Setting a combinatorial approach for future RNA-based muscle-targeted intervention in cancer cachexia

ABSTRACT BACKGROUND Cachexia is a multifactorial syndrome that occurs in multiple diseases, cancer above all. It is characterized by involuntary loss of body weight and loss of homeostatic control of energy and protein balance. Skeletal muscle is the main tissue lost and it comes together with systemic inflammation. This leads to an increase in morbidity and reduction in both tolerance and responsiveness to treatment regimens, resulting in up to 30% of deaths associated with advanced cancer. Nowadays there are no really effective treatments for cancer cachexia. Since preserving muscle mass in tumor-bearing mice prolongs survival independently on tumor growth, it is crucial to understand which are the signalling pathways involved in muscle mass loss in order to identify potential therapeutic targets to counteract cachexia development. Muscle atrophy arises when hyperactivation of proteolysis and organelle degradation exceeds rates of protein synthesis and organelle biogenesis. Proteolysis occurs via calcium-dependent proteolytic pathways and ubiquitin-mediated proteasomal and autophagic lysosomal processes that are controlled by several pathways; among all, the most important ones are Akt/FoxOs, IKK-NF-κB, IL6-JAK-Stat3 and the TGF-β/Myostatin-Smad2/3 pathways. In our study, we focused on the activity of FoxO1 and FoxO3, transcriptional factors whose activity controls the expression of crucial genes belonging to both the autophagy/lysosome system and the ubiquitin proteasome system including Atrogin1 and MuRF1, two E3-ubiquitin ligases strongly upregulated in different catabolic conditions. AIM OF THE WORK In our study, we aim at: i) generating shRNA constructs against MuRF1 and FoxO1/3; ii) performing in vivo muscle delivery and validation of shRNA oligos against MuRF1 and FoxO1/3 alone or in combination in the context of cancer-mediated muscle atrophy; iii) setting up a spatial transcriptomic approach in control and cachectic muscles transfected with shRNA oligos against MuRF1 and FoxO1/3 with the final goal of studying and comparing the transcriptome between these three experimental groups. MATERIALS AND METHODS In vivo experiments were conducted in accordance with the relevant codes of practice for the care and use of animals for scientific purposes. Cachexia was inducted in BALB/c mice by subcutaneous inoculation of C26 colon carcinoma cells, while control mice were inoculated with physiological solution; shFoxO1/3 was cloned using pSuper RNAi System, previously validated, and shMuRF1 and shScrambled oligos were cloned using the BLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP. In vitro experiments were performed transfecting shRNA constructs in C2C12 cells using Lipofectamine 3000, while in vivo tests were performed by injection and electroporation of tibialis anterior muscle of mice. RESULTS In our laboratory we successfully generated four shRNA oligos against MuRF1. We selected the most effective in vitro and tested in vivo by injection and electroporation of TA muscle of mice. Since the MuRF1 transcript was downregulated by shRNA oligo, we proceeded with the study of a combinatorial approach testing in vivo shRNA oligos against MuRF1 and FoxO1/3 alone and together in tumour-bearing mice. Both oligos were effective in counteracting muscle loss and their expression combined led to a higher trend in protecting muscle from cachexia. In the next phase, we set up a spatial transcriptomics protocol testing three different tissue fixation times, respectively 7, 12 and 24 hours. The 12 hours fixation was chosen due to the optimal results both in preservation of muscle morphology and RNA integrity. CONCLUSION All these experiments could lead to a future therapeutic approach to control cancer cachexia in patients, targeting FoxO1/3 and MuRF1 in combination, and therefore protect them from muscle loss, increase their quality of life, tolerance to anticancer therapy and reduce their mortality.