Aid TM reverse transcriptase. Real-time PCR was carried out using the ABI PRISM 7700 Sequence Detection System (Applied Bio systems) at 50°C for 2 min, 95°C for 10 min, followed by 50 cycles at 95°C for 15 s, and at 60°C for 1 min. The primers for GAPDH (224 bp) were 5'-TGAAGGTCGGAGTCAACGG-3' (sense) and 5'- CTGGAAGATGGTGATGGGATT-3' (antisense). The primers for TRAF6 (134 bp) were 5'-GCCTGGGTGACAGAGTGC-3' (sense) and 5'-AATGACTACTTATGGCTCCTTTTC-3' (antisense). The primers for ubiquitin(165 bp) were 5'-CCCTGGATGTGATGGTGTC-3' (sense) and 5'-CTCGTTGTCCCTGTTGCTG-3' (antisense). The expression of GAPDH was used to normalize that of the target genes. Each assay was done in triplicate, the average was calculate, and the expression level of TRAF6 and ubiquitin was expressed as 2–ΔΔCt, ΔCt = Ct (Target)–Ct (GAPDH).
Cells 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 proteins were fractionated using the NuPAGE 4–12% Bis-Tris gradient gel (Invitrogen) and transferred onto PVDF membrane. Membranes were blocked with 5% non-fat milk in PBS/Tween-20, and incubated with antibodies against TRAF6 (Santa Cruz), ubiquitin (Santa Cruz), and β-actin (Abcam).
In order to analyze the relationship among the expression of TRAF6 and ubiquitin and nutritional status of patients (percent weight loss, serum albumin), according to the literature, they were divided into two groups(percent weight loss ≥ 10 and <10, serum albumin ≥ 35and <35). All statistical analyses were performed using SPSS16.0 software. Measurement data were analyzed using the Student’s t test, while categorical data were studied using χ2 or Fisher exact tests. Statistical significance was set at P < 0.05.
The expression of TRAF6 in muscle of control and cancer patients
The expression of TRAF6 in muscle of cancer patients
Percent weight loss
The expression of ubiquitin in muscle of control and cancer patients
The expression of ubiquitin in muscle of cancer patients
Percent weight loss
Association between expression of TRAF6 and ubiquitin
Association between expression of TRAF6 and ubiquitin
In healthy individuals, skeletal muscle metabolism requires a balance of anabolic and catabolic processes, resulting in a continuous renewal of muscle proteins without a net change in overall muscle mass. However, in cancer cachexia and other chronic illnesses, the muscle wasting were associated with the reduced rate of protein synthesis, increased protein degradation, or a combination of both contributes. One common mechanism associated with skeletal muscle protein degradation in cancer cachexia is the activation of the adenosine triphosphate-dependent ubiquitin-proteasome proteolytic path way, this system plays a major role in muscle wasting[5, 6]. The study showed muscle ubiquitin mRNA was hyper expressed in gastric cancer patients compared to controls, the ubiquitin-proteasome proteolytic system play important role in the pathogenesis of muscle protein hyper catabolism in cancer cachexia. To investigate the role of ubiquitin expression in the skeletal muscle of gastric cancer patients. We assessed the expression of ubiquitin in 29 control muscles and 102 patient muscles. Ubiquitin was significantly upregulated in muscle of gastric cancer compared with the control muscles. Over expression of ubiquitin in muscle of gastric cancer were associated with TNM stage and weight loss.
Skeletal muscle wasting is a major reason for morbidity and mortality in many chronic disease states, disuse conditions and aging. The ubiquitin-proteasome and autophagy-lysosomal systems are the two major proteolytic pathways involved in regulation of both physiological and pathological muscle wasting. The study demonstrate that the expression level of tumor necrosis factor (α) receptor adaptor protein 6 (TRAF6), a protein involved in receptor-mediated activation of several signaling pathways, is enhanced in skeletal muscle during atrophy[9, 10]. To explore the relation of TRAF6 expression in the skeletal muscle of gastric cancer patients. We assessed the expression of TRAF6 in 29 control muscles and 102 patient muscles. TRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles, Overexpression of TRAF6 in muscle of gastric cancer were associated with TNM stage, the level of serum albumin and percent of weight loss. The study showed overexpression of TRAF6 may play important role in gastric cancer cachexia.
Paul’s study discover that TRAF6 possesses E3 ubiquitin ligase activity causing lysine-63-linked polyubiquitination of target proteins. Muscle-wasting stimuli could up regulate the expression of TRAF6 and auto-ubiquitination. Muscle-specific depletion of TRAF6 preserves skeletal muscle mass in a mouse model of cancer cachexia or denervation. Inhibition of TRAF6 also blocks the expression of the components of the ubiquitin-proteasome system (UPS) and auto phagosome formation in atrophying skeletal muscle. We also examined TRAF6 expression in skeletal muscle with gastric cancer and its correlation with ubiquitin status. We found a positive correlation between TRAF6 and ubiquitin expression, suggesting that TRAF6 may up regulates ubiquitin activity in cancer cachexia. While more investigations are required to understand its mechanisms of TRAF6 and ubiquitin in skeletal muscle. Correct the catabolic-anabolic imbalance is essential for the effective treatment of cancer cachexia.
Work was supported by Zhejiang Provincial Department of Science and Technology Research Foundation (2011C33009).
- Gullett N, Rossi P, Kucuk O, Johnstone PA: Cancer-induced cachexia: a guide for the oncologist. J Soc Integr Oncol. 2009, 7 (4): 155-169.PubMedGoogle Scholar
- Evans WJ: Skeletal muscle loss: cachexia, sarcopenia, and inactivity. Am J Clin Nutr. 2010, 91 (4): 1123S-1127S. 10.3945/ajcn.2010.28608A.View ArticlePubMedGoogle Scholar
- Evans WJ, Morley JE, Argilés J, et al: Cachexia: a new definition. Clin Nutr. 2008, 27 (6): 793-799. 10.1016/j.clnu.2008.06.013.View ArticlePubMedGoogle Scholar
- Dodson S, Baracos VE, Jatoi A, et al: Muscle wasting in cancer cachexia: clinical implications, diagnosis, and emerging treatment strategies. Annu Rev Med. 2011, 62: 265-279. 10.1146/annurev-med-061509-131248.View ArticlePubMedGoogle Scholar
- Baracos VE: Cancer-associated cachexia and underlying biological mechanisms. Annu Rev Nutr. 2006, 26: 435-461. 10.1146/annurev.nutr.26.061505.111151.View ArticlePubMedGoogle Scholar
- Tisdale MJ: Mechanisms of cancer cachexia. Physiol Rev. 2009, 89 (2): 381-410. 10.1152/physrev.00016.2008.View ArticlePubMedGoogle Scholar
- Chopard A, Hillock S, Jasmin BJ: Molecular events and signalling pathways involved in skeletal muscle disuse-induced atrophy and the impact of counter measures. J Cell Mol Med. 2009, 13 (9B): 3032-3050. 10.1111/j.1582-4934.2009.00864.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Kubrak C, Olson K, Jha N, et al: Nutrition impact symptoms: key determinants of reduced dietary intake, weight loss, and reduced functional capacity of patients with head and neck cancer before treatment. Head Neck. 2010, 32 (3): 290-300.PubMedGoogle Scholar
- Paul PK, Gupta SK, Bhatnagar S, et al: Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice. J Cell Biol. 2010, 191 (7): 1395-1411. 10.1083/jcb.201006098.PubMed CentralView ArticlePubMedGoogle Scholar
- Kumar A, Bhatnagar S, Paul PK: TWEAK and TRAF6 regulate skeletal muscle atrophy. Curr Opin Clin Nutr Metab Care. 2012, 15 (3): 233-239. 10.1097/MCO.0b013e328351c3fc.PubMed CentralView ArticlePubMedGoogle Scholar
- Onodera T, Goseki N, Kosaki G: Prognostic nutritional index in gastrointestinal surgery of malnourished cancer patients. Nippon Geka Gakkai Zasshi. 1984, 85: 1001-PubMedGoogle Scholar
- Bossola M, Muscaritoli M, Costelli P, Grieco G, Bonelli G, Pacelli F, Rossi Fanelli F, Doglietto GB, Baccino FM: Increased muscle proteasome activity correlates with disease severity in gastric cancer patients. Ann Surg. 2003, 237 (3): 384-389.PubMed CentralPubMedGoogle Scholar
- Baracos VE: Management of muscle wasting in cancer-associated cachexia: understanding gained from experimental studies. Cancer. 2001, 92 (6 Suppl): 1669-1677.View ArticlePubMedGoogle Scholar
- Bossola M, Muscaritoli M, Costelli P, et al: Increased muscle proteasome activity correlates with disease severity in gastric cancer patients. Ann Surg. 2003, 237 (3): 384-389.PubMed CentralPubMedGoogle Scholar
- Paul PK, Kumar A: TRAF6 coordinates the activation of autophagy and ubiquitin-proteasome systems in atrophying skeletal muscle. Autophagy. 2011, 7 (5): 555-556. 10.4161/auto.7.5.15102.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.