Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Serum Response Factor is required for the therapeutic effects of physical activity in cancer-cachexia

Cachexia is a multifactorial and complex syndrome associated with chronic diseases such as cancer, which leads to an involuntary and progressive loss of body weight. Cachexia is characterized by an increased level of pro-inflammatory cytokines and other humoral factors, which induce autophagy and proteolysis mediating muscle fiber atrophy. Muscle wasting interferes with the therapy of the primary disease and is a negative prognostic factor for cancer patients. Physical activity has been proposed to counteract cachexia since it improves muscle metabolism and decreases systemic inflammation by regulating the balance between anti- and pro-inflammatory cytokines. Physical activity is thought to promote the release of beneficial myokines from the exercised muscle. Exercise is associated with an improvement in the quality of life and survival in cancer patients. However, the mechanisms underlying the response to physical exercise in cancer cachexia are not known. We hypothesized that the effects of exercise are mediated by a mechano-transduction response in muscle, involving Serum Response Factor (SRF), which is a gene transcription factor induced by mechanical cues mediated by actin polymerization. Since the broad involvement of SRF in muscle homeostasis includes a pivotal role in satellite cell proliferation and recruitment to myotubes, we also hypothesized that SRF beneficial effects are extended to the satellite cell niche. To verify our hypothesis, we used an in vivo model consisting of mice injected or not with C26 tumor cells, to induce cancer cachexia. The animals were further divided into two groups, which performed or not voluntary exercise. In this way, we had four experimental groups allowing us to test any interaction between exercise and cancer cachexia. To specifically address the SRF role in the rescue of muscle mass by physical exercise in cancer cachexia, we used an SRF loss of function models, consisting of inducible, muscle-specific SRF KO mice with the Cre loxP system activated by tamoxifen injections For these mice, a different tumor, i.e., the Lewis Lung Carcinoma (LLC) was used to induce cachexia, due to immune incompatibility resulting in tumor rejection in these non-immunoexpressed mouse lines. Our results showed that the C26 tumor induces loss of body weight, muscle wasting, protein degradation, including a decrease of SRF protein, which resulted from the downregulation of its transcriptional activity. We observed that the effects of C26 tumors were counteracted by physical activity, suggesting that voluntary exercise is an effective means to counteract cachexia; we also observed that the beneficial exercise effects are linearly correlated to the SRF protein levels in muscle, suggesting a pivotal role for SRF as a mediator of the response to exercise. To demonstrate that SRF is necessary to transduce exercise effects, we applied the same exercise protocol as above to LLC tumor-bearing SRF KO mice and observed that exercise could no longer rescue cachexia, thereby indicating that SRF is essential for the organismal response to exercise. Additionally, we observed opposite effects of the tumor and exercise on Pax7+ cells, i.e., the satellite cells which are responsible for muscle regeneration and homeostasis. Since we also observed the recruitment of nuclei within the muscle fibers in response to exercise, we concluded that the myogenic program is activated by exercise, resulting in stem cell recruitment to muscle fibers, which could contribute to muscle homeostasis. Overall, our data demonstrate that the positive effects of exercise on cancer cachexia are due to an SRF-mediated mechanical response of muscle fibers, resulting in transcriptional changes activating pro-myogenic genes and in the recruitment of muscle stem cells which ultimately result in the rescue of muscle mass.