Significance of the study
The study examined the proteins and biological pathways that are altered
in C26-cancer-induced atrophied muscle. This proteomic-based analysis
provides valuable information for expanding our knowledge of the
dysregulated proteins and signalling pathways that leads to catabolic
gene expression during cancer-induced muscle wasting. The identified
enriched proteins in atrophied muscle could be employed as potential
muscle wasting markers and the dysregulated biological processes could
be utilized for therapeutic development.
Cancer-associated cachexia is a multifactorial wasting syndrome that is
characterised by skeletal muscle atrophy with or without the loss of
adipose tissue (Fearon, Strasser et al. 2011, von Haehling and Anker
2014). This debilitating condition causes significant weight loss that
cannot be reversed by conventional nutritional support and results in
functional impairment, poor quality of life, chemotherapy or/and surgery
related complications, and reduced survival (Fearon, Arends et al. 2013,
Baracos, Martin et al. 2018). Most (50-80%) of the advanced cancer
patients suffers from cachexia, however, the severity varies with the
cancer type (Lim, Brown et al. 2020). This wasting condition is
multifactorial and thought to be driven by a number of factors,
including reduced food intake, inflammation and metabolic changes
(Tisdale 2009).
Muscle wasting is a key feature of cachexia, which is attributed by
increased protein degradation and decreased protein synthesis (Wyart,
Bindels et al. 2020). Muscle plays an important role in maintaining
metabolic homeostasis of the body as it serves to be the principal depot
for amino acids required for protein synthesis and energy production
(Wolfe 2006). Muscle protein homeostasis is maintained by fine balance
between anabolism and catabolism of proteins. Disruption to this balance
results in loss of net muscle protein which leads to muscle wasting
(Mantovani, Macciò et al. 2001, Webster, Kempen et al. 2020). Cancer
cells tend to secrete various pro-inflammatory and pro-cachectic factors
via the classical secretory pathway and extracellular vesicles (EVs)
which disrupt the balance between the anabolism and catabolism of
proteins, tipping the balance toward catabolism (Chitti, Kang et al.
2023). These catabolic factors are known to activate autophagy,
apoptosis, and ubiquitin proteasome pathways that result in reduced
protein synthesis and increased protein degradation (Suzuki, Asakawa et
al. 2013, Brown, Lee et al. 2018, Webster, Kempen et al. 2020). However,
little or no clinical benefit has been reported upon targeting some of
these secreted catabolic or inflammatory factors (Advani, Advani et al.
2018, Roeland, Bohlke et al. 2020).
Critical knowledge gaps remain in understanding the muscle pathology of
how tumor-derived factors leads to disrupted skeletal muscle metabolism
and functions. To develop new therapies, it is vital to have extensive
understanding of the dysregulated proteins and signalling pathways that
results in activation of catabolism during muscle atrophy. In this
study, we evaluated the protein profile of C2C12 myotubes upon treatment
with C26-derived conditioned media (CM) that contains secretory factors
and EVs. Label-free quantitative proteomics analysis was performed using
high-resolution mass spectrometry on C2C12 myotubes treated with
C26-derived CM. Functional enrichment analysis revealed enrichment of
proteins associated with inflammation, mitochondrial dysfunction, muscle
catabolism, ROS production, and ER stress in C26-derived CM treated
C2C12 myotubes.
To examine the effect of
C26-derived CM on
myotubes, first C2C12 myoblast
were differentiated into myotubes using horse serum. Differentiated
myotubes were then treated with C26-derived CM, or differentiation media
(untreated) for 48 h (Figure 1A ). Immunofluorescence images
highlighted that treatment with C26-derived CM resulted in significant
decrease in myotube diameter compared to the untreated myotubes
(Figure 1B and C ). Further validation with Western blot
depicted a decrease in the abundance of muscle abundant protein; myosin
heavy chain (MyHC) (Figure 1D ). Additionally, the metabolic
activity of C2C12 myotubes treated with C26-derived CM was also
evaluated at 24, 48 and 72 h. The metabolic activity was found to be
significantly lower upon C26-derived CM treatment compared to untreated
C2C12 myotubes at all three time points (Figure E ).
Collectively, these data confirms that C26-derived CM induces muscle
atrophy in vitro and alters the metabolic function of the muscle
cells.
To study the protein abundance changes that occur in skeletal muscles
during C26 cancer-induced muscle atrophy,
mass spectrometry-based
label-free quantitative proteomics analysis was performed with untreated
C2C12 and C26-derived CM treated C2C12 (Figure 2A ). A total of
1901 and 1823 proteins (Supplementary table 1) were identified
in the untreated and C26-derived CM treated C2C12 cells, respectively,
and 1735 proteins were found to be commonly present in both
(Figure 2B ). For an unbiased rigorous analysis, only proteins
identified across all 3 biological replicates were considered for
further analysis. The subsequent quantitative Venn diagram indicated 166
and 88 proteins to be uniquely present in the untreated and C26-derived
CM treated C2C12 cells, respectively. Among the 1735 commonly identified
proteins between untreated and C26-derived CM treated C2C12, 472
proteins were found to be more abundant in the untreated C2C12 cells,
and 546 proteins were more abundant in C2C12 cells treated with
C26-derived CM (Figure
2C ). Overall, these results suggest that the proteome of C2C12 myotube
was altered with the treatment of C26-derived CM.
To assess the changes in the protein abundance in C2C12 myotube upon
treatment with C26-derived CM, volcano plot was employed. Volcano plot
was plotted for significant proteins with FDR correction of 0.05.
Comparison of the proteins identified in untreated C2C12 myotube to
C26-derived CM treated C2C12 myotube highlighted the significant
upregulation of lysosomal proteases Ctsl1 and Lgmn expression,
implicated in autophagy (Figure 2D ). Furthermore, AMPD3
expression was also found to be significantly higher in C26-derived CM
treated C2C12 myotube (Figure 2D ). Increased expression of
Ampd3 has been shown to significantly reduce the rate of mitochondrial
protein synthesis and thereby alter the metabolic phenotype of atrophied
muscle (Miller, Hafen et al. 2021). On the contrary, proteins involved
in muscle differentiation, proliferation, energy production and
protection against oxidative stress such as Fn1, Thbs1, Acadm, Aldh2 and
Txnrd1 were enriched in untreated C2C12 (Figure 2D ).
To better understand and visualize the changes in the protein profile of
C2C12 upon treatment with C26 CM across all 3 biological replicates, a
heatmap was generated with proteins that were statistically significant
with fold change greater than ≥2 (Figure 2E ). Proteins involved
in mitochondrial dysfunction, ER stress and cytokine production such as
Cox5b, Upp2, Cd14, Sdf2l1, and Snx7 were significantly abundant across
all 3 biological replicates of C26-derived CM treated C2C12 myotubes.
Consistently, proteins involved in muscle development and/or
regeneration such as Ncstn, Fn1, Enpp1 and Tgm2 were found to be
depleted in all 3 biological replicates of C2C12 myotubes exposed to
C26-derived CM.
Next to check the biological processes that were altered in C2C12
myotube upon C26-derived CM treatment,
functional enrichment analysis
using FunRich was performed (Pathan, Keerthikumar et al. 2015).
Statistically significant proteins with fold change greater than ≥2 was
taken into consideration. Top pathways activated in C26-derived CM
treated C2C12 were inflammatory cytokine signalling; especially signal
transduction through the interleukin-8 (IL-8), interferon-gamma (IFN-γ),
Toll-like receptor 4 (TLR4), positive regulation of NIK/NF-κB, and
endoplasmic reticulum (ER) stress mediated signalling pathways. The
results further depicted altered electron transport chain and
mitochondrial respiration (Figure 3A ). On the other hand,
muscle development or regenerative function such as Notch signalling,
platelet-derived growth factor receptor-beta signalling pathway and
negative regulation of Transforming Growth Factor-β (TGF-β) signalling
pathways were among the major downregulated biological processes in
C2C12 myotube treated with
C26-derived CM (Figure
3B ). Additionally, biological processes implicated with muscle
structural integrity such as regulation of cell shape, extracellular
matrix organization and cytoskeletal organization were also perturbed
upon C26 CM treatment (Figure 3B ). Taken together, C26-derived
CM activate the inflammatory pathways and inactivate muscle development
pathways in C2C12 myotubes.
Next, Western blotting was used to validate the proteomics data.
Consistent with the proteomics analysis, Western blot also confirmed the
activation of ER stress in C2C12 treated with C26 CM. ER stress markers
such as BiP and calnexin was found to be significantly abundant in C26
CM treated C2C12 compared to untreated C2C12 (Figure 3C and D ).
Muscle atrophy is one of the core features of cancer-associated cachexia
which appears to be driven by tumour derived secretory factors and
extracellular vesicles (Biswas and Acharyya 2020, Wang, An et al. 2022).
Given that C26 colon cancer cell is one of the most well-characterized
and extensively used model for cancer-associated cachexia, we performed
LC-MS/MS-based proteomic profiling of C2C12 myotubes after incubating
with C26-derived CM.
Mitochondrial metabolic dysfunction is an important element of the
wasting process, as it precedes the development of atrophy (Halle, Pena
et al. 2019). Consistent with this notion, the proteomic analysis
revealed an alteration in proteins associated with impairment of several
metabolic pathways which is supported by the finding from the MTS assay.
AMP deaminase 3 (Ampd3) has been reported to be one of the most
upregulated genes in atrophied muscles (Lecker, Jagoe et al. 2004).
Consistent with previously published observations, Ampd3 was found to
significantly enriched in C2C12 treated with C26-derived CM. Ampd3 is a
cytosolic enzyme that is responsible for irreversible deamination of
adenosine monophosphate (AMP) to inosine monophosphate (IMP). This
causes a reduction of mitochondrial protein synthesis and ATP
production, altering metabolism in muscle during atrophy (Winder, Holmes
et al. 2000, Ibebunjo, Chick et al. 2013, Miller, Hafen et al. 2021).
Furthermore, cytochrome c oxidase subunit 5B (Cox5b) was found to be
28-fold higher in C2C12 myotubes
upon C26-dervied CM treatment. Cox5b has been implicated with reactive
oxygen species (ROS) and nitric oxide (NO) production in hypoxic
condition which can lead to mitochondrial dysfunction and muscle wasting
(Wang and Chaudry 1996, Hinkelbein, Böhm et al. 2017, Nuoc, Kim et al.
2017). Stromal cell-derived factor 2-like 1 (Sdf2l1) is another protein
which was found to be 26.8-fold higher in C2C12 myotubes exposed to
C26-derived CM. Expression of Sdf2l1 has been reported to be induced by
ER stress (Tiwari, Schuiki et al. 2013). Several seminal studies have
shown ER stress is activated in different model of cancer-associated
cachexia and is involved in muscle atrophy (Niida, Masuda et al. 2020,
Gallot and Bohnert 2021). ER stress tend to trigger the unfolded protein
response (UPR) to restore cellular homeostasis. However, prolonged ER
stress response can activate apoptotic signals which in turn damage the
target cells (Bhattarai, Chaudhary et al. 2020). Additionally, there was
a significant abundance of lysosomal proteases such Ctsl1 and Lgmn
expression, known to be involved in autophagy. Deval, Mordier et al. has
demonstrated a positive correlation between Ctsl expression and
proteolysis intensity suggesting Ctsl can serve as early marker of
muscle wasting (Deval, Mordier et al. 2001).
Functional enrichment analysis exhibited the activation of NF-κB
signally pathways in C26-derived CM treated C2C12 myotubes. Indeed, it
is well documented that upregulation of NF-κB in skeletal muscle induces
muscle atrophy by degrading specific muscle proteins and inhibiting
regeneration of myofibers by blocking MyoD synthesis (Guttridge, Mayo et
al. 2000, Li, Malhotra et al. 2008, Thoma and Lightfoot 2018). Moreover,
inflammatory cytokine signalling pathways such as Toll-like receptor 4
signalling pathway, lipopolysaccharide-mediated signalling pathway,
positive regulation of cytokine secretion, TNF-α production, IFN-γ
production, and inflammatory response were also found to be
significantly upregulated C2C12 myotubes treated with C26 derived CM.
TLR4 is known to be a master regulator of inflammatory catabolism.
Activation of TLR4 by lipopolysaccharide (LPS) has been reported to
induce C2C12 myotube atrophy via upregulation of ubiquitin ligases such
as atrogin-1 and MuRF1 expression and formation of autophagosome (Doyle,
Zhang et al. 2011, Yadav, Dahuja et al. 2021). TNF-α is known to have
muscle catabolic properties (Layne and Farmer 1999, Greiwe, Cheng et al.
2001). TNF-α has been reported to inhibit protein synthesis in muscle by
inhibiting the expression of differentiation-specific genes (Layne and
Farmer 1999, Williamson, Kimball et al. 2005). It is also known to
promote ubiquitin-proteasomal mediated muscle catabolism by increasing
the production of mitochondrial ROS and activating NF-κB signalling
pathway (Li, Atkins et al. 1999, Li and Reid 2000).
On the other hand, proteins and biological processes that were
significantly downregulated in C2C12 upon C26-derived CM treatment were
related to muscle structural integrity such as cell shape regulation,
extracellular matrix (ECM), and cytoskeletal organization. Muscle
development or/and regenerative pathways such as Notch and
platelet-derived growth factor receptor-beta
(PDGFRβ) signalling pathways were
also significantly less abundant in
C2C12 myotubes treated with
C26-derived CM. Notably, both
PDGFRβ and notch signalling
pathways play an important roles in skeletal muscle development and
regeneration (Piñol-Jurado, Gallardo et al. 2017, Vargas-Franco, Kalra
et al. 2022). PDGFRβ has been found to be involved in muscle fibre
hypertrophy and ECM production that is needed for the muscle growth and
development (Uezumi, Fukada et al. 2014, Sugg, Korn et al. 2017). ECM is
a complex type of connective tissue crucial for regulating myogenesis
and skeletal muscle development (Huang, Ge et al. 2011). In addition to
maintaining skeletal muscle morphology and contraction, ECM also
regulate various physiological functions of skeletal muscle (Zhang, Liu
et al. 2021). Fibronectin facilitates the fusion of myoblast tubes
during myoblast differentiation and was also depleted in C2C12 myotubes
treated with C26-derived CM (Vaz, Martins et al. 2012). Deficiency of
fibronectin can cause skeletal muscle dysfunction by perturbing ECM and
myotube formation (Knoblauch, Will et al. 2007).
Taken together, this
proteomic-based study provides a better understanding of the mechanisms
leading to C26 cancer induced muscle wasting. Further investigation of
the upregulated proteins identified in C2C12 myotubes upon treatment
with C26-derived CM might serve as potential muscle wasting markers.