Inhibition of activin-like kinase 4/5 attenuates cancer cachexia associated muscle wasting

Abstract

Cancer mediated activation of the ActRIIB-ALK4/5 heterodimer by myostatin is strongly associated with muscle wasting. We investigated in vitro and in vivo the efficacy of ALK4/5 receptor blockers SB431542 and GW788388 in preventing muscle wasting, and explored synergy with IGF-I analogue LONG R3 (LR3) IGF-I. In vitro, C2C12 skeletal muscle cells were treated with vehicle, SB431542, GW788388 and LR3 IGF-I. A C26-CD2F1 cachexia model was used to induce cachexia in vivo. Mice were allocated as non-tumour bearing (NTB) or C26 tumour-bearing (C26 TB) vehicle control, treated with SB431542, LR3 IGF-I, SB431542 and LR3 IGF-I, or GW788388 (intraperitoneally or orally). In vitro, differentiation index and mean nuclei count increased using SB431542, GW788388, LR3 IGF-I. In vivo, GW788388 was superior to SB431542 in limiting loss of bodyweight, grip-strength and gastrocnemius weight. and downregulated Atrogin-1 expression comparable to NTB mice. LR3 IGF-I treatment limited loss of muscle mass, but at the expense of accelerated tumour growth. In conclusion, treatment with GW788388 prevented cancer cachexia, and downregulated associated ubiquitin ligase Atrogin-1.

Figure 1

Fusion indices of C2C12 cells treated with SB431542, GW788388 and LONG R3 IGF-I. Fusion indices of C2C12 cells on days two, four, and six for (A) different concentrations of SB431542 (SB), (B) GW788388 (GW), and (C) LONG R3 IGF-I (LR3 IGF-I) and mean nuclei count for (D) SB431542, (E) GW788388, and (F) LONG R3 IGF-I. *A statistically significant difference (p < 0.05 of two-way ANOVA followed by a post hoc Bonferroni test) was observed compared to vehicle samples on the corresponding day. Representative images of H&E stained differentiated (G) vehicle treated C2C12 cells, (H) SB431542 treated cells, (I) GW788388 treated cells, and (J) LONG R3 IGF-I treated C2C12 cells. All acquired images were taken from day 6 samples.

Figure 2

Experimental groups and timeline. Allocation of male CD2F1 mice to different treatment groups. Each box represents a day, ranging from day 0 to day 21. *Start of the experiment (day 0). ^†Inoculation on day 0 of C26 cells in Tumour-Bearing animals. ^‡Intraperitoneal. ^§Orally. Striped boxes indicate day of treatment. Black dots below boxes indicate time points of grip-strength measurement. Lines above boxes indicate time points of tumour size measurement. Black boxes indicate the end of the experiment.

Figure 3

In vivo C26 - CD2F1 cachexia model (impact on body weight, tumour size, grip-strength and muscle mass). Data depicted in red indicates C26 tumour-bearing, vehicle treated male CD2F1 mice (n = 20). Data depicted in green indicates healthy, non-tumour-bearing male CD2F1 mice (n = 20). (A) Loss of bodyweight (mean ± SEM) in vehicle-treated, C26 tumour-bearing (TB) mice (p < 0.001). (B) Estimated tumour growth rate as measured every other day, starting on day 9, via digital callipers. (C) Relation between tumour mass and relative bodyweight change. No relation could be observed for animals experiencing rapid bodyweight loss requiring early sacrifice (n = 8, Spearman’s rho = −0.119, p = 0.779). In animals not (yet) experiencing this rapid decline of bodyweight loss prior to day 21, tumour mass was negatively associated with change in bodyweight (n = 12, Spearman’s rho = −0.608, p = 0.036). (D) Untreated C26 TB male CD2F1 mice (n = 20) experienced loss of grip-strength throughout the experiments as compared with non-tumour-bearing male CD2F1 mice (n = 20). Student’s t-test was conducted performed for on the mean differences of grip-strength change. This loss of grip-strength could already be observed on day 7 (p = 0.003), but became more apparent on day 14 (p < 0.001). (E) Student’s t-test was conducted performed for on the mean weight of m. gastrocnemius, m. tibialis anterior and m. soleus. Wet muscle weight of gastrocnemius (p < 0.001), tibialis anterior (p < 0.001), and soleus (p < 0.001) muscles was significantly reduced in untreated, C26 TB male CD2F1 mice (n = 20) as compared with non-tumour-bearing male CD2F1 mice (n = 20).

Figure 4

Treatment efficacy of SB431542, GW788388 and LONG R3 IGF-I on bodyweight, grip strength, tumour weight and muscle weight at sacrifice. Bar graphs depicting the mean ± SEM (A) relative grip strength immediately prior to sacrifice, (B) relative bodyweight at sacrifice, (C) gastrocnemius (GCM) muscle weight, (D) tibialis anterior (TA) muscle weight, (E) tumour weight for non-tumour bearing (NTB) (n = 20); C26 tumour-bearing (TB) vehicle treated (n = 20); SB431542 treated (SB, n = 20); SB431542 with LONG R3 IGF-I (SB + IGF, n = 12); LONG R3 IGF-I treated (IGF, n = 12) and GW788388 (GW) treated intraperitoneally (IP, n = 8) and orally (PO, n = 8) male CD2F1 mice. Multiple group comparisons were done by one-way ANOVA with a Bonferroni’s post-hoc test. All groups were compared against NTB mice and TB vehicle treated mice. Asterisk brackets are displayed for significant results only. *p < 0.05 **p < 0.01 ***p < 0.001.

Figure 5

Scatterplot charts for individual m. gastrocnemius and m. tibialis ant. muscle weight in healthy, non-tumour bearing mice and treated tumour-bearing mice. Scatterplots of gastrocnemius (GCM) muscle tibialis anterior (TA) muscle weight for all individual tumour-bearing (TB) groups compared with the non-tumour bearing (NTB) male CD2F1 mice. The horizontal reference line indicates the lowest TA muscle weight observed in NTB mice. The vertical reference line indicates the lowest GCM muscle weight observed in NTB mice. Multiple group comparisons were done by one-way ANOVA with a Bonferroni’s post-hoc. All groups were compared against NTB male CD2F1 mice (n = 20). Differences were observed for (A) untreated, C26 TB (n = 20, reduced TA, p < 0.001; reduced GCM, p < 0.001), (B) SB431542 treated (n = 20, reduced TA, p < 0.001; reduced GCM, p < 0.001) and (C) LONG R3 IGF-I treated male CD2F1 mice (n = 12, reduced TA, p = 0.027). No differences in muscle weight were observed for (D) combined SB431542 and LONG R3 IGF-I treated (n = 12), (E) GW788388 (intraperitoneally, n = 8) and (F) GW788388 (orally, n = 8) treated male CD2F1 mice.

Figure 6

mRNA expression levels in cachectic muscle. Bar graphs depicting the mean ± SEM mRNA expression levels in gastrocnemius muscle of (A) Atrogin-1, (B) MuRF1, (C) MyoD and (D) Myogenin in non-tumour bearing (NTB, n = 20); C26 tumour-bearing (TB) vehicle treated (n = 20); tumour-bearing SB431542 treated (SB, n = 20); tumour-bearing combined SB431542 and LONG R3 IGF-I treated (SB + IGF, n = 12); tumour-bearing LONG R3 IGF-I treated (IGF, n = 12) and tumour-bearing GW788388 treated (pooled orally and intraperitoneally GW treated groups, n = 16) male CD2F1 mice. Multiple group comparisons were done by one-way ANOVA with a Bonferroni’s post-hoc test. All groups were compared against NTB mice and TB vehicle treated mice. Asterisk brackets are displayed for significant results only. *p < 0.05.