JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia.

Abstract

Cachexia, the metabolic dysregulation leading to sustained loss of muscle and adipose tissue, is a devastating complication of cancer and other chronic diseases. Interleukin-6 and related cytokines are associated with muscle wasting in clinical and experimental cachexia, although the mechanisms by which they might induce muscle wasting are unknown. One pathway activated strongly by IL-6 family ligands is the JAK/STAT3 pathway, the function of which has not been evaluated in regulation of skeletal muscle mass. Recently, we showed that skeletal muscle STAT3 phosphorylation, nuclear localization, and target gene expression are activated in C26 cancer cachexia, a model with high IL-6 family ligands. Here, we report that STAT3 activation is a common feature of muscle wasting, activated in muscle by IL-6 in vivo and in vitro and by different types of cancer and sterile sepsis. Moreover, STAT3 activation proved both necessary and sufficient for muscle wasting. In C(2)C(12) myotubes and in mouse muscle, mutant constitutively activated STAT3-induced muscle fiber atrophy and exacerbated wasting in cachexia. Conversely, inhibiting STAT3 pharmacologically with JAK or STAT3 inhibitors or genetically with dominant negative STAT3 and short hairpin STAT3 reduced muscle atrophy downstream of IL-6 or cancer. These results indicate that STAT3 is a primary mediator of muscle wasting in cancer cachexia and other conditions of high IL-6 family signaling. Thus STAT3 could represent a novel therapeutic target for the preservation of skeletal muscle in cachexia.

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doi: 10.1152/ajpendo.00039.2012
303:E410-E421, 2012. First published 5 June 2012;
Am J Physiol Endocrinol Metab
Leonidas G. Koniaris and Teresa A. Zimmers
Andrea Bonetto, Tufan Aydogdu, Xiaoling Jin, Zongxiu Zhang, Rui Zhan, Leopold Puzis,
cachexia
wasting downstream of IL-6 and in experimental cancer
JAK/STAT3 pathway inhibition blocks skeletal muscle
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JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream
of IL-6 and in experimental cancer cachexia
Andrea Bonetto,1,2Tufan Aydogdu,3Xiaoling Jin,1,2Zongxiu Zhang,1,2Rui Zhan,1,4Leopold Puzis,2
Leonidas G. Koniaris,2,3,4,5and Teresa A. Zimmers1,2,3,4,5
1Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania;2Division
of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine,
Miami, Florida;3Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, Florida;
4Program in Cancer Biology, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine,
Miami, Florida; and5Department of Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania
Submitted 23 January 2012; accepted in final form 29 May 2012
BonettoA,AydogduT,JinX,ZhangZ,ZhanR,PuzisL,Koniaris
LG, Zimmers TA. JAK/STAT3 pathway inhibition blocks skeletal
musclewastingdownstreamofIL-6andinexperimentalcancercachexia.
Am J Physiol Endocrinol Metab 303: E410–E421, 2012. First published
June 5, 2012; doi:10.1152/ajpendo.00039.2012.—Cachexia, the meta-
bolic dysregulation leading to sustained loss of muscle and adipose
tissue, is a devastating complication of cancer and other chronic
diseases. Interleukin-6 and related cytokines are associated with
muscle wasting in clinical and experimental cachexia, although the
mechanisms by which they might induce muscle wasting are un-
known. One pathway activated strongly by IL-6 family ligands is the
JAK/STAT3 pathway, the function of which has not been evaluated in
regulation of skeletal muscle mass. Recently, we showed that skeletal
muscle STAT3 phosphorylation, nuclear localization, and target gene
expression are activated in C26 cancer cachexia, a model with high
IL-6 family ligands. Here, we report that STAT3 activation is a
common feature of muscle wasting, activated in muscle by IL-6 in
vivo and in vitro and by different types of cancer and sterile sepsis.
Moreover, STAT3 activation proved both necessary and sufficient for
muscle wasting. In C2C12 myotubes and in mouse muscle, mutant
constitutively activated STAT3-induced muscle fiber atrophy and
exacerbated wasting in cachexia. Conversely, inhibiting STAT3 phar-
macologically with JAK or STAT3 inhibitors or genetically with
dominant negative STAT3 and short hairpin STAT3 reduced muscle
atrophy downstream of IL-6 or cancer. These results indicate that
STAT3 is a primary mediator of muscle wasting in cancer cachexia
and other conditions of high IL-6 family signaling. Thus STAT3 could
represent a novel therapeutic target for the preservation of skeletal
muscle in cachexia.
Janus-activated kinase; interleukin-6; T cell protein tyrosine phospha-
tase/protein tyrosine phosphatase, nonreceptor type 2; suppressors of
cytokine signaling 3; electroporation; atrophy; burn; sepsis; lipopoly-
saccharide; incb018424; signal transducer and activator of transcrip-
tion 3 inhibitory peptide
CACHEXIA, FROM “KAKOS” MEANING “BAD” and “hexis” meaning “to
have (fut.),” is a devastating syndrome that involves the loss of
both muscle and adipose tissue and can be associated with
many disease states, such as certain types of cancer, congestive
heart failure, diabetes, kidney failure, and HIV/AIDS (15, 16).
The whole body wasting that occurs in the presence of cancer
has been acknowledged clinically since the time of the ancient
Greeks, who coined the term. Clinically, cachexia is defined as
an unintentional 10% loss of body weight over a 12-mo period
that is directly associated with an underlying disease (39). The
progressive loss of adipose tissue and skeletal muscle despite
adequate feeding results in weakness, reduced ambulation,
diminished quality of life, poor response to therapy, and often
death due to respiratory failure or infection. In cancer alone,
cachexia afflicts some 80% of all patients and is itself respon-
sible for 25–30% of all cancer-related deaths. Currently, there
are no approved effective treatments for muscle wasting in
cancer. Understanding the molecular mechanisms responsible
for muscle wasting is necessary to develop targeted therapies
for these most vulnerable patients.
Both host and tumor-derived factors have been shown ex-
perimentally to contribute to muscle wasting (4). Members of
the TGF? superfamily, including TGF? itself, activin, GDF-
15, and the muscle-specific family member myostatin can
cause weight loss and muscle loss through SMAD pathway
activation whether or not their levels are increased in cancer (6,
35, 63). As well, inflammatory cytokines, including tumor
necrosis factor (TNF)/cachectin, IL-1? and -?, interferon-?,
and IL-6 and related ligands, have been implicated in cachexia
either through experimental manipulation using mouse models
or by association of serum levels in patients with cachexia (3).
In addition to producing anorexia, certain cytokines are known
to directly induce proteolysis or lipolysis in vitro or in vivo. In
the case of TGF? family members, muscle wasting is down-
stream of SMAD activation (35), whereas TNF-induced mus-
cle wasting is mediated largely through NF-?B (11, 45). The
pathways through which other cytokines initiate muscle loss
are not described.
IL-6 is a multifunctional cytokine involved in a variety of
host defenses and pathological processes (40). IL-6-secreting
cells can induce wasting of both muscle and fat stores and
ultimately death (23, 55). Serum IL-6 levels are elevated in
most experimental models of cachexia, and IL-6 is responsible
at least in part for the muscle wasting seen in mice with
colon-26 cancer cachexia as well as in Yomoto uterine cancer
cachexia (49–52). Suramin, an antagonist of IL-6 binding to its
receptor, markedly decreased the rate of cachexia in C26 tumor
bearers (50), whereas IL-6-neutralizing antibodies have been
shown to slow cachexia in Yomoto-bearing mice (52). Inhibi-
tion of IL-6-using antibody against the soluble receptor slows
muscle wasting in ApcMinmice (57). Moreover, IL-6 has been
demonstrated to be a very sensitive predictor of weight loss in
a number of series, including patients with advanced small-cell
Address for reprint requests and other correspondence: T. A. Zimmers, Thomas
Jefferson University, Kimmel Cancer Center, 233 South 10th St., BLSB 306,
Philadelphia, PA 19107 (e-mail: tzimmers@kimmelcancercenter.org).
Am J Physiol Endocrinol Metab 303: E410–E421, 2012.
First published June 5, 2012; doi:10.1152/ajpendo.00039.2012.
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lung cancer (46) and colon cancer (12, 17), and elevated IL-6
levels are associated with reduced survival in a variety of
cancer types (48). In addition to IL-6 itself, other IL-6 family
cytokines have also been implicated in muscle wasting, includ-
ing ciliary neutrophic factor (CNTF) (14, 27) and leukemia
inhibitor factor (LIF) (1, 38).
IL-6 and related ligands activate signaling by binding li-
gand-specific ?-receptors [IL-6 receptor-?, a.k.a. gp80 in the
case of IL-6 and CNTF-R and oncostatin M (OSM)-R in the
case of CNTF and OSM] in either membrane-bound or soluble
forms (7, 29). These ligand receptor complexes bind ubiqui-
tously expressed type I cytokine receptor IL-6 signal transduc-
er/gp130, inducing activation of three major pathways: the
signal transducers and activators of transcription 1 and 3
(STAT1/3), ERK, and phosphatidylinositol 3-kinase/Akt path-
ways (19). For the former, binding of gp130 induces activation
of associated Janus kinases (JAKs), which subsequently recruit
SH2-containing proteins, including STAT3. Phosphorylation
of STAT3 on tyrosine 705 results in dimerization, nuclear
localization, DNA binding, and target gene regulation. Signal-
ing can be antagonized at the level of JAK or STAT3 by
feedback inhibition through STAT3 target genes, including
suppressors of cytokine signaling (SOCS) proteins, particularly
SOCS3 (26). Dephosphorylation of STAT3 is mediated by T
cell protein tyrosine phosphatase/protein tyrosine phosphatase,
non receptor type 2 (TC-PTP/PTPN2) in the nucleus and
cytoplasm (58).
Of pathways stimulated by IL-6 ligands, the ERK pathway
has been implicated previously in cancer cachexia and muscle
wasting (44), and Akt is the primary anabolic pathway for
skeletal muscle (22). Although STAT3 activation has been
reported in muscle of mice with cachexia (5, 8), no functional
data on STAT3 on modulating muscle size yet exist. Thus here
we sought to determine the role of IL-6-induced and cancer-
induced JAK/STAT3 pathway activation on skeletal muscle
size and wasting.
MATERIALS AND METHODS
Cachexia models. All animal studies were approved by the Uni-
versity of Miami and Thomas Jefferson University Institutional Ani-
mal Care and Use Committees. For delivery of sustained high levels
of IL-6, athymic nude mice (Harlan Laboratories, Indianapolis, IN)
were injected intramuscularly with Chinese hamster ovary (CHO)
cells expressing recombinant human IL-6 or with similarly selected
cells expressing no recombinant protein. Mice were then euthanized at
different time points (4, 8, 12, and 16 days) after injection of CHO
cells. C57BL/6J male mice were implanted s.c. with Alzet osmotic
minipumps delivering 1 ?g/h recombinant murine IL-6 for 7 days. For
cancer cachexia, CD2F1 female mice (Harlan) were injected intras-
capularly with 106C26 (colon-26) adenocarcinoma cells (Donna
McCarthy). C57BL/6J female mice were injected with 106B16F10
melanoma cells (ATCC, Manassas, VA) or 106Lewis lung carcinoma
cells (Denis Guttridge). ApcMinmice (The Jackson Laboratory, Bar
Harbor, ME) were maintained in our colony. All tumor-bearing mice
were euthanized when weight loss was ?10%. For sterile sepsis,
C57BL/6J wild-type or IL-6-null (B6.129S2-IL6tm1Kopf/J) male mice
(Jackson) were given LPS (1 ?g/h for 7 days) by osmotic pump
implanted sucutaneously. IL-6 serum levels were measured by IL-6
Quantikine M ELISA (R & D Systems).
Gene transfer. Gene transfer in skeletal muscle was performed as
described (43). Briefly, on day 0, legs were shaved and tibialis
muscles were pretreated by injection of 25 ?l of 0.5 U/?l hyaluron-
idase through the skin. Two hours later, 50 ?g of plasmid DNA in
PBS was injected in the tibialis anterior muscle using a Hamilton
syringe. Next, three pulses (20 ms each at 75 V/cm, 1 pulse/s) were
applied through the skin to the dorsal aspect of the lower limb using
a square pulse generator (ECM-830) and stainless-steel paddle elec-
trodes (BTX-Harvard Apparatus). The polarity was then inverted, and
three more pulses were delivered to the muscle. Cotransfection of an
expression vector carrying a green fluorescent protein under the
control of the cytomegalovirus promoter (pCMV-GFP) at 1/10 the
molarity of experimental construct or empty vector was performed to
identify transfected fibers. Transfections were performed the same day
as tumor inoculation for the CHO cell and C26 tumor-bearing exper-
iments, and mice were euthanized 9 days later for CHO/nude mouse
experiments and 12 days later for C26 tumor-bearing experiments.
Cell culture. C2C12 skeletal myoblasts (ATCC) were grown in
high-glucose DMEM supplemented with 10% FBS, 100 U/ml peni-
cillin, 100 mg/ml streptomycin, 100 mg/ml sodium pyruvate, and 2
mM L-glutamine and maintained at 37°C in 5% CO2. Differentiation
to myotubes was induced by shifting confluent cultures to DMEM
supplemented with 2% horse serum and replacing the medium every
2nd day for 5 days. At 5 days, myotubes were treated with recombi-
nant murine IL-6 (R & D Systems) 10 or 100 ng/ml in the presence or
absence of 1 nM Velcade (Selleck Chemicals), 50 ?M STAT3
inhibitor peptide (Calbiochem) (62), 400 nM JAK1/2 inhibitor
(INCB018424; ChemieTek) (56), or specific adenoviral constructs. In
the case of adenovirus, 24 h after infection the cells were washed and
challenged with IL-6 for 48 h, and then cells were fixed and measured.
Plasmid and adenoviral constructs. Plasmids were STAT3 Y705F
Flag pRC/CMV (dnSTAT3; plasmid 8709), Stat3-C Flag pRc/CMV
(cSTAT3; plasmid 8,722), and pCMV-GFP [plasmid 11,153 (37)]
from Addgene, short hairpin (sh)STAT3 plasmid RMM3981-
97059840 from Open Biosystems, and pcDNA3.1 (pCMV-empty)
from Invitrogen. Replication defective Ad-CMV-GFP (no. 1060) was
purchased (Vector Biolabs, Philadelphia, PA). Ad-cSTAT3-GFP, Ad-
dnSTAT3, Ad-shSTAT3, Ad-PTPN2-GFP, and Ad-dnSTAT3-GFP
were made using human recombinant adenovirus serotype 5 (D1/E3)
construct, in which GFP and genes of interest are expressed from
separate promoters (Vector Biolabs).
RNA extraction and quantitative real-time PCR. Total RNA was
extracted from flash-frozen quadriceps using TRIzol (Sigma-Aldrich),
as described previously (64). cDNA was used as a template in
real-time PCR reactions with QuantiTect SYBR-Green PCR Master
Mix (BioRad) and run on a Bio-Rad MyIQ machine. Relative gene
expression was normalized by dividing the specific expression value
by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression
value and calculated using the 2???CTmethod (45a). Primer sequences
are as reported (8).
Western blotting analysis and antibodies. Proteins in muscle and
C2C12myotubes were assayed by Western blotting of total lysates, as
reported (8). Antibodies were rabbit monoclonal phospho-STAT3
(Tyr705), GFP and GAPDH, and rabbit polyclonal STAT3 from Cell
Signaling, mouse monoclonal gp130 from R & D Systems, mouse
monoclonal Flag and ?-actin from Sigma, and rabbit polyclonal
fibrinogen from Dako.
Electrophoretic mobility shift assay. Nuclear extracts were prepared
from flash-frozen muscle as reported (8, 9), using 3=-biotinylated-STAT3
oligonucleotides (5=-GATCCTTCTGGGAATTCCTAGATC-3=, 3=-CT-
AGGAAGACCCTTAAGGATCTAG-5=) from IDT and the Light Shift
Chemiluminescent electrophoretic mobility shift assay (EMSA) kit
(Pierce).
Morphological studies (fiber size, immunofluorescence, immuno-
histochemistry). For morphometric studies in Fig. 1, images were ob-
tained in brightfield. For studies involving adenovirus, direct GFP
fluorescence was used. For inhibitor studies, C2C12 myotubes were
fixed in ice-cold acetone-methanol and incubated with an anti-myosin
heavy chain antibody (1:1,000; Millipore) and an AlexaFluor 488-
labeled secondary antibody (Invitrogen). In all cases, analysis of
GFP-positive fiber size was by measuring average fiber diameter of
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long multinucleate fibers, avoiding regions of clustered nuclei on a
calibrated image using National Institutes of Health (NIH) Image J
1.43 software. For histology and morphometry of muscle, 8-?m-thick
cryosections of tibialis muscles were fixed in formaldehyde vapor. For
immunofluorescence analysis, cryosections of either gastrocnemius or
tibialis muscles were fixed in 3% formaldehyde and then incubated
with pSTAT3 primary antibody (1:50) and AlexaFluor 594-
conjugated secondary antibody (Invitrogen). For immunohistochem-
istry (IHC) analysis, muscle sections were fixed in cold acetone and
then in 4% BSA-PBS containing 3% H2O2 and incubated with
pSTAT3 primary antibody (1:50) and then Real Envision Detection
System using DAB? chromogen (Dako). All samples were observed
under an Olympus IX70 fluorescence microscope, and calibrated
images were recorded for morphometric examination. Morphometry
was determined from a section taken at the midbelly. All GFP-positive
fibers were measured by tracing the perimeter of each individual fiber
using a Cintiq pen tablet input device (Wacom) and Image J for
calculation of the cross-sectional area.
Statistical analysis. All results are expressed as means ? SE except
where noted. Western blots show independent samples and are represen-
tative of at least two trials. Significance of the differences was evaluated
by analysis of variance, followed by Tukey’s posttest.
Fig. 1. IL-6-induced and tumor-induced muscle wasting
were associated with STAT3 activation in vivo and in
vitro. A: Chinese hamster ovary (CHO)/IL-6 cells in-
jected in athymic nude mice caused loss of body weight
[final body weight (FBW)] and muscle weight, including
tibialis anterior and gastrocnemius vs. CHO/control mice
(n ? 8/group, euthanized on day 12). Data are represen-
tative of ?10 experiments. B: Western blotting analysis
of muscle from mice in A reveals increased Y705-
STAT3 in the gastrocnemius (GSN), quadriceps (Quad),
and tibialis anterior of mice treated with CHO/IL-6 cells
(?IL-6) vs. CHO/controls (?IL-6). Blot is representa-
tive of 4 independently assayed samples from each group
on day 8, but virtually identical results were observed on
days 4 and 12 (not shown). C: muscle wasting was
progressive over time with CHO/IL-6 cells vs. CHO/
controls. Gastronemius is shown (n ? 4–6/point).
D: quantitative real-time RT-PCR (qPCR) shows ele-
vated expression of STAT3 target genes and atrogin-1 in
quadriceps from mice with cachexia and CHO/IL-6 from
days 8, 12, and 16 normalized to CHO/control samples
(n ? 3/condition, sampled in triplicate). E and F: recom-
binant IL-6 administered by osmotic pumps (1 ?g/h for
7 days) induced a marked reduction in quadriceps weight
(n ? 4–6/group; E) and increased pY705-STAT3 by
Western blotting analysis of mice euthanized after 7 days
of treatment (F). This experiment is 1 of 2. Fold change
vs. control group (PBS) for the pSTAT3/GAPDH ratio is
shown under the blots (*P ? 0.05). G: quadriceps
pY705-STAT3 was increased in experimental models of
cancer cachexia when weight loss in the tumor-bearing
group was equal to 10% of starting body weight for C26,
B16-F10, and Lewis lung carcinoma (LLC)-tumor bear-
ing mice and ApcMinmice or after 7 days of chronic LPS
administration by osmotic pump. Note that IL-6-null
(IL6?/?) mice showed less pSTAT3 with LPS pumps. Fold
change vs. control group for the pSTAT3/STAT3 ratio is
reported under each of the blots. *P ? 0.05; **P ? 0.01;
***P ? 0.001.
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RESULTS
IL-6 induced muscle wasting and STAT3 activation in mice.
To model the sustained high levels of IL-6 observed in cancer,
sepsis, burn, and other conditions associated with muscle
wasting, we administered IL-6 to mice by using two ap-
proaches. In the first, we injected athymic nude mice with CHO
cells expressing human IL-6 vs. control CHO cells expressing
no recombinant protein (65). In the second, we implanted
osmotic pumps delivering recombinant murine IL-6 in
C57BL/6J mice. CHO/IL-6 treatment led to blood levels of
80–100 ng/ml IL-6, as reported previously (65). Compared
with CHO/control mice, which maintained tumor-free body
mass vs. starting body mass over the course of the experiment,
CHO/IL-6 injected mice grew markedly wasted, with a signif-
icant loss of body mass and proportionately greater loss of
muscle mass (Fig. 1A). All muscles were affected. Consistent
with direct signaling of IL-6 on muscle, pY705-STAT3 was
markedly elevated in gastrocnemius, quadriceps, and tibialis
anterior muscles of CHO/IL-6 mice (Fig. 1B). Muscle weight
loss was progressive with longer exposure to IL-6 (Fig. 1C).
Expression of the RNA for STAT3 was elevated along with
expression of the STAT3 target genes SOCS3, fibrinogen, and
LBP1, the latter two of which are acute-phase reactants (Fig.
1D). Consistent with muscle wasting, we also observed mark-
edly elevated expression of Atrogin-1, a muscle-specific ubiq-
uitin ligase expressed in virtually all conditions of muscle
wasting. Administration of recombinant murine IL-6 by pump
over 7 days also led to weight loss, systemic muscle wasting
(quadriceps is shown; Fig. 1E), and increased pY705-STAT3
in skeletal muscle (Fig. 1F). Thus IL-6 alone is sufficient to
induce systemic muscle STAT3 activation and wasting.
STAT3 is activated in muscle in cancer cachexia and sepsis.
High IL-6 levels have been reported to correlate with the
degree of muscle wasting in cancer patients and mouse models
of cachexia. Previously, we have reported that C26 tumor-
dependent cachexia is associated with high serum levels of
several IL-6 family ligands (IL-6, IL-11, LIF, and OSM) along
with robust activation of the STAT3 pathway in skeletal muscle
(Ref. 8 and Fig. 1G). Baltgalvis et. al. (5) have reported elevated
pY705-STAT3 in ApcMinmice with cachexia due to intestinal
cancer. To survey the potential universality of STAT3 activa-
tion in muscle wasting, we assayed muscle from mice with
?10% weight loss due to B16.F10 melanoma (31), Lewis lung
adenocarcinoma (LLC) (60), and, to compare with prior data,
ApcMinand C3-1-TAg prostate cancer (36). All but the last
showed increased pY705-STAT3 in skeletal muscle, suggest-
ing that STAT3 activation is a common feature in many types
of experimental cancer cachexia (Fig. 1G). Sepsis is also
associated with high serum IL-6, and here we show that
chronic administration of LPS in mice by osmotic minipump
for 7 days led to 10% weight loss (data not shown) and
increased levels of pY705-STAT3 in quadriceps. STAT3 acti-
vation was reduced in LPS pump-treated Il6-null mice, sug-
gesting that IL-6 contributes to but is not solely responsible for
STAT3 activation in cachexia of sepsis (Fig. 1G). It should be
noted that the degree of STAT3 phosphorylation was not
consistent across the cachexia models. Generally, those known
to be associated with high IL-6 and more aggressive wasting,
including CHO/IL-6, C26, ApcMin, and sepsis, showed repro-
ducibly higher pSTAT3 levels than did those that are not
classically associated with IL-6, namely LLC and B16.F10
melanoma, as well as sepsis in the absence of IL-6.
IL-6 induced wasting and STAT3 activation in muscle
cultures. We next sought to determine the direct effects of IL-6
on muscle using the C2C12myotube model. To study effects on
myofiber size per se vs. differentiation and fusion, this and all
subsequent experiments were performed on C2C12myotubes at
4 or 5 days after the onset of differentiation, when fibers cover
?80% of the culture area. Under these conditions, 48 h of
recombinant murine IL-6 (10 or 100 ng/ml) exposure resulted
in a significant reduction in fiber diameter (?36%, P ? 0.001)
compared with controls (Fig. 2A). C2C12myofiber wasting was
accompanied by increased pY705-STAT3 at both 10 and 100
ng/ml and at 1, 24, and 48 h after the initial stimulation with
IL-6. The difference between control and IL-6-induced
pSTAT3 levels lessened over time, consistent with the induc-
tion of feedback inhibitory pathways, including expression of
SOCS3 (Ref. 26 and Fig. 2B).
IL-6 has been shown conflictingly to both induce proteolysis
and also induce protein synthesis and protein accumulation in
the C2C12myotube model (2, 13). To determine whether IL-6
induced C2C12 fiber atrophy results from activation of the
ubiquitin-proteasome pathway, we incubated myotubes in the
presence of 1 nM Velcade and IL-6. In fact, IL-6-induced
atrophy was reduced but not abolished in the presence of the
proteasome inhibitor (Fig. 2C), suggesting that part of the IL-6
effect is likely through this pathway.
STAT3 activation is sufficient to induce muscle wasting in
vitro and in vivo. Given the positive relationship between
elevated IL-6, STAT3 phosphorylation, and muscle wasting,
we next sought to determine whether STAT3 activation was
sufficient to induce muscle wasting. Thus we infected C2C12
myofibers with a recombinant adenovirus expressing a mutant,
constitutively activated STAT3 (cSTAT3) known to possess
increased DNA binding/transcriptional activity (10). Under
conditions in which total STAT3 RNA was increased approx-
imately twofold by quantitative real-time PCR, Ad-cSTAT3-
GFP infection resulted in a 30% reduction in fiber diameter vs.
Ad-GFP and induced the expression of known STAT3 target
genes, including SOCS3 and the STAT3-mediated acute-phase
response gene fibrinogen. Consistent with wasting, atrogin-1
expression was also markedly increased (Fig. 3A). Thus
STAT3 activation is by itself sufficient to induce muscle fiber
wasting in cell culture.
To assay STAT3 activity in vivo, we used direct injection
and electroporation of a CMV-cSTAT3 plasmid into the tibia-
lis anterior of CD2F1 mice. CMV/empty vector was electro-
porated into the contralateral leg as an internal control. Coin-
jection of CMV/GFP was used to mark transfected fibers.
Transfection of cSTAT3 was sufficient to induce a marked
reduction in fiber cross-sectional area in non-tumor-bearing
mice (?22% vs. empty vector controls, P ? 0.001). Further-
more, cSTAT3 transfection exacerbated muscle fiber atrophy
in the presence of the C26 tumor, reducing cross-sectional area
an additional 35% compared with C26 plus empty vector alone
(P ? 0.001; Fig. 3B). These results indicate that STAT3
activation is sufficient for muscle fiber atrophy in vivo as well.
Inhibition of STAT3 prevents IL-6-induced myofiber atrophy in
vitro. Next, we sought to determine whether STAT3 is not only
sufficientbutalsonecessarytoinducefiberatrophy.Thedominant
negative STAT3 (dnSTAT3) mutation results in a phenylalanine
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substitution at Tyr705, thus preventing IL-6-induced STAT3
dimerization and nuclear translocation (30). Infection of C2C12
myotubes with Ad-dnSTAT3-GFP resulted in myofiber hypertro-
phy vs. Ad-GFP alone in the absence of exogenous IL-6 (?15%,
P ? 0.001) and completely blocked myofiber atrophy induced by
IL-6 (?26% vs. IL-6 Ad-GFP, P ? 0.001) (Fig. 4A). The
hypertrophic effect of dnSTAT3 at baseline was likely due to
inhibition of endogenous IL-6 signaling because IL-6 is expressed
by C2C12 myotubes (20). Ad-shSTAT3-GFP induced baseline
hypertrophy more robustly than dnSTAT3 (?25%, P ? 0.001)
and prevented IL-6-mediated wasting (?33% vs. IL-6 Ad-
shScramble, P ? 0.001) (Fig. 4B).
We next sought to inhibit STAT3 pharmacologically. C2C12
myotubes were treated with a cell-permeable STAT3 SH2
domain mimetic peptide (SIP) (62). SIP is a potent and selec-
tive inhibitor of STAT3 SH2 domain/phosphotyrosine interac-
tions in cancer cells (62). The 29-mer cell-permeable peptide is
derived from the STAT3 SH2 domain, can replicate STAT3
biochemical properties, binds with high affinity to known
STAT3-binding phosphotyrosine peptide motifs, and prevents
Fig. 2. IL-6 induced loss of C2C12myofiber mass and activated STAT3 in a proteasome-dependent manner. A: IL-6 treatment resulted in loss of C2C12myofiber
diameter after 48 h. Black bars show representative myotube diameter (n ? 158–200 fibers/condition from 3 independent wells). Data are representative of ?8
experiments. ***P ? 0.001. B: in C2C12myotubes, increased pSTAT3 was observed after 1 h of treatment with 10 or 100 ng/ml IL-6 (left) and after 1, 24, and
48 h of 100 ng/ml IL-6. Fold change vs. respective control group for the pSTAT3/STAT3 ratio is reported under each set of blots. Data are representative of
?5 trials. C: Velcade/bortezomib (1 nM) cotreatment for 48 h prevented IL-6-induced muscle atrophy (n ? 170–210 fibers/well from 3 independent wells for
each condition).
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activation of endogenous STAT3. C2C12 myotubes were
treated for 48 h with 50 ?M STAT3 inhibitory peptide in the
presence or absence of 100 ng/ml IL-6. STAT3 inhibitory
peptide resulted in mild hypertrophy at baseline (?5% vs.
PBS, P ? 0.001) and a partial reduction in loss of fiber
diameter with IL-6 treatment (Fig. 5A).
Testing one step upstream in the pathway, we treated C2C12
cells with the JAK1/2 inhibitor INCB018424, which has been
shown to reduce pSTAT3 in peripheral blood of patients with
myelofibrosis. INCB018424 (400 nM) completely blocked IL-6-
induced wasting, whereas it induced only a mild hypertrophy at
baseline (Fig. 5B). This activity was associated with significant
reductions in pY705-STAT3 levels at every time point (Fig. 5C).
Taken together, these data using genetic and pharmacological
inhibition of STAT3 indicate that STAT3 activation is necessary
for IL-6-mediated myofiber wasting in vitro.
STAT3 inhibition reduced muscle wasting in vivo down-
stream of IL-6. We next sought to determine whether blocking
STAT3 activity could reduce IL-6-induced muscle wasting in
vivo. First, the tibialis anterior muscles of athymic nude mice
were subjected to electroporation with CMV/dnSTAT3 and
CMV/empty vector in opposite legs, and then mice were
injected with either CHO/control or CHO/IL-6 cells. As de-
scribed previously, 14 days later CHO/control mice had gained
body weight compared with initial body weight, whereas
CHO/IL-6 mice had lost body weight and muscle mass (data
not shown) along with muscle fiber diameter (Fig. 6). CMV-
dnSTAT3 transgenesis resulted in an increase in muscle fiber
diameter vs. CMV/empty vector in CHO/control mice (?6%,
P ? 0.001). In the setting of CHO/IL-6 treatment, where
muscle diameter was reduced 24% vs. CHO/control;CMV/
empty vector, CMV-dnSTAT3 partially blocked IL-6 induced
wasting (8% vs. CHO/IL-6;CMV/empty vector, P ? 0.001).
STAT3 inhibition reduced muscle wasting in vivo downstream of
C26cachexia.Inthemorephysiologicalmodelofcancercachexia,
C26 adenocarcinoma, dnSTAT3 resulted in more robust inhibi-
tion of wasting (Fig. 7A). In non-tumor-bearing mice, CMV-
dnSTAT3 induced an 11% increase in fiber diameter (P ? 0.001).
C26 tumors induced a 28% loss in muscle fiber diameter (P ?
0.001) vs. non-tumor-bearing CMV/empty vector controls,
Fig. 3. STAT3 was sufficient to induce muscle fiber atrophy both in vitro and in vivo. A: constitutively active STAT3 [Ad-cSTAT3-green fluorescent protein
(GFP)] resulted in decreased C2C12myofiber diameter 48 h after infection (n ? 150–200 fibers/condition from 3 independent wells) and increased transcription
of known STAT3 target genes as well as atrogin-1 (n ? 3 wells/group in triplicate). B: cytomegalovirus (CMV)-cSTAT3 transfection reduced cross-sectional
area (CSA) in the tibialis anterior muscle of non-tumor-bearing and C26-bearing CD2F1 mice. Mice were transfected on the same day as PBS, or tumor cells
were injected and then euthanized 12 days later. Only positively transfected fibers (i.e., green fibers coexpressing pCMV-GFP) were measured (n ? 650–1,900
fibers/condition; n ? 8 tumor-bearing and non-tumor-bearing mice). Both experiments have been performed ?3 times. **P ? 0.01; ***P ? 0.001.
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whereas CMV-dnSTAT3-expressing fibers were 24% larger.
These results on fiber size were associated with decreased STAT3
activity. Immunohistochemical and immunofluorescent staining
for pSTAT3 were reduced by dnSTAT3 transfection in both
non-tumor-bearing and tumor-bearing mice (Fig. 7B). Moreover,
EMSA of nuclear extracts from tibialis anterior of dnSTAT3-
transfected C26 tumor-bearing mice showed markedly reduced
STAT3 DNA binding activity vs. empty vector tumor-bearing
controls (Fig. 7C). Western blotting analysis of total lysates from
transfected muscles confirmed an overall increased level of total
STAT3 and a comigrating protein detected with anti-Flag antibody,
both of which are consistent with the expression of the Flag-tagged
dnSTAT3 protein in CMV-dnSTAT3 transfected mice, along with
expression of GFP from the reporter plasmid (Fig. 7D).
As in vitro, we also assayed the efficacy of shSTAT3 in
blocking wasting (Fig. 7E). In this experiment, a CMV-
shSTAT3 plasmid or, as a control, CMV-shScramble plasmid
was transfected into contralateral tibialis anterior muscles of
mice that were then injected with C26 cells or an equivalent
volume of saline. CMV-shSTAT3 resulted in mild muscle
hypertrophy in the absence of a C26 tumor (?10%, P ? 0.001)
and complete inhibition of muscle fiber wasting in the setting
of C26 cachexia (?18%, P ? 0.001). Thus inhibition of
STAT3 locally in skeletal muscle reduces or blocks muscle
wasting due to IL-6 treatment and cancer cachexia, indicating
that STAT3 activation in skeletal muscle is largely or entirely
responsible for muscle wasting downstream of IL-6 or cancer.
DISCUSSION
Given the preponderance of data implicating IL-6 and re-
lated ligands in muscle wasting of cancer cachexia, we sought
to determine the signaling mechanism in skeletal muscle re-
sponsible for that activity. Specifically, we hypothesized that
STAT3 activation in skeletal muscle would lead to loss of
muscle mass and that STAT3 inhibition would abolish IL-6
and cancer-induced wasting. Indeed, our results in cell culture
and in mice show that STAT3 activation is itself both neces-
Fig. 4. STAT3 activity was necessary for muscle atrophy. A: infection of adenovirus expressing dominant negative STAT3 (Ad-dnSTAT3-GFP) resulted in increased
fiber diameter in control and IL-6-treated C2C12 myotubes (n ? 200–300 fibers from 3 independent wells/condition). B: Ad-shSTAT3-GFP also increased baseline
myofiber size and inhibited IL-6-dependent fiber atrophy (n ? 200–300 fibers from 3 independent wells/condition). Data represent several independent experiments in
which adenovirus was applied for 24 h and then washed out, and IL-6 was applied for 48 h and cells were fixed and measured. ***P ? 0.001.
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Fig. 5. Pharmacological inhibition of JAK/STAT3 prevented IL-6-dependent myofiber atrophy. A: STAT3 inhibitor peptide, 50 ?M, induced modest myofiber
hypertrophy and prevented IL-6-induced fiber atrophy after 48 h of cotreatment (n ? 200–300 fibers from 3 independent wells/condition). B: the JAK inhibitor
INCB018424 (400 nM) induced modest hypertrophy in control myotubes and completely blocked IL-6-mediated myofiber atrophy after 48 h of cotreatment (n ?
200–300 fibers from 3 independent wells/condition). Data represent 3 separate experiments. C: Western blotting analysis of whole C2C12 culture extracts show
markedly reduced pSTAT3 with INCB018424 treatment at 1, 24, and 48 h. A and B are representative of at least 2 experiments each in which inhibitor and IL-6
were added at the same time and cells were incubated for 48 h and then fixed and measured. ***P ? 0.001.
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sary and sufficient for muscle wasting. This is true both for
muscle wasting downstream of IL-6 as well as in a mixed
cytokine model of C26 tumor-induced cachexia. Moreover,
STAT3 was widely activated in other cancers, including Lewis
lung carcinoma, ApcMincolon cancer, B16 melanoma, and in
our model of sterile sepsis.
Indeed, although we focus in large part on IL-6-mediated
wasting in this study, the C26 model of cachexia is also
characterized by high levels of three other ligands that activate
the gp130/JAK/STAT3 axis, namely CNTF, OSM, and LIF
(8). Each of these binds a specific ?-receptor before activating
gp130, and all are likely responsible in part for the induction of
STAT3 phosphorylation. The integration of these ligands upon
STAT3 suggests that STAT3 inhibition per se might be a more
powerful approach than antibody inhibition of individual cy-
tokines to reducing muscle wasting in cancers where several
cytokines are elevated. Moreover, given the similar activation
in other cancers, our results might also be generalizable to any
condition characterized by high levels of IL-6 family cyto-
kines, including, for example, burn injury and sepsis.
This causative role of STAT3 in muscle wasting is less
obvious than it might seem upon casual inspection. IL-6 and
related ligands activate several signaling pathways, including
two major pathways, ERK and STAT1/3 (33). ERK has been
implicated previously in C26 cachexia (44) and might have
been entirely responsible for muscle loss. Inhibition of STAT1
blocks cathepsin induction by interferon-? in cultures (21);
however, the role of STAT1 in the setting of cachexia has not
been investigated. Given that STAT3 inhibition blocks C26
tumor-induced wasting entirely, the STAT3 pathway appar-
ently takes precedence over ERK or STAT1 activation. How-
ever, STAT3 inhibition does not completely abolish IL-6-
induced wasting in vivo, suggesting that either the inhibition
was incomplete, these other pathways might also be important
drivers of muscle wasting, or both. Paradoxically, IL-6 and
other ligands have also been shown to result in activation of
Akt, the primary anabolic pathway in skeletal muscle. Whether
and how this might contribute to muscle wasting is currently
unclear but is consistent with prior reports of increased protein
synthesis in IL-6-treated C2C12cells (2) and in muscle of mice
(43) and patients with cachexia (28).
Our results are also somewhat surprising in light of other
known roles for IL-6 and STAT3 in skeletal and cardiac
muscle. In humans, acute muscle damage elicits IL-6-mediated
STAT3 localization to satellite cell nuclei (54), whereas in
mice IL-6/STAT3 is necessary for satellite cell-mediated mus-
cle hypertrophy (47). It is not immediately clear how effects on
satellite cells might contribute to wasting. However, these roles
of IL-6 on satellite cells do not exclude a role for IL-6/STAT3
in mature muscle fibers. Speculatively, the coupling of satellite
cell proliferation and fiber wasting locally might serve to
promote the fusion of myoblasts and repair of muscle by the
opening of space in the syncytium, a process that might
become pathogenic when activated systemically. Also surpris-
ing is that our observations in striated muscle are counter to
those in cardiac muscle. Whereas the roles of IL-6 in promot-
ing heart failure and preserving heart function after injury are
complex, context, and species specific, STAT3 activation in
the heart promotes both hypertrophy and cell survival (32, 33,
41, 59). Indeed, STAT3 is required for specification of cardi-
omyocytes from stem cells, murine germline deletion of
STAT3 results in embryonic lethality at the time of heart
development, and cardiomyocyte-specific ablation of STAT3
results in heart failure (7, 18). The mechanisms responsible for
these apparently opposite effects of STAT3 in cardiac vs.
skeletal muscle have not yet been explored.
Although our results firmly place STAT3 downstream of
IL-6 and cancer in causing muscle wasting, the mechanisms by
which STAT3 induces atrophy are still unknown. We hypoth-
esize that STAT3 activation of transcription is responsible,
given that dnSTAT3 largely inhibits nuclear but not mitochon-
drial effects of STAT3 (24). The number, identity, and activity
of the muscle-specific STAT3 target genes mediating wasting
are under active investigation in our laboratory. Among these
might be members of the ubiquitin-proteasome pathway, be-
cause our data indicate that IL-6-mediated wasting can be
reduced in the presence of proteasome inhibitor. A second
distinct mechanism potentially leading to muscle wasting is
Fig. 6. STAT3 inhibition prevented IL-6-dependent muscle wasting in mice. Expression of dnSTAT3 through CMV-dnSTAT3 transfection of tibialis anterior
muscle on the day of CHO injection resulted in basal hypertrophy in CHO/control mice and reduced muscle wasting in CHO/IL-6 mice vs. the CMV-empty vector
transfected controls 9 days later (n ? 450–1,200 fibers from n ? 8 mice/condition, repeated twice). ***P ? 0.001.
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Fig. 7. STAT3 inhibition using dnSTAT3 or short hairpin (sh)STAT3 prevented C26 tumor-induced muscle atrophy in mice. Mice were transfected on the day
of tumor inoculation and euthanized 12 days later. A: CMV-dnSTAT3 transfection of tibialis anterior muscle resulted in basal hypertrophy in non-tumor-bearing
mice and reduced muscle wasting in C26 tumor-bearing mice vs. CMV-empty vector transfected controls (n ? 250–500 fibers from n ? 8 mice/condition,
repeated twice). B: immunohistochemistry (brown staining; top) and immunofluorescence (bottom) for pY705-STAT3 in tibialis anterior muscles electroporated
with CMV-dnSTAT3. Note pSTAT3 nuclear localization is reduced in the presence of dnSTAT3 (brown staining in immunohistochemistry, red staining in
immunofluorescence analyses). C: CMV-dnSTAT3 transfection decreased STAT3 DNA-binding activity in nuclear extracts derived from transfected tibialis
muscle in mice with C26 cachexia, as shown by the electrophoretic mobility shift assay (EMSA; n ? 6/condition). §Unlabeled probe control. D: Western blotting
analysis of total lysate from tibialis of control (non-tumor-bearing) and C26 tumor-bearing mice transfected with CMV-dnSTAT3 or empty vector shows
expression of the Flag-taged-dnSTAT3 and GFP, with GAPDH as loading control (n ? 4). E: CMV-shSTAT3 increased muscle CSA and prevented C26-induced
fiber wasting in the tibialis muscle vs. CMV-shScramble (n ? 1,650–2,750 fibers from n ? 8 mice/condition, repeated twice). ***P ? 0.001.
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STAT3-mediated induction of the acute-phase response in
muscle, which we observed previously in C26 tumor-induced
cachexia (8). With the robust induction of acute-phase tran-
scripts, including fibrinogen, haptoglobin, and many other
acute-phase response genes, freed amino acids derived from
proteolysis of structural proteins would be synthesized into
acute-phase response proteins that are subsequently secreted.
Ultimately, this would drain protein reserves in skeletal muscle
and thus represents a causal mechanism for muscle wasting in
cancer. Indeed, Tisdale (53) and Fearon et al. (17) proposed
that, in cancer cachexia, persistent elevations in serum acute-
phase response proteins could result in muscle atrophy.
In the broader context, IL-6 family ligands represent a subset
of all factors associated with or known to induce muscle
wasting. Among the other factors is TNF, which might induce
cachexia partly through inducing IL-6 expression (2) and
perhaps as well through directly activating STAT3 (25). TNF-
induced NF-?B might also promote nuclear retention of
STAT3 and vice versa (34), although none of these proposed
relationships has been examined explicitly in cancer cachexia.
The TGF?/myostatin pathway has been shown to induce ex-
pression of IL-6 (61), suggesting that myostatin might also
work indirectly through IL-6/STAT3, although this must be
experimentally confirmed. Given such interactions, STAT3
inhibition could be additive or synergistic with the targeting of
those other pathways.
In conclusion, our findings identify STAT3 as a causative
factor and potential therapeutic target in muscle wasting during
conditions of high IL-6 family ligands. Currently, there is
much effort directed at developing STAT3 inhibitors for the
purpose of targeting its oncogenic effects (42). An opportune
side effect of such strategies might be the preservation of
skeletal muscle. Given the large number of preclinical and
clinical studies using such approaches, investigators should
consider quantifying the effects of their JAK and STAT3
inhibitors on muscle mass and function.
ACKNOWLEDGMENTS
We thank Drs. Donna O. McCarthy and Denis Guttridge for cell lines, Dr.
Connie Cepko for the pCMV-GFP plasmid, and Dr. James Darnell for the
STAT3 plasmids.
Present address of T. Aydogdu: Sanford Burnham Research Institute, La
Jolla, CA 92037.
Present address of L. Puzis: Department of Anesthesiology, Stony Brook
University, Stony Brook, NY 11794.
GRANTS
This work was supported by American Cancer Society Research Scholar
Grant TBE-111831 to T. A. Zimmers, with support from the Wendy Will Case
Cancer Fund and the Amit family, and by NIH Grants R01-CA-122596 and
R01-GM-092758 and Pennsylvania Department of Health CURE Grant TJU
No. 080-37038-AI0801. This work was conducted using the resources of the
Kimmel Cancer Center Bioimaging and Laboratory Animal facilities.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
A.B., L.G.K., and T.A.Z. did the conception and design of the research;
A.B., T.A., X.J., Z.Z., R.Z., L.P., and T.A.Z. performed the experiments; A.B.,
T.A., and T.A.Z. analyzed the data; A.B., T.A., L.G.K., and T.A.Z. interpreted
the results of the experiments; A.B., T.A., and T.A.Z. prepared the figures;
A.B. and T.A.Z. drafted the manuscript; A.B. and T.A.Z. edited and revised the
manuscript; A.B., T.A., X.J., Z.Z., R.Z., L.P., L.G.K., and T.A.Z. approved the
final version of the manuscript.
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