DOI: 10.18738/t8/rfrqe2
title: Muscle proteome changes during cancer

description: 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 week treatment of weekly intramuscular injections of either 100 mg testosterone (CAT+T, n = 9; 3M/6F) or placebo/saline (CAT+P, n = 10; 7 M/3F). Biopsies were performed on the vastus lateralis before (PRE) and after (POST) the 7 week 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 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 MS analysis to represent 59 unique proteins. Among the biological processes and pathways identified, CAT+P impacted muscle contraction, catabolic processes, and oxygen transport, while CAT+T impacted transcription regulation, muscle differentiation, muscle development, and contraction.
CONCLUSIONS. 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.
description: This archive contains the supplemental data for the associated manuscript.
This is an ancillary analysis to a previously published manuscript (https://doi.org/10.1002/jcsm.12295). 
The associated spreadsheet contains information for proteomic analysis of tissue samples from cancer patients before and after receiving 2 months of cancer treatment augmented with either adjunct testosterone or placebo. 
The complete demographic information and study protocol is outlined in the associated manuscript.
Briefly:
Cancer patients receiving treatment were randomized to receive 2 months of either adjunct testosterone or placebo.
Skeletal muscle biopsies were collected before and after 2 months of treatment.
Fractionated skeletal muscle protein extracts were separated using 2D gel electrophoresis.
Gels were stained and imaged to detect differences in total protein abundance (Sypro Ruby dye; SYP), phosphorylation (ProQ Diamond dye; PQD), and nitrosylation (Bodipy Fl‐maleimide vs ascorbate ratio of ratios).
Protein spots significantly altered in pairwise comparison between pre and post treatment timepoints were extracted and identified by mass spectrometry utilizing the Uniprot protein database for identification.
Separate gene set enrichment analysis was performed on proteins that were significantly altered in either abundance, phosphorylation, or nitrosylation using Enrichr online platform and the GO 2021 and KEGG databases.

The associated spreadsheet contains separate tabs for:
The raw image intensity data for individually identified protein spots for abundance (Sypro Ruby dye; SYP), phosphorylation (ProQ Diamond dye; PQD). Tab: [SYP&PQD_raw]
The raw image intensity data for individually identified protein spots for nitrosylation (NA/ASC). Tab: [NA&ASC_raw]
Calculation of ratio-of-ratios to assess nitrosylation (SNOFlo). Tab: [SNO]
Calculation of altered abundance and phosphorylation. Tab: [ABND+PHOS]
Combined altered proteins for abundance, phosphorylation, and nitrosylation. Tab: [DATA SHEET]
Corresponding spot number, isoelectric point, and molecular weight. Tab: [pI_MW]
Database estimate of protein ID. Tab: [MS DATA]
Uniprot accession number and protein description. Tab: [Uniprot]
Combined summary data including protein identification, change in abundance, isoelectric point, molecular weight, spot #, designation of protein structure, and fold change/significance for abundance, phosphorylation, and nitrosylation. Tab:[SUMMARY]