Molecular determinants contributing to cAMP signaling dysfunction in a cancer cachexia in vitro model

Cancer cachexia is a wasting syndrome characterized by a severe loss of skeletal muscle mass and strength, associated with shortening of patient’s survival and worsening of the quality of life. Despite its high frequency, cancer cachexia molecular causes are still poorly understood. Soluble factors released by the tumor define a situation of chronic systemic inflammation, leading to skeletal muscle mitochondrial dysfunction and protein degradation, thus driving the wasting syndrome. Interestingly, β-adrenergic-dependent cyclic adenosine monophosphate (cAMP) signaling activation improves mitochondrial functionality through a PKA/CREB-dependent mechanism. Notably, our previous results demonstrate that the pro-cachectic environment impairs skeletal muscle cAMP signaling, thus contributing to mitochondrial dysfunction and muscle wasting. The pro-cachectic environment consists of a complex cocktail of cytokines and soluble factors, including IL6, LIF, TNFα, and TGFβ/Activins, which activate downstream transcriptional effectors such as STAT3, Nf-κB, and Smad2/3. In this thesis, we explored the hypothesis that such cytokines may contribute to defective cAMP signaling and mitochondrial dysfunction. Thus, we assay whether the different cytokines impair β-adrenergic-induced activation of cAMP signalling pathway, through several cellular, biochemical, and molecular assays. Our in vitro results show that TGFβ inhibits isoproterenol-induced cAMP production (FRET assay), CREB phosphorylation, and expression of CREB-dependent genes in both C2C12 myoblasts and myotubes, thus suggesting that activation of Smad2/3 transcription factors play a key role in cAMP signaling dysfunction. Nevertheless, both IL6 and the cocktail of cytokines inhibit isoproterenol-induced CREB-phosphorylation and CREB-dependent gene expression. Altogether, these data indicate that multiple cytokines contribute differently to the defective cAMP/PKA/CREB signaling.