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.