Discussion
Muscle wasting is one of the hallmarks of cancer cachexia (Penna,
Ballaro, Beltra, De Lucia & Costelli, 2018). STAT3 signaling plays a
crucial role in the regulation of skeletal muscle generation, muscle
mass, and repair processes. It is generally believed that transient
STAT3 activation in muscles is beneficial to muscle regeneration and
hypertrophy, but prolonged STAT3 activation in muscles has been shown to
be responsible for muscle wasting (Zimmers, Fishel & Bonetto, 2016).
Increased STAT3 activation has been observed in skeletal muscles in
diverse models of cancer cachexia, including models based on IL-6
administration, C26 colon adenocarcinoma, LLC lung carcinoma, B16
melanoma, and ApcMin/+-induced intestinal cancer (Baltgalvis, Berger,
Pena, Davis, Muga & Carson, 2008; Bonetto et al., 2012b; Pretto et al.,
2015; Puppa, Gao, Narsale & Carson, 2014; Silvaet al., 2015). However,
the clinical association, the factors triggering abnormal STAT3
activation in cachectic muscles as well as the molecular mechanism of
muscle atrophy induced by consistent STAT3 activation still need to be
delineated. Here, for the first time, our data confirm the clinical
association of abnormal STAT3 activation correlated with the development
of cachectic muscle wasting by measuring the biopsy specimens from liver
cancer patients.
Most STAT3 molecules exist in the cytoplasm in an unphosphorylated form,
and their transcriptional regulatory effects rely on the phosphorylation
of the tyrosine 705 residue. The upstream activators of STAT3 include
IL-6 family cytokines, growth factors such as EGF, oncogenes such as the
Src family kinases, and other biological factors (Yu et al., 1995;
Zhong, Wen & Darnell, 1994; Song et al., 2018). Although the excessive
release of IL-6 family cytokines, including IL-6, LIF, OSM, and CNTF,
has been linked to the pathological development of cancer cachexia
(Fearon, 2013). Multiple studies on STAT3 signaling have demonstrated
that the stimulation of non-malignant cells with IL-6 family cytokines
can trigger only transient STAT3 activation, which is quickly terminated
by negative regulators such as PIAS, suppressor of cytokine signaling
(SOCS-1, 2, and 3), and cellular phosphatases (SHP-1/2, DUSP22, PRPRD,
PTPRT, and PTPN1/2) (Johnson, O’Keefe & Grandis, 2018; Ram & Waxman,
1997; Sengupta, Schmitt & Ivashkiv, 1996). Therefore, it is reasonable
to speculate that apart from IL-6 family cytokines, other regulatory
factors may also be involved in maintaining the persistent STAT3
activation in cachectic muscles in cancer.
HSP90 is an ATP-dependent molecular chaperone; it has been reported to
be one of the most abundant molecules within cells, accounting for
nearly 2% of all cellular proteins (Joshi, Wang, Araujo, Sharma,
Brodsky & Chiosis, 2018). A wide variety of stressful stimuli,
including heat shock, UV radiation, and micro-organismal infections,
could promote intracellular HSP90 synthesis. During cancer progression,
the expression level of HSP90 increases by 2- to 10-fold in tumor cells
(Joshi, Wang, Araujo, Sharma, Brodsky & Chiosis, 2018). Elevated HSP90
expression has been found in various types of human tumors and is often
correlated with increased tumor cell proliferation, inadequate responses
to chemotherapy, and poor survival (Kazarian et al., 2017; Ono et al.,
2018; Whitesell & Lindquist, 2005; Yun, Kim, Lim & Lee, 2019). Over
200 oncogenic proteins have been identified as clients of HSP90 (Trepel,
Mollapour, Giaccone & Neckers, 2010). We and others have demonstrated a
direct interaction between STAT3 and HSP90 in multiple cell types,
including cancer cells, epithelial cells, fibroblasts, and endothelial
cells (Song et al., 2017; Shah, Patel, Fried & Sehgal, 2002). Here, in
this study, we found an increased interaction of HSP90 and STAT3 in
skeletal muscle from cachexia patients and experimental mice model.
Administration of HSP90 inhibitors, which blocked the aberrant STAT3
activation, could successfully alleviate cachexia-related muscle wasting
phenotype.
Interestingly, we revealed that the direct interaction of HSP90 with
STAT3 could enhance JAK2-mediated STAT3 phosphorylation, thus induce
muscle atrophy. The increased HSP90-STAT3 interaction likely causes the
prolonged STAT3 activation and muscle atrophy since both HSP90 inhibitor
or STAT3 siRNA could successfully alleviate the pathological muscle
wasting phenotype both in vivo and in vitro ; suggesting
that persistent activation of STAT3 rather than other signaling pathway
is the major cause of the induction of muscle wasting in cancer
cachexia. A previous study by Zhang et al. reported that tumor released
HSP90 contained exosome could trigger muscle wasting by activation of
TLR4, to validate whether TLR4 mediates the protective effect of HSP90
inhibitors in cachectic muscle wasting, we repeated the experiment by
using the LPS induced the C2C12 myotube atrophy model, however, to our
surprise, we did not observe the obvious protective effects; this result
suggested that the protective effect of HSP90 inhibitors in cachectic
muscle wasting is not fully TLR4 dependent. TLR4 signaling might be
involved in the initial stage of STAT3 activation, but the tumor induced
a prolonged STAT3 activation, which is necessary for triggering the
muscle wasting as demonstrated in our mechanism study by over-expression
of the consistent activated form of STAT3 (STAT3-C) in C2C12 myotube,
required the participation of HSP90 as a molecular chaperone by the
interaction of STAT3. Therefore, it is reasonable to speculate that the
enhanced HSP90-STAT3 interaction seems to be the critical event for the
induction of persistent STAT3 activation and cachectic muscle atrophy.
An additional goal of this study was to delineate the mechanism by which
the prolonged STAT3 activation induces muscle wasting. The pathogenesis
of muscle wasting in cancer cachexia has been demonstrated to be the
result of an imbalance between the anabolic and catabolic pathways. The
STAT3 pathway has been found to be a crucial pathway involved in this
process. During the catabolic response in skeletal muscles, prolonged
STAT3 activation could disturb the sensitivity of the insulin/IGF-1/AKT
signaling pathway by enhancing the expression of inhibitory SOCS
proteins (Mashili, Chibalin, Krook & Zierath, 2013; Ueki, Kondo &
Kahn, 2004). During the anabolic response, current models indicate that
STAT3 could enhance the expression of myostatin via activation of
CAAT/enhancer-binding protein (C/EBP) (Zhang et al., 2013). Myostatin is
a TGF-beta family cytokine that can repress muscle growth and disturb
myogenesis. Our study observed the elevated expression of myostatin
accompanied by prolonged STAT3 activation, which fully supports our
conclusion. However, the most interesting mechanism revealed by our
study is that the prolonged activation of STAT3 in skeletal muscle
derived from either C26 tumor-bearing cachectic mice or C2C12 myotubes
treated with C26 CM was correlated with elevated expression of
UPS-related muscle-specific E3 ubiquitin ligases, such as Atrogin-1 and
MuRF1. In addition, the transfection-induced overexpression of
constitutively activated STAT3 (STAT3-C) also triggered E3 ligases,
indicating that constitutive activation of STAT3 itself might be enough
to induce muscle wasting by activating E3 ligases mediated protein
degradation pathways. However, the pathway linking STAT3 and the
activation of the protein degradation pathway has never been evaluated.
FOXO1, as a key member of the forkhead box protein O (FOXO)
transcription factor family, has been found to be widely expressed in
various types of metabolism-associated organs, such as the liver and
skeletal muscles. In skeletal muscles, the FOXO1 transcription factor
represents a master regulator of muscle growth, regeneration, and
atrophy. Increased FOXO1 expression and activation have been found in
skeletal muscle in energy-deprived states such as fasting, cancer, and
severe diabetes (O’Neill et al., 2019; Reed, Sandesara, Senf & Judge,
2012). Enhanced FOXO1 activation in skeletal muscle triggered the muscle
atrophy by activation of the UPS and lysosomal proteolysis through the
upregulation of FOXO downstream target genes, including atrogin-1 and
MuRF1 (Milan et al., 2015). Genetic data demonstrated that STAT3
expression and activation are correlated with the intact function of
FOXO transcription factors; however, whether FOXO1 is necessary for
STAT3-induced muscle wasting or vice versa is still not known. In this
study, we demonstrated for the first time that STAT3 could regulate the
expression and nuclear translocation of FOXO1 in the skeletal muscle,
subsequently up-regulated E3 ligases and FOXO1-dependent triggering of
muscle wasting, which could partly explain the pathological mechanism by
which consistent STAT3 activation in the skeletal muscle could trigger
muscle atrophy. Since the bioinformatics study demonstrated that there
are several STAT3 consensus motifs in FOXO1 promoter regions, our
subsequent ChIP analysis indicated that STAT3 could bind to the FOXO1
promoter and directly regulate the FOXO1 gene transcription in skeletal
muscles. To test the pathogenic effects of STAT3 on muscle atrophy, a
constitutively active recombinant STAT3 mutant expression plasmid,
STAT3-C, was adopted in our study. Our results showed that the
overexpression of the constitutively activated form of STAT3 in skeletal
muscle could mimic the effect of the C26 cell-conditioned medium on
muscle wasting phenotype induction. Knocking down the expression of
FOXO1 in STAT3-C-transfected or C26 cell-conditioned medium-treated
myotubes almost completely abolished the activation of the
ubiquitin-proteasome system and eliminated muscle wasting, suggesting
that STAT3 activation-induced muscle atrophy is indeed FOXO1 dependent.
In summary, our study demonstrated that HSP90 inhibitors could
effectively alleviate cancer cachexia-induced skeletal muscle wastingin vivo ; these effects might be associated with the abnormal
activation of the JAK2/STAT3/FOXO1 and ubiquitin-proteasome pathways.
The close monitoring of HSP90 levels in cancer patients and the
development of strategies to block HSP90 activity could potentially
prevent or reverse cachexia development.