- Email: [email protected]
The expression of genes in the ubiquitin-proteasome proteolytic pathway is increased in skeletal muscle from patients with cancer
The expression of genes in the ubiquitin-proteasome proteolytic pathway is increased in skeletal muscle from patients with cancer Arthur Williams, MD, Xiaoyan Sun, PhD, Josef E. Fischer, MD, and Per-Olof Hasselgren, MD, Cincinnati, Ohio
Background: The intracellular mechanisms of muscle cachexia in patients with cancer are not known. To assess the role of the ubiquitin-proteasome proteolytic pathway in cancer-induced muscle breakdown, we determined messenger RNA levels for ubiquitin and several 20S proteasome subunits in muscle from patients undergoing surgery for cancer. Methods: A biopsy specimen was obtained from the rectus abdominis muscle in patients undergoing laparotomy for cancer (n = 6) or noncancer disease (n = 6). Tissue levels of mRNA for ubiquitin and the 20S proteasome subunits HC3, HC5, HC7, and HC9 were determined by dot blot analysis. Results: The mRNA levels for ubiquitin and the 20S proteasome subunits were 2 to 4 times higher in muscle from patients with cancer than in muscle from control patients. Conclusion: This is the first report of increased expression of genes in the ubiquitin-proteasome proteolytic pathway in muscle tissue from patients with cancer. Cancer-induced muscle catabolism may at least in part reflect ubiquitin-proteasome–dependent protein breakdown. (Surgery 1999:126:744-50.) From the Department of Surgery, University of Cincinnati, Cincinnati, Ohio
ALTHOUGH CANCER CACHEXIA REFLECTS depletion of both adipose and muscle tissue, muscle atrophy is particularly important as a prognostic factor in the overall survival of the patient with cancer. The catabolic response in muscle results in muscle wasting and fatigue and severely influences the quality of life in patients with cancer. When respiratory muscles are involved, pulmonary complications, including pneumonia from aspirations, are common. In addition, there is evidence that the response to chemotherapy is impaired in patients with cachexia.1 It has been estimated that almost one third of deaths in patients with cancer are related to muscle cachexia.2 A better understanding of mechanisms involved in cancer-induced muscle catabolism therefore has important clinical implications. The cancer-related catabolic response in skeleSupported in part by NIH grant DK 37908 and by grants from the Shriners of North America, Tampa, Fla. Presented at the 56th Annual Meeting of the Central Surgical Association, St Louis, Mo, Mar 4-6, 1999. Reprint requests: Per-Olof Hasselgren, MD, Department of Surgery, University of Cincinnati, 231 Bethesda Ave, ML 0558, Cincinnati, OH 45267-0558. Copyright © 1999 by Mosby, Inc. 0039-6060/99/$8.00 + 0
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tal muscle is primarily caused by stimulated protein breakdown, in particular myofibrillar protein breakdown, although inhibited protein synthesis may also contribute.3 Intracellular protein breakdown is regulated by multiple proteolytic pathways, including lysosomal, calcium-dependent, and ubiquitin-proteasome–dependent mechanisms.4 Recent studies in rats and mice with experimental tumors suggest that muscle proteolysis in cancer is regulated mainly by the ubiquitin-proteasome pathway and is associated with the upregulated expression of several genes in this pathway.5-8 It is not known whether similar mechanisms are involved in cancer-induced muscle catabolism in humans. The purpose of the present study was to determine mRNA levels for ubiquitin and subunits of the 20S proteasome, the catalytic core of the ubiquitin-proteasome pathway,9 in muscle tissue from patients undergoing surgery for cancer. PATIENTS AND METHODS A biopsy specimen was obtained from the rectus abdominis muscle during the initial phase of the operation in patients undergoing laparotomy for cancer or noncancer disease. After skin incision and dissection through the subcutaneous fat, the anterior sheet of the rectus abdominis muscle was opened with scissors, and a muscle biopsy speci-
Surgery Volume 126, Number 4
Fig 1. Dot blots (upper panel) and quantification (lower panel) of mRNA for ubiquitin (Ubi) in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
men weighing approximately 1 g was obtained. The biopsy specimen was immediately frozen in liquid nitrogen and stored at –70°C until analysis. After the muscle biopsy specimen had been obtained, small bleeding vessels were carefully controlled with ligatures and cautery, and the operation continued in a routine fashion. No complications occurred from the biopsy procedure. A written consent was obtained from all patients. The study was approved by the Institutional Review Board at the University of Cincinnati. Muscle mRNA levels for ubiquitin and the 20S proteasome subunits HC3, HC5, HC7, and HC9 were measured by dot blot hybridization. The hybridization was performed under stringent conditions in 50% formamide, 5 × SSPE (1 × SSSP = 0.75 mol/L NaCl, 0.05 mol/L NaH2 PO4, 0.005 mol/L EDTA), 5 × Denhardt’s solution, 1% SDS and 200 µg/mL denatured salmon sperm DNA at 56°C. cDNA probes for ubiquitin and the 20S proteasome subunits were randomly labeled with α-32P-dCTP (DuPont, Boston, Mass). RNA was extracted from the muscle tissue by the guanidinium thiocyanate-phenol-chloroform method10 with an RNA STAT-60 kit (Tel-Test “B”
Williams et al 745
Fig 2. Dot blots (upper panel) and quantification (lower panel) of mRNA for HC3 in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
Inc, Friendship, Texas). Different amounts (20 µg, 10 µg, and 5 µg) of RNA were loaded onto a nylon membrane (Nytra membrane; Schleicher & Schnell, Keene, NH) with a Minifold II slot-blot filtration manifold (BioRad, Hercules, Calif) and fixed to the membrane by ultraviolet cross-linking for 5 minutes. Prehybridization was performed at 56°C for 4 hours in a buffer consisting of 50% formamide, 1% SDS, 5 × Denhardt’s solution, 5 × SSPE, and 200 µg/mL denatured salmon sperm DNA. Hybridization was carried out overnight at 56°C in the same buffer 32P-labeled cDNA probe. After 2 posthybridization washes in 2 × SSPE and 0.2% SDS for 5 minutes at room temperature and in 0.1 × SSPE and 0.1% SDS for 15 minutes at 68°C, the membranes were transferred to Phosphor Image Cassettes (Molecular Dynamics, Sunnyvale, Calif) for quantitation. Control for equal loading was obtained by dividing the signal intensity of the specific mRNA by the signal intensity of messenger RNA for the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Complement DNA probes for human ubiquitin, HC3, HC5, HC7, and HC9 were generated by
746 Williams et al
Fig 3. Dot blots (upper panel) and quantification (lower panel) of mRNA for HC5 in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
reverse transcriptase polymerase chain reaction as described previously.11 The primers used to generate the probes were based on gene sequences reported previously12-14 (Table I). The specificity of the probes was determined by Northern blotting. All probes hybridized a single band corresponding to the expected size (not shown). Results are presented as means ± SEM. The Student t test was used for statistical analysis. RESULTS Six patients (4 men, 2 women; mean age, 67 years; range, 53-76 years) undergoing laparotomy for cancer (pancreas, 1 patient; colon, 1 patient; esophagus, 1 patient; colorectal liver metastases, 3 patients) and 6 control patients (5 men, 1 woman; mean age, 54 years; range, 22-92 years) undergoing laparotomy for benign disease (colon stricture after previous diverticulitis, 2 patients; biliary stricture, 1 patient; duodenal adenoma, 1 patient; colon dysplasia, 1 patient; peptic ulcer, 1 patient) were included in the study. Three of the patients with cancer reported a weight loss of 9%, 9%, and
Surgery October 1999
Fig 4. Dot blots (upper panel) and quantification (lower panel) of mRNA for HC7 in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
13%, respectively. The remainder of the patients had not experienced weight loss. Dot blot analysis showed that mRNA levels for ubiquitin were approximately 3 times higher in muscle from patients with cancer than in muscle from control patients (Fig 1). The mRNA levels for HC3 and HC5 were 3 to 4 times higher in patients with cancer than in control patients (Figs 2 and 3) and those for HC7 and HC9 were doubled in patients with cancer (Figs 4 and 5). DISCUSSION The present study is the first report of upregulated expression of the ubiquitin gene and several 20S proteasome subunit genes in muscle tissue from patients with cancer. The results suggest that muscle cachexia in patients with cancer may at least in part be regulated by the ubiquitin-proteasome pathway. The ubiquitin-proteasome mechanism of protein breakdown was reviewed recently elsewhere.15 Proteins degraded by this mechanism are conjugated to multiple molecules of ubiquitin after they are
Williams et al 747
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Table I. Sequences of 5´ and 3´ primers used to generate cDNA probes Gene
Primer
Ubiquitin
5´ 3´
HC3
5´ 3´
HC5
5´ 3´
HC7
5´ 3´
HC9
5´ 3´
GAPDH Fig 5. Dot blots (upper panel) and quantification (lower panel) of mRNA for HC9 in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
recognized and degraded by a large proteolytic complex, the 26S proteasome. The catalytic core of the 26S proteasome is the 20S proteasome, a particle consisting of 4 stacked rings, with each ring made up of 7 subunits.9 The subunits of the outer rings are called α-subunits, and the subunits of the inner rings are called β-subunits. The functions of the α-subunits include interaction between the 20S proteasome and various regulators, whereas the catalytic sites are located on the inner side of some of the β-subunits. Among the 20S proteasome subunits studied in the present report, HC3 and HC9 are α-subunits and HC5 and HC7 are β-subunits. The present results are similar to those in previous reports in which upregulated gene expression of several components of the ubiquitin-proteasome pathway was found in muscle of rats and mice with experimental tumors.5-8 In some of those studies, evidence was found that cancer stimulates other proteolytic mechanisms as well, including lysosomal and calcium-dependent protein degradation, but the ubiquitin-proteasome-dependent proteolysis is probably the most important mechanism of cancer-related muscle cachexia. In addition to cancer, previous reports suggest
5´ 3´
Sequence TAA GAC CAT CAC CCT CGA GG TGG ATG TTG TAG TCA GAC AGGG ATG GCG GAG CGC GGG TAC AGC TAT GCT ATC GCA GCC AAG TAA TC TTG CAG CTG CGA TTT TCG CCC TAC GTC CTT CCT TAA GGA AAC AGT TTC TCA TCG GTA TCC AAG GCC CC TAG GTG CCC TGT TTG GGG AAG ACC ACT ATA TTT TCT CCA GAA G CTA TTT ATC CTT TTC TTT CTG TTC ACA TCG CTC AGA CAC CAT G GAA GGC CAT GCC AGT GAG GTT
that the ubiquitin-proteasome pathway is involved in the regulation of muscle protein breakdown in a number of other catabolic conditions, including sepsis,11 burn injury,16 starvation,17 metabolic acidosis,18 denervation,19 and treatment with glucocorticoids.20 Most of those studies were performed in experimental animals and to the best of our knowledge, only 6 reports (including the present one) have been published in which the ubiquitinproteasome pathway was examined in human muscle tissue21-25 (Table II). Upregulated gene expression of the ubiquitin-proteasome pathway was found in muscle from patients with head trauma,21 sepsis,23 AIDS,25 and cancer (present study); whereas in patients with Duchenne muscular dystrophy22 and Cushing’s syndrome,24 mRNA levels for several of the proteins and enzymes in the ubiquitin-proteasome pathway were not elevated. The study in patients with Cushing’s syndrome contrasts to animal experiments in which evidence of increased ubiquitin-proteasome–dependent muscle proteolysis was found after treatment of rats with glucocorticoids20 indicating that there may not be an absolute correlation between experimental animals and humans with regards to the role of the ubiquitin-proteasome pathway in muscle cachexia.
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Table II. Studies of the ubiquitin-proteasome pathway in human skeletal muscle
Condition Head trauma Muscular dystrophy Sepsis Cushing’s syndrome AIDS Cancer
Gene expression of the ubiquitinproteasome pathway
Mansoor et al, 199621 Combaret et al, 199622 Tiao et al, 199723 Rallière et al, 199724 Llovera et al, 199825 Present study
Increased Unchanged Increased Unchanged Increased Increased
The mediators of cancer-induced muscle cachexia are not fully known. There is evidence that proinflammatory cytokines, in particular tumor necrosis factor, are involved in the regulation of protein breakdown in cancer and in other catabolic conditions.5 Other studies suggest that a tumor product, different from cytokines and with a molecular weight of approximately 24 kDa, mediates muscle protein breakdown in patients with certain types of cancer.8 This molecule, which is a sulphated glycoprotein, stimulated protein breakdown in incubated muscles from mice, suggesting a direct effect on muscle proteolysis.8 Although the present results of increased mRNA levels for ubiquitin and 20S proteasome subunits are consistent with the concept that muscle catabolism in patients with cancer may be regulated by the ubiquitin-proteasome pathway, the results need to be interpreted with caution for several reasons. First, muscle protein breakdown rates were not measured in the present study. In fact, only 3 of the patients with cancer reported weight loss, suggesting that the expression of the ubiquitin-proteasome pathway may be upregulated before protein breakdown is increased, or at least before increased protein breakdown results in loss of muscle mass. Second, it is not known from the present study whether the increased steady state levels of mRNA reflected stimulated transcription of the respective genes or increased stability of the transcripts or a combination of these changes. Finally, increased mRNA levels for ubiquitin and 20S proteasome subunits do not necessarily mean that protein levels were elevated or that the function of the proteins was increased. Despite these limitations, the present results are important because they suggest that the catabolic response to cancer may be regulated at the gene level in human muscle tissue. An important implication of the findings is that changes in the ubiquitin-proteasome pathway noted previously in rats and mice with experimental tumors5-8 reflect changes in patients with cancer. Therefore tumor models in experimental animals are probably valid
Reference
to explore mediators and mechanisms of cancerrelated muscle cachexia and to test different therapeutic interventions aimed at inhibiting the catabolic response to cancer. REFERENCES 1. van Eys J. Nutrition and cancer: physiological interrelationships. Annu Rev Nutr 1985;5:435-61. 2. Warren S. The immediate cause of death in cancer. Am J Med Sci 1932;184:610-3. 3. Smith KL, Tisdale MJ. Increased protein degradation and decreased protein synthesis in skeletal muscle during cancer cachexia. Br J Cancer 1993;67:680-5. 4. Attaix D, Taillandier D. The critical role of the ubiquitinproteasome pathway in muscle wasting in comparison to lysosomal and calcium-dependent systems. Adv Mol Cell Biol 1998;27:235-66. 5. Llovera M, Carbo N, Garcia-Martinez C, Costelli P, Tessitori L, Baccino FM, et al. Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour-bearing rats. Biochem Biophys Res Commun 1996;24:653-5. 6. Baracos VE, De Vivo C, Hoyle DHR, Goldberg AL. Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma. Am J Physiol 1995;268:E996-E1006. 7. Temparis S, Asensi M, Taillandier D, Aurrousseau E, Larbaud D, Obled A, et al. Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats. Cancer Res 1994;54:5568-73. 8. Lorite MJ, Thompson MG, Drake JL, Carling G, Tisdale MJ. Mechanism of muscle protein degradation induced by a cancer cachectic factor. Br J Cancer 1998;78:850-6. 9. Lupas A, Baumeister W. The 20S proteasome. In: Peters JM, Harris JR, Finley D, editors. Ubiquitin and the biology of the cell. New York: Plenum Press; 1998. p. 127-46. 10. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. 11. Tiao G, Fagan J, Samuels N, James JH, Hudson K, Lieberman M, et al. Sepsis stimulates nonlysosomal, energydependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle. J Clin Invest 1994;94:2255-64. 12. Tso JY, Sun XH, Kao T, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucl Acids Res 1985;13:2485-502. 13. Tamura T, Lee DH, Osaka F, Fujiwara T, Shin S, Chung CH, et al. Molecular cloning and sequence analysis of cDNAs for five major subunits of human proteasomes (multi-catalytic proteinase complexes). Biochim Biophys Acta 1991;1039:95-102. 14. Nothwang HG, Tamusa T, Tanaka K, Ichikasa A. Sequence
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15.
16.
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21.
22.
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25.
analyses and inter-species comparisons of three novel human proteasomal subunits, HsN3, HsC7-I and HsC10-II, confine potential proteolytic active-site residues. Biochim Biophys Acta 1994;1219:361-8. Hasselgren PO, Fischer JE. The ubiquitin-proteasome pathway. Review of a novel intracellular mechanism of muscle protein breakdown during sepsis and other catabolic conditions. Ann Surg 1997;225:307-16. Fang CH, Tiao G, James H, Ogle C, Fischer JE, Hasselgren PO. Burn injury stimulates multiple proteolytic pathways in skeletal muscle, including the ubiquitin-energy-dependent pathway. J Am Coll Surg 1995;180:161-70. Wing SS, Goldberg AL. Glucocorticoids activate the ATPubiquitin-dependent proteolytic system in skeletal muscle during fasting. Am J Physiol 1993;264:E668-76. Mitch WE, Medina R, Grieber S, May RC, England BK, Price SR, et al. Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphatedependent pathway involving ubiquitin and proteasomes. J Clin Invest 1994;93:2127-33. Medina R, Wing SS, Haas A, Goldberg AL. Activation of the ubiquitin-ATP-dependent proteolytic system in skeletal muscle during fasting and denervation atrophy. Biomed Biochim Acta 1991;50:347-56. Tiao G, Fagan J, Lieberman M, Wang JJ, Fischer JE, Hasselgren PO. Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids. J Clin Invest 1996;97:339-48. Mansoor O, Beaufrere B, Boirie Y, Ralliere C, Taillandier D, Aurousseau E, et al. Increased mRNA levels for components of the lysosomal, calcium-activated and ATP-ubiquitindependent proteolytic pathways in skeletal muscle from head trauma patients. Proc Natl Acad Sci USA 1996;93:2714-8. Combaret L, Taillandier D, Voisin L, Samuels SE, Boespflug-Tanguy O, Attaix D. No alteration in gene expression of components of the ubiquitin-proteasome proteolytic pathway in dystrophin-deficient muscles. FEBS Lett 1996;393:292-6. Tiao G, Hobler S, Meyer TA, Luchette FA, Fischer JE, Hasselgren PO. Sepsis is associated with increased mRNAs of the ubiquitin proteasome proteolytic pathway in human skeletal muscle. J Clin Invest 1997;99:163-8. Ralliere C, Tauveron I, Taillandier D, Guy L, Boiteux JP, Giraud B, et al. Glucocorticoids do not regulate the expression of proteolytic genes in skeletal muscle from Cushing syndrome patients. J Clin Endocrinol Metab 1997;82:31614. Llovera M, Garcia-Martinez C, Agell N, Lopez-Soriano FJ, Authier FJ, Gherardi RK, et al. Ubiquitin and proteasome gene expression is increased in skeletal muscle of slim AIDS patients. Int J Mol Med 1998;2:69-73.
DISCUSSION Dr J. F. Moley (St Louis, Mo). The underlying mechanisms of the cancer-associated cachexia have not yet been elucidated completely. A number of factors may contribute, including decreased food intake, excessive uptake of substrate by tumor, circulating factors released by the tumor or from necrotic areas of the tumor, and possibly neurogenic factors stimulated by the tumor. Positron emission tomography scanning has again drawn attention to the excessive glucose use by cancers.
Williams et al 749
These scans, which rely on the uptake of a glucose analog, FDG, by a tumor, do show malignancies and their metastases as very bright spots against the normal background and focus our attention on substrate use by cancers. There does, however, appear to be excessive protein depletion in patients with cancer. We published a study 15 years ago of patients with cancer cachexia, comparing them with patients with starvation and either normal volunteers or patients with anorexia nervosa, and did find a significant decrease in lean body mass in these patients compared with patients with cancer cachexia. This study provides some confirmation that intracellular protein catabolism is involved in the cachexia that occurs in patients with cancer. I am somewhat surprised by the dramatic results that are noted in this small number of patients with relatively mild weight loss. Was the investigator blinded as to what samples were analyzed in the laboratory? Why was the GADPH normalization used to determine or quantify expression of the mRNA species? Is that standard? Why was the reverse transcriptase approach not used? Why was the expression not normalized to the protein content of the cell? The ubiquitin pathway appears to be a downstream pathway. Do you have any idea as to what it is that tags that protein initially and causes the increase in availability of the substrate for the ubiquitin pathway in these cancer cells? I guess ultimately that is the key question. Dr Williams. We were surprised also to see such dramatic results in a small subset of patients, with varying tumor biologic evidence from a varied population of tumors. Our studies would suggest that the ubiquitin system probably is upregulated before the weight loss experienced by these patients. The dot blots were performed blinded. As far as why we used GAPDH, it is a standard housekeeping gene that we use routinely when doing these kind of experiments. In regards to the downstream regulation or whether this is the rate-limiting component of skeletal muscle protein breakdown in cancer, it is hard to say. Evidence from our laboratory and others has shown that perhaps the ubiquitin system is not actively breaking down the skeletal muscle proteins but perhaps is cleaning up what has already been broken down. So we do not have a good handle on what the role of ubiquitin is in this whole process, and further studies are needed. Dr Christopher R. McHenry (Cleveland, Ohio). I think it is worth reemphasizing that this is a novel report and that it is the first to demonstrate increased gene expression in the ubiquitin-proteasome proteolytic pathway in muscle tissue from patients with cancer. One of the criticisms of the study is obviously the small sample size. Another concern is whether the increase in gene expression that you have demonstrated is just a func-
750 Williams et al
tion of a patient having malignancy rather than having malignancy-induced cachexia? Did you compare the messenger RNA levels for ubiquitin and the 20S proteasome subunits in your 3 patients who had cancer and weight loss versus the 3 patients who had no weight loss? If so, was there any difference? Why would you expect there to be increased gene expression in patients with cancer who have not lost any weight? Finally, are there any therapeutic interventions that may downregulate gene expression in the ubiquitinproteasome proteolytic pathway that you could investigate to see whether cancer cachexia could be ameliorated? Dr Williams. I agree that the sample size is a problem. Further investigations and further enrollments are being performed currently. We did not find any correlation between the amount of weight loss and the level of ubiquitin or any of the proteasome subunits. That may represent the fact that this is a process that happens long before any noticeable weight loss occurs.
Surgery October 1999
As far as therapy is concerned, this protein is called ubiquitin for a reason. It is found almost everywhere and is involved in a number of regulatory phenomena, including acquisition of memory, antigen presentation, skeletal muscle breakdown, and a host of other issues. So finding an intervention is going to be a little bit difficult because we are going to have to find a skeletal muscle–specific intervention. There are a number of different sites at which this process could be modified, and we are currently trying to find one that might be specific for skeletal muscle. Dr Donald L. Kaminski (St Louis, Mo). Could you explain your controls a little better? For example, do you have control data from patients who lost weight from noncancer-related causes? Dr Williams. No, we do not have any data from patients who were dieting or from any weight loss from the benign process.
Background: The intracellular mechanisms of muscle cachexia in patients with cancer are not known. To assess the role of the ubiquitin-proteasome proteolytic pathway in cancer-induced muscle breakdown, we determined messenger RNA levels for ubiquitin and several 20S proteasome subunits in muscle from patients undergoing surgery for cancer. Methods: A biopsy specimen was obtained from the rectus abdominis muscle in patients undergoing laparotomy for cancer (n = 6) or noncancer disease (n = 6). Tissue levels of mRNA for ubiquitin and the 20S proteasome subunits HC3, HC5, HC7, and HC9 were determined by dot blot analysis. Results: The mRNA levels for ubiquitin and the 20S proteasome subunits were 2 to 4 times higher in muscle from patients with cancer than in muscle from control patients. Conclusion: This is the first report of increased expression of genes in the ubiquitin-proteasome proteolytic pathway in muscle tissue from patients with cancer. Cancer-induced muscle catabolism may at least in part reflect ubiquitin-proteasome–dependent protein breakdown. (Surgery 1999:126:744-50.) From the Department of Surgery, University of Cincinnati, Cincinnati, Ohio
ALTHOUGH CANCER CACHEXIA REFLECTS depletion of both adipose and muscle tissue, muscle atrophy is particularly important as a prognostic factor in the overall survival of the patient with cancer. The catabolic response in muscle results in muscle wasting and fatigue and severely influences the quality of life in patients with cancer. When respiratory muscles are involved, pulmonary complications, including pneumonia from aspirations, are common. In addition, there is evidence that the response to chemotherapy is impaired in patients with cachexia.1 It has been estimated that almost one third of deaths in patients with cancer are related to muscle cachexia.2 A better understanding of mechanisms involved in cancer-induced muscle catabolism therefore has important clinical implications. The cancer-related catabolic response in skeleSupported in part by NIH grant DK 37908 and by grants from the Shriners of North America, Tampa, Fla. Presented at the 56th Annual Meeting of the Central Surgical Association, St Louis, Mo, Mar 4-6, 1999. Reprint requests: Per-Olof Hasselgren, MD, Department of Surgery, University of Cincinnati, 231 Bethesda Ave, ML 0558, Cincinnati, OH 45267-0558. Copyright © 1999 by Mosby, Inc. 0039-6060/99/$8.00 + 0
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tal muscle is primarily caused by stimulated protein breakdown, in particular myofibrillar protein breakdown, although inhibited protein synthesis may also contribute.3 Intracellular protein breakdown is regulated by multiple proteolytic pathways, including lysosomal, calcium-dependent, and ubiquitin-proteasome–dependent mechanisms.4 Recent studies in rats and mice with experimental tumors suggest that muscle proteolysis in cancer is regulated mainly by the ubiquitin-proteasome pathway and is associated with the upregulated expression of several genes in this pathway.5-8 It is not known whether similar mechanisms are involved in cancer-induced muscle catabolism in humans. The purpose of the present study was to determine mRNA levels for ubiquitin and subunits of the 20S proteasome, the catalytic core of the ubiquitin-proteasome pathway,9 in muscle tissue from patients undergoing surgery for cancer. PATIENTS AND METHODS A biopsy specimen was obtained from the rectus abdominis muscle during the initial phase of the operation in patients undergoing laparotomy for cancer or noncancer disease. After skin incision and dissection through the subcutaneous fat, the anterior sheet of the rectus abdominis muscle was opened with scissors, and a muscle biopsy speci-
Surgery Volume 126, Number 4
Fig 1. Dot blots (upper panel) and quantification (lower panel) of mRNA for ubiquitin (Ubi) in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
men weighing approximately 1 g was obtained. The biopsy specimen was immediately frozen in liquid nitrogen and stored at –70°C until analysis. After the muscle biopsy specimen had been obtained, small bleeding vessels were carefully controlled with ligatures and cautery, and the operation continued in a routine fashion. No complications occurred from the biopsy procedure. A written consent was obtained from all patients. The study was approved by the Institutional Review Board at the University of Cincinnati. Muscle mRNA levels for ubiquitin and the 20S proteasome subunits HC3, HC5, HC7, and HC9 were measured by dot blot hybridization. The hybridization was performed under stringent conditions in 50% formamide, 5 × SSPE (1 × SSSP = 0.75 mol/L NaCl, 0.05 mol/L NaH2 PO4, 0.005 mol/L EDTA), 5 × Denhardt’s solution, 1% SDS and 200 µg/mL denatured salmon sperm DNA at 56°C. cDNA probes for ubiquitin and the 20S proteasome subunits were randomly labeled with α-32P-dCTP (DuPont, Boston, Mass). RNA was extracted from the muscle tissue by the guanidinium thiocyanate-phenol-chloroform method10 with an RNA STAT-60 kit (Tel-Test “B”
Williams et al 745
Fig 2. Dot blots (upper panel) and quantification (lower panel) of mRNA for HC3 in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
Inc, Friendship, Texas). Different amounts (20 µg, 10 µg, and 5 µg) of RNA were loaded onto a nylon membrane (Nytra membrane; Schleicher & Schnell, Keene, NH) with a Minifold II slot-blot filtration manifold (BioRad, Hercules, Calif) and fixed to the membrane by ultraviolet cross-linking for 5 minutes. Prehybridization was performed at 56°C for 4 hours in a buffer consisting of 50% formamide, 1% SDS, 5 × Denhardt’s solution, 5 × SSPE, and 200 µg/mL denatured salmon sperm DNA. Hybridization was carried out overnight at 56°C in the same buffer 32P-labeled cDNA probe. After 2 posthybridization washes in 2 × SSPE and 0.2% SDS for 5 minutes at room temperature and in 0.1 × SSPE and 0.1% SDS for 15 minutes at 68°C, the membranes were transferred to Phosphor Image Cassettes (Molecular Dynamics, Sunnyvale, Calif) for quantitation. Control for equal loading was obtained by dividing the signal intensity of the specific mRNA by the signal intensity of messenger RNA for the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Complement DNA probes for human ubiquitin, HC3, HC5, HC7, and HC9 were generated by
746 Williams et al
Fig 3. Dot blots (upper panel) and quantification (lower panel) of mRNA for HC5 in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
reverse transcriptase polymerase chain reaction as described previously.11 The primers used to generate the probes were based on gene sequences reported previously12-14 (Table I). The specificity of the probes was determined by Northern blotting. All probes hybridized a single band corresponding to the expected size (not shown). Results are presented as means ± SEM. The Student t test was used for statistical analysis. RESULTS Six patients (4 men, 2 women; mean age, 67 years; range, 53-76 years) undergoing laparotomy for cancer (pancreas, 1 patient; colon, 1 patient; esophagus, 1 patient; colorectal liver metastases, 3 patients) and 6 control patients (5 men, 1 woman; mean age, 54 years; range, 22-92 years) undergoing laparotomy for benign disease (colon stricture after previous diverticulitis, 2 patients; biliary stricture, 1 patient; duodenal adenoma, 1 patient; colon dysplasia, 1 patient; peptic ulcer, 1 patient) were included in the study. Three of the patients with cancer reported a weight loss of 9%, 9%, and
Surgery October 1999
Fig 4. Dot blots (upper panel) and quantification (lower panel) of mRNA for HC7 in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
13%, respectively. The remainder of the patients had not experienced weight loss. Dot blot analysis showed that mRNA levels for ubiquitin were approximately 3 times higher in muscle from patients with cancer than in muscle from control patients (Fig 1). The mRNA levels for HC3 and HC5 were 3 to 4 times higher in patients with cancer than in control patients (Figs 2 and 3) and those for HC7 and HC9 were doubled in patients with cancer (Figs 4 and 5). DISCUSSION The present study is the first report of upregulated expression of the ubiquitin gene and several 20S proteasome subunit genes in muscle tissue from patients with cancer. The results suggest that muscle cachexia in patients with cancer may at least in part be regulated by the ubiquitin-proteasome pathway. The ubiquitin-proteasome mechanism of protein breakdown was reviewed recently elsewhere.15 Proteins degraded by this mechanism are conjugated to multiple molecules of ubiquitin after they are
Williams et al 747
Surgery Volume 126, Number 4
Table I. Sequences of 5´ and 3´ primers used to generate cDNA probes Gene
Primer
Ubiquitin
5´ 3´
HC3
5´ 3´
HC5
5´ 3´
HC7
5´ 3´
HC9
5´ 3´
GAPDH Fig 5. Dot blots (upper panel) and quantification (lower panel) of mRNA for HC9 in muscle from control patients (open bar) and patients with cancer (filled bar). Results are means ± SEM with 6 patients in each group. *P < .05 vs control.
recognized and degraded by a large proteolytic complex, the 26S proteasome. The catalytic core of the 26S proteasome is the 20S proteasome, a particle consisting of 4 stacked rings, with each ring made up of 7 subunits.9 The subunits of the outer rings are called α-subunits, and the subunits of the inner rings are called β-subunits. The functions of the α-subunits include interaction between the 20S proteasome and various regulators, whereas the catalytic sites are located on the inner side of some of the β-subunits. Among the 20S proteasome subunits studied in the present report, HC3 and HC9 are α-subunits and HC5 and HC7 are β-subunits. The present results are similar to those in previous reports in which upregulated gene expression of several components of the ubiquitin-proteasome pathway was found in muscle of rats and mice with experimental tumors.5-8 In some of those studies, evidence was found that cancer stimulates other proteolytic mechanisms as well, including lysosomal and calcium-dependent protein degradation, but the ubiquitin-proteasome-dependent proteolysis is probably the most important mechanism of cancer-related muscle cachexia. In addition to cancer, previous reports suggest
5´ 3´
Sequence TAA GAC CAT CAC CCT CGA GG TGG ATG TTG TAG TCA GAC AGGG ATG GCG GAG CGC GGG TAC AGC TAT GCT ATC GCA GCC AAG TAA TC TTG CAG CTG CGA TTT TCG CCC TAC GTC CTT CCT TAA GGA AAC AGT TTC TCA TCG GTA TCC AAG GCC CC TAG GTG CCC TGT TTG GGG AAG ACC ACT ATA TTT TCT CCA GAA G CTA TTT ATC CTT TTC TTT CTG TTC ACA TCG CTC AGA CAC CAT G GAA GGC CAT GCC AGT GAG GTT
that the ubiquitin-proteasome pathway is involved in the regulation of muscle protein breakdown in a number of other catabolic conditions, including sepsis,11 burn injury,16 starvation,17 metabolic acidosis,18 denervation,19 and treatment with glucocorticoids.20 Most of those studies were performed in experimental animals and to the best of our knowledge, only 6 reports (including the present one) have been published in which the ubiquitinproteasome pathway was examined in human muscle tissue21-25 (Table II). Upregulated gene expression of the ubiquitin-proteasome pathway was found in muscle from patients with head trauma,21 sepsis,23 AIDS,25 and cancer (present study); whereas in patients with Duchenne muscular dystrophy22 and Cushing’s syndrome,24 mRNA levels for several of the proteins and enzymes in the ubiquitin-proteasome pathway were not elevated. The study in patients with Cushing’s syndrome contrasts to animal experiments in which evidence of increased ubiquitin-proteasome–dependent muscle proteolysis was found after treatment of rats with glucocorticoids20 indicating that there may not be an absolute correlation between experimental animals and humans with regards to the role of the ubiquitin-proteasome pathway in muscle cachexia.
748 Williams et al
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Table II. Studies of the ubiquitin-proteasome pathway in human skeletal muscle
Condition Head trauma Muscular dystrophy Sepsis Cushing’s syndrome AIDS Cancer
Gene expression of the ubiquitinproteasome pathway
Mansoor et al, 199621 Combaret et al, 199622 Tiao et al, 199723 Rallière et al, 199724 Llovera et al, 199825 Present study
Increased Unchanged Increased Unchanged Increased Increased
The mediators of cancer-induced muscle cachexia are not fully known. There is evidence that proinflammatory cytokines, in particular tumor necrosis factor, are involved in the regulation of protein breakdown in cancer and in other catabolic conditions.5 Other studies suggest that a tumor product, different from cytokines and with a molecular weight of approximately 24 kDa, mediates muscle protein breakdown in patients with certain types of cancer.8 This molecule, which is a sulphated glycoprotein, stimulated protein breakdown in incubated muscles from mice, suggesting a direct effect on muscle proteolysis.8 Although the present results of increased mRNA levels for ubiquitin and 20S proteasome subunits are consistent with the concept that muscle catabolism in patients with cancer may be regulated by the ubiquitin-proteasome pathway, the results need to be interpreted with caution for several reasons. First, muscle protein breakdown rates were not measured in the present study. In fact, only 3 of the patients with cancer reported weight loss, suggesting that the expression of the ubiquitin-proteasome pathway may be upregulated before protein breakdown is increased, or at least before increased protein breakdown results in loss of muscle mass. Second, it is not known from the present study whether the increased steady state levels of mRNA reflected stimulated transcription of the respective genes or increased stability of the transcripts or a combination of these changes. Finally, increased mRNA levels for ubiquitin and 20S proteasome subunits do not necessarily mean that protein levels were elevated or that the function of the proteins was increased. Despite these limitations, the present results are important because they suggest that the catabolic response to cancer may be regulated at the gene level in human muscle tissue. An important implication of the findings is that changes in the ubiquitin-proteasome pathway noted previously in rats and mice with experimental tumors5-8 reflect changes in patients with cancer. Therefore tumor models in experimental animals are probably valid
Reference
to explore mediators and mechanisms of cancerrelated muscle cachexia and to test different therapeutic interventions aimed at inhibiting the catabolic response to cancer. REFERENCES 1. van Eys J. Nutrition and cancer: physiological interrelationships. Annu Rev Nutr 1985;5:435-61. 2. Warren S. The immediate cause of death in cancer. Am J Med Sci 1932;184:610-3. 3. Smith KL, Tisdale MJ. Increased protein degradation and decreased protein synthesis in skeletal muscle during cancer cachexia. Br J Cancer 1993;67:680-5. 4. Attaix D, Taillandier D. The critical role of the ubiquitinproteasome pathway in muscle wasting in comparison to lysosomal and calcium-dependent systems. Adv Mol Cell Biol 1998;27:235-66. 5. Llovera M, Carbo N, Garcia-Martinez C, Costelli P, Tessitori L, Baccino FM, et al. Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour-bearing rats. Biochem Biophys Res Commun 1996;24:653-5. 6. Baracos VE, De Vivo C, Hoyle DHR, Goldberg AL. Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma. Am J Physiol 1995;268:E996-E1006. 7. Temparis S, Asensi M, Taillandier D, Aurrousseau E, Larbaud D, Obled A, et al. Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats. Cancer Res 1994;54:5568-73. 8. Lorite MJ, Thompson MG, Drake JL, Carling G, Tisdale MJ. Mechanism of muscle protein degradation induced by a cancer cachectic factor. Br J Cancer 1998;78:850-6. 9. Lupas A, Baumeister W. The 20S proteasome. In: Peters JM, Harris JR, Finley D, editors. Ubiquitin and the biology of the cell. New York: Plenum Press; 1998. p. 127-46. 10. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. 11. Tiao G, Fagan J, Samuels N, James JH, Hudson K, Lieberman M, et al. Sepsis stimulates nonlysosomal, energydependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle. J Clin Invest 1994;94:2255-64. 12. Tso JY, Sun XH, Kao T, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucl Acids Res 1985;13:2485-502. 13. Tamura T, Lee DH, Osaka F, Fujiwara T, Shin S, Chung CH, et al. Molecular cloning and sequence analysis of cDNAs for five major subunits of human proteasomes (multi-catalytic proteinase complexes). Biochim Biophys Acta 1991;1039:95-102. 14. Nothwang HG, Tamusa T, Tanaka K, Ichikasa A. Sequence
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analyses and inter-species comparisons of three novel human proteasomal subunits, HsN3, HsC7-I and HsC10-II, confine potential proteolytic active-site residues. Biochim Biophys Acta 1994;1219:361-8. Hasselgren PO, Fischer JE. The ubiquitin-proteasome pathway. Review of a novel intracellular mechanism of muscle protein breakdown during sepsis and other catabolic conditions. Ann Surg 1997;225:307-16. Fang CH, Tiao G, James H, Ogle C, Fischer JE, Hasselgren PO. Burn injury stimulates multiple proteolytic pathways in skeletal muscle, including the ubiquitin-energy-dependent pathway. J Am Coll Surg 1995;180:161-70. Wing SS, Goldberg AL. Glucocorticoids activate the ATPubiquitin-dependent proteolytic system in skeletal muscle during fasting. Am J Physiol 1993;264:E668-76. Mitch WE, Medina R, Grieber S, May RC, England BK, Price SR, et al. Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphatedependent pathway involving ubiquitin and proteasomes. J Clin Invest 1994;93:2127-33. Medina R, Wing SS, Haas A, Goldberg AL. Activation of the ubiquitin-ATP-dependent proteolytic system in skeletal muscle during fasting and denervation atrophy. Biomed Biochim Acta 1991;50:347-56. Tiao G, Fagan J, Lieberman M, Wang JJ, Fischer JE, Hasselgren PO. Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids. J Clin Invest 1996;97:339-48. Mansoor O, Beaufrere B, Boirie Y, Ralliere C, Taillandier D, Aurousseau E, et al. Increased mRNA levels for components of the lysosomal, calcium-activated and ATP-ubiquitindependent proteolytic pathways in skeletal muscle from head trauma patients. Proc Natl Acad Sci USA 1996;93:2714-8. Combaret L, Taillandier D, Voisin L, Samuels SE, Boespflug-Tanguy O, Attaix D. No alteration in gene expression of components of the ubiquitin-proteasome proteolytic pathway in dystrophin-deficient muscles. FEBS Lett 1996;393:292-6. Tiao G, Hobler S, Meyer TA, Luchette FA, Fischer JE, Hasselgren PO. Sepsis is associated with increased mRNAs of the ubiquitin proteasome proteolytic pathway in human skeletal muscle. J Clin Invest 1997;99:163-8. Ralliere C, Tauveron I, Taillandier D, Guy L, Boiteux JP, Giraud B, et al. Glucocorticoids do not regulate the expression of proteolytic genes in skeletal muscle from Cushing syndrome patients. J Clin Endocrinol Metab 1997;82:31614. Llovera M, Garcia-Martinez C, Agell N, Lopez-Soriano FJ, Authier FJ, Gherardi RK, et al. Ubiquitin and proteasome gene expression is increased in skeletal muscle of slim AIDS patients. Int J Mol Med 1998;2:69-73.
DISCUSSION Dr J. F. Moley (St Louis, Mo). The underlying mechanisms of the cancer-associated cachexia have not yet been elucidated completely. A number of factors may contribute, including decreased food intake, excessive uptake of substrate by tumor, circulating factors released by the tumor or from necrotic areas of the tumor, and possibly neurogenic factors stimulated by the tumor. Positron emission tomography scanning has again drawn attention to the excessive glucose use by cancers.
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These scans, which rely on the uptake of a glucose analog, FDG, by a tumor, do show malignancies and their metastases as very bright spots against the normal background and focus our attention on substrate use by cancers. There does, however, appear to be excessive protein depletion in patients with cancer. We published a study 15 years ago of patients with cancer cachexia, comparing them with patients with starvation and either normal volunteers or patients with anorexia nervosa, and did find a significant decrease in lean body mass in these patients compared with patients with cancer cachexia. This study provides some confirmation that intracellular protein catabolism is involved in the cachexia that occurs in patients with cancer. I am somewhat surprised by the dramatic results that are noted in this small number of patients with relatively mild weight loss. Was the investigator blinded as to what samples were analyzed in the laboratory? Why was the GADPH normalization used to determine or quantify expression of the mRNA species? Is that standard? Why was the reverse transcriptase approach not used? Why was the expression not normalized to the protein content of the cell? The ubiquitin pathway appears to be a downstream pathway. Do you have any idea as to what it is that tags that protein initially and causes the increase in availability of the substrate for the ubiquitin pathway in these cancer cells? I guess ultimately that is the key question. Dr Williams. We were surprised also to see such dramatic results in a small subset of patients, with varying tumor biologic evidence from a varied population of tumors. Our studies would suggest that the ubiquitin system probably is upregulated before the weight loss experienced by these patients. The dot blots were performed blinded. As far as why we used GAPDH, it is a standard housekeeping gene that we use routinely when doing these kind of experiments. In regards to the downstream regulation or whether this is the rate-limiting component of skeletal muscle protein breakdown in cancer, it is hard to say. Evidence from our laboratory and others has shown that perhaps the ubiquitin system is not actively breaking down the skeletal muscle proteins but perhaps is cleaning up what has already been broken down. So we do not have a good handle on what the role of ubiquitin is in this whole process, and further studies are needed. Dr Christopher R. McHenry (Cleveland, Ohio). I think it is worth reemphasizing that this is a novel report and that it is the first to demonstrate increased gene expression in the ubiquitin-proteasome proteolytic pathway in muscle tissue from patients with cancer. One of the criticisms of the study is obviously the small sample size. Another concern is whether the increase in gene expression that you have demonstrated is just a func-
750 Williams et al
tion of a patient having malignancy rather than having malignancy-induced cachexia? Did you compare the messenger RNA levels for ubiquitin and the 20S proteasome subunits in your 3 patients who had cancer and weight loss versus the 3 patients who had no weight loss? If so, was there any difference? Why would you expect there to be increased gene expression in patients with cancer who have not lost any weight? Finally, are there any therapeutic interventions that may downregulate gene expression in the ubiquitinproteasome proteolytic pathway that you could investigate to see whether cancer cachexia could be ameliorated? Dr Williams. I agree that the sample size is a problem. Further investigations and further enrollments are being performed currently. We did not find any correlation between the amount of weight loss and the level of ubiquitin or any of the proteasome subunits. That may represent the fact that this is a process that happens long before any noticeable weight loss occurs.
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As far as therapy is concerned, this protein is called ubiquitin for a reason. It is found almost everywhere and is involved in a number of regulatory phenomena, including acquisition of memory, antigen presentation, skeletal muscle breakdown, and a host of other issues. So finding an intervention is going to be a little bit difficult because we are going to have to find a skeletal muscle–specific intervention. There are a number of different sites at which this process could be modified, and we are currently trying to find one that might be specific for skeletal muscle. Dr Donald L. Kaminski (St Louis, Mo). Could you explain your controls a little better? For example, do you have control data from patients who lost weight from noncancer-related causes? Dr Williams. No, we do not have any data from patients who were dieting or from any weight loss from the benign process.