Alcohol Worsens Cancer Cachexia and Anabolic Signaling in C26 Tumor Bearing Mice

Abstract

Cancer cachexia results in a loss of skeletal muscle mass, leading to increased morbidity and mortality. Similarly, chronic alcohol consumption leads to skeletal muscle loss and dysfunction. It is unknown, whether prior alcohol intake or its continued use after cancer establishment, exacerbates cancer cachexia. Anabolic pathways are central to the maintenance of muscle mass and are notoriously decreased by alcohol intake. Among these pathways, the mTORC1 pathway and subsequent rates of protein synthesis are of central importance to the maintenance of muscle health. Purpose To determine if prior or concurrent alcohol consumption worsens skeletal muscle anabolic signaling (i.e. puromycin and mTOR pathway) during cancer cachexia. Methods Male CD2F1 mice (n=42), 12‐weeks old, were randomized into 6 groups (n=6‐8/gr): control (CON), control‐cancer (CON‐C), prior EtOH (PRIOR), prior EtOH‐cancer (PRIOR‐C), continuous EtOH (EtOH), and continuous EtOH w/cancer (EtOH‐C). Mice were acclimated to a control liquid diet, after which the EtOH groups transitioned to the Lieber DeCarli EtOH diet at 20% kcal EtOH for 6 weeks. At week 7, PRIOR mice weaned off EtOH and remained on the control diet until sacrifice. Also at week 7, cancer mice were injected subcutaneously on the right flank with C26 colon carcinoma cells (1x106 cells) and consumed their assigned diet for 14 days while tumors developed. After a 4‐hour fast, puromycin was injected and 30 minutes later the gastrocnemius (GAS) was excised. GAS protein was isolated and Western Blots performed. Data were analyzed via 1‐way ANOVA with post hoc testing when appropriate. Data are presented relative to CON (set to 100%) as mean ± SE, with protein expression normalized either to ponceau staining or total levels of the given protein. Results Body weight at sacrifice was lower in the PRIOR‐C and EtOH‐C (p<0.05) indicating an alcohol effect. Tumor weight and tibia length were not different between groups. GAS weight was reduced by cancer in all groups, and most notably in PRIOR‐C and EtOH‐C mice (p<0.001). Protein synthesis, assessed by puromycin, decreased in all cancer groups, with a ~70% reduction in PRIOR‐C and ~82% reduction in EtOH‐C relative to CON. Similarly, phosphorylation of S6K1 Thr389, rpS6 Ser240/244 and Ser235/236 was significantly reduced in PRIOR‐C and EtOH‐C relative to CON (p<0.05), indicating a greater reduction in these markers of mTORC1 activity with alcohol intake. Surprisingly, no differences in 4E‐BP1 Ser65, mTOR Ser2448, or mTOR Ser2481 were observed across groups. PRAS40 Thr246 was increased by cancer alone as well as in EtOH‐C mice (35% vs. CON) (p<0.05). Increases in eEF2 Thr56 were also detected in PRIOR‐C (50%) and EtOH‐C (83%) (p<0.05), possibly indicating impaired peptide chain elongation. No changes were noted in markers of protein breakdown including ubiquitinated proteins, and autophagic proteins ULK1 Ser757 and LC3B‐II/I. Conclusions The intake of alcohol both prior to, and during tumor development lead to greater decreases in GAS weight, protei...

Molecular Mechanisms of Cachexia: A Review

“…In the context of cellular development, mTORC1 plays a crucial role in regulating the activity of unc-51-like autophagy-activating kinase 1 (ULK1) and ATG13. This regulation leads to a reduction in catabolic autophagy and an enhancement in protein synthesis mediated by 4E-BPs and p70S6K [49,50]. In another related pathway, MSTN makes its negative impact on muscle mass regulation by inducing ActRIIB, which phosphorylates SMAD2 and SMAD3, inhibiting Akt protein [51,52].…”

“…The development of antibodies capable of inhibiting the actions of both myostatin and activin could, therefore, serve as a potential approach to mitigate muscle atrophy [143][144][145]. The crucial carcinogenic pathway PI3K/AKT/mTOR-also responsible for protein biosynthesis-is also effectively suppressed by myostatin and activin [49,50]. In contrast, Insulin-like growth factor 1 (IGF-1) is an important activator of the PI3K/Akt signaling pathway, which plays a crucial role in protein synthesis and the promotion of muscle growth [45,146].…”