Inhibition of N-Terminal Acetyltransferase C Mitigates Endoplasmic Reticulum Stress-Mediated Muscle Atrophy in Cancer Cachexia.

Abstract

<h4>Background</h4>Cancer cachexia is a complex syndrome marked by weight loss and muscle wasting, significantly impacting patient quality of life and survival. Mechanistically, it is characterized by suppressed protein synthesis and enhanced muscle catabolism, with the role of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) becoming increasingly evident. This study aimed to explore ER stress-tolerant factors in muscle wasting and evaluate their potential to prevent muscle loss in cancer cachexia.<h4>Methods</h4>A genome-wide CRISPR screening was conducted in the context of ER stress-mediated growth inhibition of C2C12 myoblasts. The candidate genes resistant to ER stress were further evaluated in C2C12 myotubes treated with conditioned medium of Lewis lung adenocarcinoma (LLC) cells. Twelve-week-old male mice were administered LLC cells and shRNA against Naa35 via adeno-associated virus. Four weeks later, tibialis anterior (TA) muscles were analysed for muscle mass, grip strength and molecular changes with quantitative polymerase chain reaction, western blotting and histological analysis.<h4>Results</h4>CRISPR screening identified Naa35, Naa38 and Naa30, all three components of N-terminal acetyltransferase C, as key molecules for resistance to ER stress. The atrophic muscles of mice bearing LLC demonstrated an elevation of UPR, as well as 1.64-fold upregulation of Naa35 protein (p = 0.0072). Among the three branches of the UPR, an ATF6 inhibitor, AEBSF, abolished upregulation of Naa35, Naa38 and Naa30, and an ATF6 activator, AA147, induced Naa35 expression in a dose-dependent manner (p < 0.001). In cells treated with LLC conditioned medium, Naa35 knockdown reduced the amount of cathepsin K (CTSK) protein, which subsequently resulted in the CTSK-mediated proteolysis of insulin receptor substrate 1. In LLC-bearing mice, Naa35 knockdown led to a 65.4% reduction in CTSK protein expression (p < 0.001) and preservation of the phosphorylation levels of protein kinase B (p < 0.0324) and anabolic-related S6 kinase (p < 0.0375). Concurrently, the expression of catabolism-related genes was repressed (MuRF1, p < 0.0015; MAFbx1, p < 0.0265). These alterations were associated with the restoration of TA muscle mass (2.52 ± 0.19 vs. 3.72 ± 0.45 mg/g, p = 0.0004), fibre area (1741 ± 992 vs. 2099 ± 1264 mm², p < 0.0001), grip strength in all four limbs (0.0328 ± 0.0076 vs. 0.0506 ± 0.0130 N/g, p = 0.0295) and wire mesh hanging time (496 ± 331 vs. 1038 ± 370 s, p = 0.0406).<h4>Conclusions</h4>Inhibition of N-terminal acetyltransferase C prevents ER stress-induced muscle wasting via the downregulation of CTSK and subsequent activation of the anabolic pathway. This suggests that N-terminal acetyltransferase C is a potential therapeutic target for combating muscle wasting in cancer cachexia.