Genome-wide profiling by Next Generation Sequencing reveals protective effects of dietary EPA+DHA on chemotherapy-induced muscle toxicity in a clinically relevant model of colorectal cancer
Introduction: Tumor induced skeletal muscle loss is well recognized in the cancer domain. Chemotherapy-induced loss of skeletal muscle mass (muscle atrophy) is also a challenging side effect of cancer treatment. Retaining muscle mass is key to tolerance to chemotherapy and outcomes, hence interventions to preserve muscle mass warrant investigations. We aimed to investigate the protective effects of dietary fish oil (EPA+DHA) on chemotherapy-induced muscle atrophy through a comprehensive  mRNA expression analysis of gastrocnemius muscle using Next Generation RNA Sequencing (NGS) to delineate the underlying molecular mechanisms of muscle homeostasis. Methods: Female Fischer 344 rats were fed either a control diet for the entire study or switched to a diet containing fish oil (2.0 g /100 g of diet) at the start of chemotherapy. All rats received tumor implantation and tumor was allowed to grow for 2weeks. The various groups included tumor-bearing rats on a control diet (TUMOR) euthanized prior to chemotherapy administration, tumor-bearing rats that received two cycles of chemotherapy (irinotecan + 5-fluorouracil; FOLFIRI) while on a control diet (CONTROL) or fish oil diet (FO). After two cycles of FOLFIRI, RNA was extracted from gastrocnemius muscle and subjected to NGS. Differential Expression of Genes (DEG) was performed using DEseq2, with a fold-change cut-off = 1.5 and p-value < 0.05. Ingenuity Pathways Analysis (IPA) was used for functional annotation of DEGs, canonical pathways, and upstream regulators. Results: Transcriptomic analysis revealed distinct alterations in skeletal muscle gene expression profiles in response to chemotherapy and fish oil supplementation. In the comparison between CONTROL and TUMOR groups, 272 genes exhibited differential expression (DE), with 55.15% upregulated and 44.85% downregulated. Conversely, in the comparison between the FO and CONTROL groups, 274 genes showed DE, with 72.99% upregulated and 27.01% downregulated. Biological functional analysis reveals that Chemotherapy led to the alteration of genes involved in the proliferation of muscle cells (p<10-7), connective tissue disorder (p<10-6),  apoptosis (p<10-3),  and neurodevelopmental disorders (p<10-3). The provision of FO exclusively altered immune-related functions, as seen by the downregulation of several genes in the leukocyte extravasation pathway (-log p-value 5.92, z-score -2.83). Moreover, upstream analysis predicted the inhibition of key transcription factors involved in myogenic regeneration by chemotherapy treatment in rats on a control diet, while cytokines that drive inflammation were predicted to be inhibited by FO.  Conclusion: Chemotherapy negatively impacts processes involved in muscle homeostasis, including muscle cell proliferation and regeneration. Furthermore, the provision of fish oil primarily influenced inflammation pathways by the down-regulation of genes involved in the leukocyte extravasation pathway. Our findings provide novel insights into the molecular mechanisms underlying chemotherapy-induced muscle toxicity and the potential therapeutic benefits of dietary fish oil supplementation in mitigating muscle atrophy. Overall design: RNA sequencing was performed from muscle of rats bearing tumor, treated with chemotherapy with control diet or fish oil diet.