C26 cell conditioned medium blocks myostatin growth while mechanical stimulation counteracts this negative effect

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Benefit of exercise practice during cancer cachexia

Recently, it was shown in tumor-bearing mice performing wheel running that natural killer cells were recruited to attack the tumor and decreased its mass, thus contributing to remove a primary cause of cachexia. Nonetheless, it is clear that exercise has direct effects on muscle cells and tissues, in both physiological and pathological conditions (Pigna et al., 2016; Padilha et al., 2017; Aversa et al., 2017).
Epidemiological studies and one clinical trial showed that physical activity that occurs post-diagnosis improves prognosis, rather that exercise habits established before disease (breast and colorectal cancers): indeed, physical activity after diagnosis lowers the risk of both cancer-specific and overall mortality, ameliorates patients’ quality of life and increases survival (Holmes et al., 2005; Meyerhardt et al., 2006; Irwin et al., 2008; Holick et al., 2008).

Molecular pathway involved in exercise response

It is worth noting that exercise used as a support to medical treatments may vary significantly in intensity and metabolic impact. Resistance exercise training is defined as multiple repetitions of static or dynamic muscular contractions performed against a high load or resistance. Resistance training increases muscle mass in healthy subjects and attenuates muscle wasting associated with ageing. Endurance exercise training consists of performing low-to-medium intensity exercise for long periods of time. Endurance training (such as running, cycling, or swimming) involves the use of several large groups of muscles and tends to be aerobic. Adaptations to endurance exercise include improved oxygen delivery to muscles and their increased oxidative capacity (Hakkinen et al., 2005).
The IGF1/AKT/FoxO pathway, known to induce muscle hypertrophy via protein synthesis and a reduction of protein degradation, is activated by both training types. Aerobic exercise induces mitochondrial biogenesis via AMPK activity, which, in turn, induces PGC1a activation leading to potentiated ATP synthesis. At day 1 after overload, which mimics resistance training, the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway is activated. This pathway can activate mTOR to induce protein synthesis. However, ERK1/2 are also activated by endurance training via integrins like integrins 1 and 7 which form a dimere, a mechanotransductor on cytoplasmic membrane.

Regulation of SRF activity by Rho/actin/MRTFs pathway

SRF by itself is a low transactivator and it needs coactivators such as ternary complex factors (TCF) or Myocardin-related transcription factors (MRTFs) to induce the transcription of SRF target genes (Cen et al., 2003; Schroter et al., 1990). TCF family members are activated via phosphorylation by MAPK like ERK, JNK or p38. The phosphorylation of TCF induces the inhibition of interaction between it C domain and ETS domain, which, in turn, increases DNA affinity (Posern et al., 2006; Yang et al., 1999). TCF forms a complex with SRF and a Serum response element (SRE) in the promoter of target genes. In this way, the ETS domain recognizes an EBS region near of CArG box in promoter of SRF target genes.
MRTFs show high homology with myocardin, another cardiac-muscle specific SRF cofactor. The two MRTFs, MRTFA (MAL/MKL1/SAC) and MRTF-B (MAL16/MKL2), are ubiquitary. Their subcellular localization is regulated by actin dynamics. Some studies show that MRTFA is localized most of the time in the cytoplasm and that stimulation induces its accumulation in the nucleus (Miralles et al., 2003). Modulation of actin dynamics mediates signal-induced SRF transcriptional activity: stabilization of filamentous actin (F-actin) by the actin-binding drug, jasplakinolide, is sufficient to activate SRF without extracellular stimuli, but overexpression of actin inhibits SRF (Sotiropoulos et al., 1999). The Rho family of Ras-related GTPases (RhoA), controls the polymerization of the actin cytoskeleton in response to extracellular signals, but also myosin-based contractility, focal adhesion formation, transformation and cytokineses (Olson et al., 2010). RhoA regulates formines like mDia and ROCK activity (Rho-associated protein kinase). mDia controls profiline and stimulate its polymerization. ROCK controls LIMK activity (LIM domain kinase), which induces actin polymerisation by repression of cofiline activity (Miralles et al., 2003; Pollard et al., 2003, dos Remedios et al., 2003). Profilin and cofilin regulate concentration of monomeric actin (Globular-actin/G-actin) and transport of G-actin between nuclei and cytoplasm. In fact, the association of cofilin with importin 9 allows the import of monomeric actin in the nucleus; on the contrary, interaction between profiling and exportin 6 induces G actin exportation to cytoplasm (Dopie et al., 2012). Striated muscle activator of Rho signalling (STARS) controls MRTFA localization directly by actin polymerisation and indirectly via the activation of RhoA (Kuwahara et al., 2005; Zheng et al., 2009).

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C26 cells conditioned medium blocks myotubes growth while mechanical stimulation counteracts this negative effect

We established cell cultures containing a mixed population of myotubes and unfused myoblasts, obtained by 4d culturing in differentiation medium (DM). In the order to study the effects of tumor-derived factors on muscle cell culture, C26 cell conditioned medium was obtained and used to treat C2C12 mixed cultures. In preliminary experiments, two-day treatment with a DM containing 20% of C26 conditioned medium (CM) induced myotube atrophy and hampered myoblast differentiation (data not shown). Control medium or CM were, therefore, used to treat C2C12 mixed cultures, in the absence (static condition, SC) or presence (dynamic condition, DC) of mechanical stimulation consisting of daily sessions of longitudinal stretch for 6 hours per day, as described in M&M (Figure 9).

Table of contents :

INDEX
THE LIST OF ABBREVIATIONS
THE THESIS EXPLAINED
INTRODUCTION
1. Muscle mass homeostasis in the adult
1.1 Skeletal muscle regeneration
1.2 Muscle mass regulation.
2. Cancer effects on muscle mass
3. Exercise effect on cancer cachexia
3.1 Benefit of exercise practice during cancer cachexia.
3.2 Molecular pathway involved in exercise response
4. Serum response factor
4.1SRF expression
4.2SRF target genes
4.3 Regulation of SRF activity by Rho/actin/MRTFs pathway
4.4SRF Knock-out in skeletal muscle cells
OBJECTIVES
RESULTS.
5. C26 cell conditioned medium effects on C2C12 myotube
5.1 C26 cell conditioned medium blocks myostatin growth while mechanical stimulation counteracts this negative effect
5.2 Mechanical cues rescue the myogenic differentiation program via MRFs activation
6. C26 cell conditioned medium and mechanical cues affect myogenic differentiation via secreted factor by modulating secreted factors
6.1 Mechanical cues restore Activin A and Follistatin balance favouring myogenic differentiation
6.2 Follistatin is not sufficient to counteract myotube atrophy due to conditioned medium
7. SRF activity counteracts the negative effect of C26 cell conditioned medium on myogenic differentiation
7.1 Mechanical cues activate the Rho/actin/MRTFs pathway
7.2 MRTF-A translocation to the nucleus is stimulated by mechanical cues
7.3 SRF expression increases in presence of mechanical cues
7.4 A knock-down of SRF is sufficient to block the myogenic differentiation rescue associated to mechanical cues
DISCUSSION
MATERIAL AND METHODS
8. Cell culture
8.1 C26 conditioned medium protocol
8.2 Flexcell
9. Infection by adenovirus MRTF-A-GFP
10. Cell treatment by recombinant follistatin
11. SRF Knock-down
12. Immunofluorescence
PhD in Morphogenesis and Tissue Engineering
PhD in Physiology, physiopathology and therapeutics
13. RNA extraction, cDNA synthesis and Q-PCR
14. ELISA
15. Western Blot
15.1 Western Blot
15.2 Western Blot of F and G-actin
16. Imaging and quantification
17. Statistical analysis
REFERENCES

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