Figuring out Nfix role in Cancer-Associated Cachexia: a novel player in Muscle Wasting

Cancer-associated cachexia is a debilitating disorder characterized by unintentional body weight loss and skeletal muscle wasting, accounting for 20-30% of cancer related death1-2. Muscle atrophy derives from an imbalance between protein synthesis and protein degradation, favouring the latter. This involves a reduction of pro-anabolic pathways alongside an increase in the pro-catabolic signaling and upregulation of atrogenes3. Nuclear Factor I X (NFIX) is a transcription factor that plays a pivotal role in skeletal muscle. Indeed, it is crucial during development4 when it determines the switch from embryonic to foetal myogenesis, but it is also important in adulthood, controlling tissue homeostasis and timing of skeletal muscle regeneration after acute damage5. Interestingly, NFIX regulates the expression of two growth factors involved in the control of muscle mass. It upregulates Insulin Growth Factor 14, which promotes muscle growth, and inhibits Myostatin5, a negative regulator of muscle mass. This regulatory balance is particularly relevant in cancer cachexia, and so we asked if NFIX could be a novel central player in this disorder. Therefore, NFIX protein levels were assessed in the Tibialis Anterior (TA) of one of the most severe models of cancer cachexia, induced by the subcutaneous injection of C26 colon adenocarcinoma cells in BalB/C mice. A significant downregulation of NFIX expression was observed. Similarly, both Nfix mRNA and NFIX protein levels were significantly reduced in an in vitro model of cancer cachexia, generated by treating C2C12 myotubes with the Conditioned Medium (CM) from C26 cells. To evaluate if NFIX overexpression might be a new approach to counteract cachexia, C2C12 were transduced with a lentiviral vector containing NFIX under the CMV promoter. Preliminary results demonstrated that NFIX overexpression exerts a protective effect against atrophy in in vitro culture. Along with the deciphering of NFIX function in skeletal muscle during cancer cachexia, it is crucial to determine whether NFIX modulation could affect tumour properties. This is particularly important when considering NFIX overexpression as new therapeutic approach, as any proposed intervention must avoid giving benefits to tumour growth or invasion. Interestingly, silencing NFIX, using a specific short hairpin RNA, in C26 resulted in decreased proliferation and increased migration. Moreover, CM from C26 shNfix cells induces a more pronounced atrophic phenotype in C2C12 myotubes, suggesting that the absence of Nfix may confer a pro-tumorigenic advantage while exacerbating muscle wasting In summary, the study of in-vitro and in-vivo models of cancer cachexia identify NFIX overexpression as a promising therapeutic strategy to preserve of skeletal muscle mass during cancer-associated cachexia, without conferring advantages to the tumour.