br AAA expression On the whole although cancer induced
AAA expression. On the whole, although cancer-induced muscle wasting is associated with in-creased autophagy, tissue-specific inhibition seems unable to counteract the depletion.
Muscle wasting is improved by formoterol while maintaining autophagy
Changing perspective by focusing on anti-wasting treatments, autophagy was measured in TB mice treated with formoterol, a selective β2-agonist that effectively counteracted cachexia in both TB mice and cancer patients [15,16]. Consistently, in mice
Fig. 1. Muscle specific autophagy inhibition does not spare cancer-induced wasting. (a) Beclin-1 knockdown in the tibialis anterior (TA) muscle of control (C, n = 6) and C26-bearing male mice (C26, n = 7). (b) TA and gastrocnemius (GSN) mass and (c) TA fiber cross-sectional area (CSA) in the above-mentioned groups. (d) Immunofluorescence images of TA muscle sections stained for p62 (red) and nuclei (blue). (e) LC3B and p62 Western blotting analyses in TA homogenates. *p b 0.05 versus SCR; **p b 0.01 versus SCR.
2676 Autophagy and PD-98059 in cancer cachexia
Fig. 1 (legend on previous page)
Autophagy and mitochondria in cancer cachexia 2677
bearing the C26 tumor, formoterol counteracted the loss of body weight, muscle mass and strength, without protecting from adipose wasting (Fig. 2a). In agreement with previous observations , Beclin-1 and LC3B-II levels increased in C26 TB mice (Fig. 2b), while treatment with formoterol only moderately reduced LC3B-II levels (Fig. 2b), not allowing to draw conclusions on formoterol activity on the autophagy flux. An autophagy flux study was then performed in TB mice at early cachexia stages, treating systemically for 2 days with colchi-cine, in order to avoid premature animal death (see Ref. ). The results showed that the protection against muscle wasting exerted in the C26 hosts by formoterol was not associated with reduced autophagy flux (Fig. 2c).
TP53INP2-mediated autophagy exacerbates cancer-induced muscle wasting
Provided that cancer-induced muscle wasting could not be prevented by tissue-specific autophagy
inhibition and that the formoterol-mediated muscle-sparing effect was not achieved by reduced autoph-agy flux, the next question was whether increasing the autophagy flux would impact on muscle wasting in TB mice. Along this line, Beclin-1 overexpression was excluded since the expression was already strongly increased in the muscle of TB mice. The choice was to modulate TP53INP2/DOR, a positive regulator of autophagy that, however, was previous-ly reported to be down-regulated in muscle-wasting conditions . Muscle TP53INP2 transcript levels were reduced in both C26 and LLC hosts (Fig. 3a) as well as in cachectic cancer patients (Fig. 3b). In particular, TP53INP2 levels were unchanged in non-cachectic patients, suggesting the presence of an adaptive response to cope with an excessive stress-induced autophagy.
A gain of function experiment performed using transgenic mice overexpressing TP53INP2 in skeletal muscle (Tg mice, hereafter) showed that body weight loss was enhanced in Tg TB mice (Fig. 3c, d), despite that food intake and tumor
Fig. 2. Formoterol counteracts muscle wasting while maintaining autophagy flux. (a) Body weight changes at the end of the experiment, voluntary grasping strength and tissue masses (GSN, gastrocnemius; TA, tibialis anterior; WAT, gonadal white adipose tissue) in controls (C), C26-bearing male mice (C26) and C26 receiving formoterol (C26 For), n = 6 for all groups. (b) Beclin-1 and LC3B Western blotting analyses in GSN homogenates of the above-mentioned animals. (c) LC3B Western blotting analyses in GSN homogenates of mice receiving colchicine (Col) or vehicle for 2 days before sacrifice. Different letters indicate statistically different results. Statistical significance is set at p b 0.05.
2678 Autophagy and mitochondria in cancer cachexia
Fig. 3. TP53INP2 is repressed in atrophic muscles, and muscle overexpression exacerbates body weight loss.
(d) Body weight changes at the end of the experiment in the above-mentioned animal groups. Different letters indicate statistically different results. Statistical significance is set at p b 0.05.
burden remained comparable to wild-type (WT) tumor hosts (Fig. S2).
The exacerbation of body wasting is likely due to excessive muscle protein catabolism. MRI analysis of body composition and the evaluation of tissue weight at necropsy showed a marked loss of lean and muscle mass, respectively, in wild-type (WT) TB mice, that worsened in Tg tumor hosts (Fig. 4a, b). As for the fat mass, a biphasic trend was observed. In the early stages, Tg TB mice showed an accumulation of fat that was progressively lost reaching the same WT levels, confirmed at the end point by the WAT mass (Fig. 4c, d).