Autophagy is induced in the skeletal muscle of cachectic cancer patients

Abstract

Basal rates of autophagy can be markedly accelerated by environmental stresses. Recently, autophagy has been involved in cancer-induced muscle wasting. Aim of this study has been to evaluate if autophagy is induced in the skeletal muscle of cancer patients. The expression (mRNA and protein) of autophagic markers has been evaluated in intraoperative muscle biopsies. Beclin-1 protein levels were increased in cachectic cancer patients, suggesting autophagy induction. LC3B-I protein levels were not significantly modified. LC3B-II protein levels were significantly increased in cachectic cancer patients suggesting either increased autophagosome formation or reduced autophagosome turnover. Conversely, p62 protein levels were increased in cachectic and non-cachectic cancer patients, suggesting impaired autophagosome clearance. As for mitophagy, both Bnip3 and Nix/Bnip3L show a trend to increase in cachectic patients. In the same patients, Parkin levels significantly increased, while PINK1 was unchanged. At gene level, Beclin-1, p-62, BNIP3, NIX/BNIP3L and TFEB mRNAs were not significantly modulated, while LC3B and PINK1 mRNA levels were increased and decreased, respectively, in cachectic cancer patients. Autophagy is induced in the skeletal muscle of cachectic cancer patients, although autophagosome clearance appears to be impaired. Further studies should evaluate whether modulation of autophagy could represent a relevant therapeutic strategy in cancer cachexia.

Figure 1. Beclin-1 and LC3B expression in rectus abdominis muscle of cancer and control patients.

(A) Beclin-1 and (B) LC3B mRNA levels were evaluated by real-time PCR (control, n = 11; non cachectic cancer patients, n = 17; cachectic cancer patients, n = 12); (C) Beclin-1 and (D) LC3BI and LC3BII protein levels were evaluated by western blotting (control, n = 9; non cachectic cancer patients, n = 16; cachectic cancer patients, n = 10): representative western blots for Beclin-1, LC3BI, LC3BII and GAPDH (loading control) are shown on the lower panel and densitometric quantifications of Beclin-1, LC3B-I and LC3B-II protein levels normalized to GAPDH are shown on the upper panel. Representative pattern for Beclin-1, group ‘Cancer no cachexia’ consists of two non adjacent lanes on the same blot. The whole blot is reported in Supplementary Figure S2. Data (mean ± SEM) are expressed as percentage of controls. Significance of the differences: *p < 0.05 vs controls; ^#p < 0.05 vs cancer no cachexia.

Figure 2. p62 expression in rectus abdominis muscle of cancer and control patients.

(A) p62 mRNA levels were evaluated by real-time PCR (control, n = 11; non cachectic cancer patients, n = 17; cachectic cancer patients, n = 12); (B) p62 protein levels were evaluated by western blotting (control, n = 8; non cachectic cancer patients, n = 15; cachectic cancer patients, n = 10): representative western blots for p62 and GAPDH (loading control) are shown on the lower panel and densitometric quantifications of p62 protein levels normalized to GAPDH are shown on the upper panel. Data (mean ± SEM) are expressed as percentage of controls. Significance of the differences: *p < 0.05 vs controls.

Figure 3. Bnip3 and Nix/Bnip3L expression in rectus abdominis muscle of cancer and control patients.

(A) Bnip3 and (B) Nix/Bnip3L mRNA levels were evaluated by real-time PCR (Bnip3: control, n = 8; non cachectic cancer patients, n = 16; cachectic cancer patients, n = 10; Nix/Bnip3L: control, n = 9; non cachectic cancer patients, n = 16; cachectic cancer patients, n = 11); (C) Bnip3 and (D) Nix/Bnip3L protein levels were evaluated by western blotting (Bnip3: control, n = 11; non cachectic cancer patients, n = 17; cachectic cancer patients, n = 10; Nix/Bnip3L: control, n = 3; non cachectic cancer patients, n = 5; cachectic cancer patients, n = 5): representative western blots for Bnip3, Nix/Bnip3L and GAPDH (loading control) are shown on the lower panel and densitometric quantifications of Bnip3 and Nix/Bnip3L protein levels normalized to GAPDH are shown on the upper panel. Data (mean ± SEM) are expressed as percentage of controls.

Figure 4. PINK1 and Parkin expression in rectus abdominis muscle of cancer and control patients.

(A) PINK1 and (B) Parkin mRNA levels were evaluated by real-time PCR (PINK1: control, n = 9; non cachectic cancer patients, n = 16; cachectic cancer patients, n = 11; Parkin: control, n = 9; non cachectic cancer patients, n = 16; cachectic cancer patients, n = 11); (C) PINK1 and (D) Parkin protein levels were evaluated by western blotting (PINK1: control, n = 7; non cachectic cancer patients, n = 8; cachectic cancer patients, n = 9; Parkin: control, n = 7; non cachectic cancer patients, n = 8; cachectic cancer patients, n = 10): representative western blots for PINK1, Parkin and GAPDH (loading control) are shown on the lower panel and densitometric quantifications of PINK1 and Parkin protein levels normalized to GAPDH are shown on the upper panel. Data (mean ± SEM) are expressed as percentage of controls. Significance of the differences: *p < 0.05 vs controls; ^#p < 0.05 vs cancer no cachexia.

Figure 5. TFEB expression in rectus abdominis muscle of cancer and control patients.

(A) TFEB mRNA levels were evaluated by real-time PCR (control, n = 11; non cachectic cancer patients, n = 16; cachectic cancer patients, n = 12); (B) TFEB nuclear protein levels were evaluated by western blotting (control, n = 4; non cachectic cancer patients, n = 5; cachectic cancer patients, n = 3): representative western blots for TFEB and Lamin A (loading control) are shown on the lower panel and densitometric quantifications of TFEB protein levels normalized to Lamin A are shown on the upper panel. Data (mean ± SEM) are expressed as percentage of controls.