Cancer and Associated Therapies Impact the Skeletal Muscle Proteome

Abstract

Introduction: Both cancer and cancer associated therapies (CAT; including chemotherapy or concurrent chemoradiation) disrupt cellular metabolism throughout the body, including the regulation of skeletal muscle mass and function. Adjunct testosterone therapy during standard of care chemotherapy and chemoradiation modulates CAT-induced dysregulation of skeletal muscle metabolism and protects lean body mass during CAT. However, the extent to which the skeletal muscle proteome is altered under these therapeutic conditions is unknown. Objective: We probed the skeletal muscle proteome of cancer patients as an ancillary analysis following a randomized, double-blind, placebo-controlled phase II trial investigating the effect of adjunct testosterone on body composition in men and women with advanced cancers undergoing CAT. Methods: Men and women diagnosed with late stage (≥IIB) or recurrent head and neck or cervical cancer who were scheduled to receive standard of care CAT were administered an adjunct 7 weeks treatment of weekly intramuscular injections of either 100 mg testosterone (CAT+T, n = 7; 2M/5F) or placebo/saline (CAT+P, n = 6; 4M/2F). Biopsies were performed on the vastus lateralis before (PRE) and after (POST) the 7 weeks treatment. Extracted proteins were separated with 2-dimensional gel electrophoresis (2DE), and subjected to analyses of total protein abundance, phosphorylation and S-nitrosylation. Proteoforms showing significant 1.5 fold differences (t-test p ≤ 0.05) between PRE and POST timepoints were identified by mass spectroscopy (MS), and lists of altered proteins were subjected to Gene Set Enrichment Analysis (GSEA) to identify affected pathways. Results: A total of 756 distinct protein spots were identified. Of those spots, 102 were found to be altered in terms of abundance, phosphorylation, or S-nitrosylation, and identified by mass spectroscopy analysis to represent 58 unique proteins. Among the biological processes and pathways identified, CAT+P predominantly impacted metabolic processes, cell assembly, oxygen transport, and apoptotic signaling, while CAT+T impacted transcription regulation, muscle differentiation, muscle development, and contraction. Conclusion: Cancer and CAT significantly altered the skeletal muscle proteome in a manner suggestive of loss of structural integrity, reduced contractile function, and disrupted metabolism. Proteomic analysis suggests that the addition of adjunct testosterone minimized the structural and contractile influence of cancer and its associated therapies.

Keywords: atrophy; cachexia; nitrosylation; phosphorylation; post-translational modifications; proteome; testosterone.

FIGURE 1

Heat map. Heat map depicting the fold change in abundance, phosphorylation, and S-nitrosylation of skeletal muscle proteins in cancer patients following 7 weeks of treatment with testosterone (T) or placebo (P). Proteoforms marked with “X” changed significantly (p < 0.05) from pre to post treatment. Proteoforms are labeled (central column) as either intact (I), fragments (F), or aggregate (A) based on identification and predicted molecular weight as described in the text.

FIGURE 2

Gene set enrichment analysis (GSEA). (A) Volcano Plots. Volcano plots of protein spots identified by 2D electrophoresis represented as -log10 p-value vs. log2 of fold change from pre to post. Proteins were deemed significantly altered with a p < 0.05 and a log2 fold change >1.5 (increased) or < −1.5 (decreased). Analyses identified significant differences in abundance (red), phosphorylation (blue), and S-nitrosylation (purple) between pre and post treatment biopsies for a subset of proteins (significantly altered proteins are colored). (B) Gene Ontology (GO). Significantly altered proteins were used to identify affected gene sets using the 2021 GO Biological Process 2021 database. The top 10 pathways identified by Enrichr combined score (see text for description) are displayed. Pathways that were identified as significantly altered (adjusted p < 0.05) are denoted by a dark colored bar for abundance (red), phosphorylation (blue), and S-nitrosylation (purple). The number and identity of significantly altered proteins and total number of proteins in the identified pathway are denoted on each bar. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG). Significantly altered proteins were used to identify affected pathways using the 2021 KEGG pathway database. The top 10 pathways identified by Enrichr combined score (see text for description) are displayed. Pathways that were identified as significantly altered (adjusted p < 0.05) are denoted by a dark colored bar for abundance (red), phosphorylation (blue), and S-nitrosylation (purple). The number and identity of significantly altered proteins and total number of proteins in the identified pathway are denoted on each bar.