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STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia.

Bonetto A, Aydogdu T, Kunzevitzky N, Guttridge DC, Khuri S, Koniaris LG, Zimmers TA - PLoS ONE (2011)

Bottom Line: In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response.These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer.These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood.

View Article: PubMed Central - PubMed

Affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

ABSTRACT

Background: Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia.

Methodology/principal findings: Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer.

Conclusions/significance: These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood.

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Fibrinogen is produced and released by the skeletal muscle following activation of the IL-6/STAT3 pathway.A, Western blotting analysis and quantitation of fibrinogen in C2C12 myotubes infected with Ad-cSTAT3-GFP or Ad-GFP as control. Fibrinogen expression was increased consistent with the increase in the levels of STAT3. **P<0.01, ***P<0.001 vs. GFP. B, Western blotting analysis and quantitation of fibrinogen expression in C2C12 treated with IL-6 (100 ng/ml) for 1, 24, 48 h. GAPDH was used as loading control. Increased expression of fibrinogen was observed at each time point after IL-6 treatment. Data (means ± SEM) are expressed as relative densitometry value. ***P<0.001 vs. respective controls. C, Fibrinogen levels by ELISA of the conditioned medium of C2C12 exposed to IL-6 for 30 min, 1, 6, 24, 48 h. Fibrinogen levels were significantly elevated after 6, 24 and 48 h of IL-6 treatment. Data (means ± SEM) are expressed as ng/ml. **P<0.01, ***P<0.001 vs. controls (C).
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pone-0022538-g008: Fibrinogen is produced and released by the skeletal muscle following activation of the IL-6/STAT3 pathway.A, Western blotting analysis and quantitation of fibrinogen in C2C12 myotubes infected with Ad-cSTAT3-GFP or Ad-GFP as control. Fibrinogen expression was increased consistent with the increase in the levels of STAT3. **P<0.01, ***P<0.001 vs. GFP. B, Western blotting analysis and quantitation of fibrinogen expression in C2C12 treated with IL-6 (100 ng/ml) for 1, 24, 48 h. GAPDH was used as loading control. Increased expression of fibrinogen was observed at each time point after IL-6 treatment. Data (means ± SEM) are expressed as relative densitometry value. ***P<0.001 vs. respective controls. C, Fibrinogen levels by ELISA of the conditioned medium of C2C12 exposed to IL-6 for 30 min, 1, 6, 24, 48 h. Fibrinogen levels were significantly elevated after 6, 24 and 48 h of IL-6 treatment. Data (means ± SEM) are expressed as ng/ml. **P<0.01, ***P<0.001 vs. controls (C).

Mentions: Despite the induction of fibrinogen and SAA mRNA in muscle, the protein levels of acute phase proteins in skeletal muscle extracts theoretically could be due to contaminating plasma. In order to further test our hypothesis that fibrinogen is produced directly from skeletal muscle following activation of the IL-6/STAT3 pathway, we infected C2C12 murine myotube cultures with a recombinant adenovirus expressing a constitutively activated form of STAT3, cSTAT3 [53], along with GFP as a marker. Western blotting of C2C12 extracts 48h after infection demonstrated significant elevation of fibrogen in Ad-cSTAT3-GFP cultures versus Ad-GFP cultures (+86% vs. GFP, p<0.01; Figure 8A). In order to determine whether fibrinogen was produced after IL-6 challenging, C2C12 myotubes were exposed to murine recombinant IL-6 for up to 48 h. This resulted into an overall increase in fibrinogen, both in the cellular compartment by Western blotting (Figure 8B) and in the culture medium by ELISA (Figure 8C). These experiments show that even in the absence of other cell types and tissues, skeletal muscle cells respond to IL-6 and activation of STAT3 by synthesizing acute phase protein RNAs and proteins for secretion.


STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia.

Bonetto A, Aydogdu T, Kunzevitzky N, Guttridge DC, Khuri S, Koniaris LG, Zimmers TA - PLoS ONE (2011)

Fibrinogen is produced and released by the skeletal muscle following activation of the IL-6/STAT3 pathway.A, Western blotting analysis and quantitation of fibrinogen in C2C12 myotubes infected with Ad-cSTAT3-GFP or Ad-GFP as control. Fibrinogen expression was increased consistent with the increase in the levels of STAT3. **P<0.01, ***P<0.001 vs. GFP. B, Western blotting analysis and quantitation of fibrinogen expression in C2C12 treated with IL-6 (100 ng/ml) for 1, 24, 48 h. GAPDH was used as loading control. Increased expression of fibrinogen was observed at each time point after IL-6 treatment. Data (means ± SEM) are expressed as relative densitometry value. ***P<0.001 vs. respective controls. C, Fibrinogen levels by ELISA of the conditioned medium of C2C12 exposed to IL-6 for 30 min, 1, 6, 24, 48 h. Fibrinogen levels were significantly elevated after 6, 24 and 48 h of IL-6 treatment. Data (means ± SEM) are expressed as ng/ml. **P<0.01, ***P<0.001 vs. controls (C).
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Related In: Results  -  Collection

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pone-0022538-g008: Fibrinogen is produced and released by the skeletal muscle following activation of the IL-6/STAT3 pathway.A, Western blotting analysis and quantitation of fibrinogen in C2C12 myotubes infected with Ad-cSTAT3-GFP or Ad-GFP as control. Fibrinogen expression was increased consistent with the increase in the levels of STAT3. **P<0.01, ***P<0.001 vs. GFP. B, Western blotting analysis and quantitation of fibrinogen expression in C2C12 treated with IL-6 (100 ng/ml) for 1, 24, 48 h. GAPDH was used as loading control. Increased expression of fibrinogen was observed at each time point after IL-6 treatment. Data (means ± SEM) are expressed as relative densitometry value. ***P<0.001 vs. respective controls. C, Fibrinogen levels by ELISA of the conditioned medium of C2C12 exposed to IL-6 for 30 min, 1, 6, 24, 48 h. Fibrinogen levels were significantly elevated after 6, 24 and 48 h of IL-6 treatment. Data (means ± SEM) are expressed as ng/ml. **P<0.01, ***P<0.001 vs. controls (C).
Mentions: Despite the induction of fibrinogen and SAA mRNA in muscle, the protein levels of acute phase proteins in skeletal muscle extracts theoretically could be due to contaminating plasma. In order to further test our hypothesis that fibrinogen is produced directly from skeletal muscle following activation of the IL-6/STAT3 pathway, we infected C2C12 murine myotube cultures with a recombinant adenovirus expressing a constitutively activated form of STAT3, cSTAT3 [53], along with GFP as a marker. Western blotting of C2C12 extracts 48h after infection demonstrated significant elevation of fibrogen in Ad-cSTAT3-GFP cultures versus Ad-GFP cultures (+86% vs. GFP, p<0.01; Figure 8A). In order to determine whether fibrinogen was produced after IL-6 challenging, C2C12 myotubes were exposed to murine recombinant IL-6 for up to 48 h. This resulted into an overall increase in fibrinogen, both in the cellular compartment by Western blotting (Figure 8B) and in the culture medium by ELISA (Figure 8C). These experiments show that even in the absence of other cell types and tissues, skeletal muscle cells respond to IL-6 and activation of STAT3 by synthesizing acute phase protein RNAs and proteins for secretion.

Bottom Line: In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response.These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer.These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood.

View Article: PubMed Central - PubMed

Affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

ABSTRACT

Background: Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia.

Methodology/principal findings: Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer.

Conclusions/significance: These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood.

Show MeSH
Related in: MedlinePlus