Role of mRNA splicing regulation in cancer cachexia

We intend to investigate the mechanisms by which tumor-derived soluble factors induce cachexia, i.e. the loss of muscle mass and contractile function. The proposal stands on the following findings: i) tumor-derived factors stimulate the activity of cAMP PDE4D/B isoforms, thus inducing skeletal muscle resistance to tonic beta2-adrenergic signaling sustained by continuous release of norepinephrine from sympathetic autonomous neurons, which in the skeletal muscle keeps mitochondrial function and ATP production by promoting mitochondrial biogenesis. ii) Rolipram, a PDE4-specific inhibitor, rescues defective contractile strength and mitochondrial biogenesis and function of cachectic mice, thus underscoring the link between cAMP signaling and mitochondrial homeostasis iii) an emerging body of evidence indicate that up-regulation of mRNA alternative splicing (AS) is tightly associated to muscle wasting in cancer-induced cachexia, denervation- and microgravity-induced atrophy and in ageing associated sarcopenia. These data suggest that AS activation is an adaptive response to energy stress, i.e. loss of ATP, aimed to restore energetic fitness by restricting energy consumption and by stimulating energy production through activation of catabolic pathways. Thus, we may raise the hypothesis that tumor-induced beta2-adrenergic resistance impairs both mitochondrial biogenesis and restoration of energetic fitness, thus locking AS in a hyperactive pro-catabolic state, which leads to irreversible cachexia. We will test this hypothesis by investigating the following specific aims: i) to perform an AS analysis by carrying out an RNA-Seq in skeletal muscle of control, cachectic and Rolipram treated cachectic mice. As Rolipram partially restores oxygen consumption and energetic fitness, we expect that most of energy stress-induced aberrant AS is normalized. ii) Identification of altered AS events, will allow to build mRNA splicing maps of aberrant AS events in cachectic muscle which may either be restored by Rolipram (energy-driven AS) or not restored (energy-independent AS). We expect that the former would include genes executing the energy-producing catabolic pathways, while the latter would include genes involved in regulatory/signaling network responsible for the tumor-induced irreversible AS deregulation iii) we will investigate the role of tumor-induced altered AS in stimulating PDE4 activity by favoring the generation of the more active “long PDE4D/B isoforms”. We expect that PDE4D/B activation, putatively through an energy independent AS event driving the expression of PDE4D/B more active splice variant, would act upstream to prevent the rescue of energetic fitness by impairing mitochondrial biogenesis and fusion. iv) to identify the molecular mechanisms responsible for the tumor-induced activation of the AS events, not reversed by Rolipram, which act as master coordinators of the initial events triggering the establishment of cachexia.