HSP90 and STAT3 were required for C26 CM-induced myotube
atrophy.
To assess the involvement of HSP90 in cancer cachexia-related muscle
wasting, we adopted the in vitro cachectic cell model by
culturing the C2C12 myotubes with C26 tumor cell-derived conditional
medium (C26 CM) (Figure S1b,c). Western blot assay confirms the
pathological phenotype of cachectic muscle atrophy based on the loss of
MHC expression and the enhanced expression of Atrogin-1, MuRF1, and
myostatin (Figure 2a). H&E staining further demonstrated the myotube
shrinking induced by C26 CM (Figure 2c). Notably, we observed a dramatic
increase in the HSP90-STAT3 interaction in C2C12 myotubes upon C26 CM
treatment (Figure 2b); this is consistent with the in vivo data,
although the increased expression of HSP90 in skeletal muscle was not
obvious in C26 CM induced muscle atrophy (Figure 2a). To determine
whether the atrophy effect was HSP90-dependent, we knocked down HSP90 in
C2C12 myotubes. The results showed that the administration of RNAi
against HSP90 or STAT3 prevented the decrease in the myotube size,
myotube diameter, and myotube area induced by C26 CM (Figure 2c, d
left). These results were further confirmed by the measurement of the
transcriptional expression of MuRF-1 and atrogin-1 (Figure 2d right).
Consistently, the myotube atrophy-associated phenotypes, including the
down-regulated expression of MHC and the up-regulated expression of
myostatin, MuRF-1, and atrogin-1, were largely reversed with the knocked
down of HSP90 (Figure 2e left). Similar effects were observed when STAT3
was knocked down in C2C12 myotubes (Figure 2e right), indicating that
the enhanced HSP90-STAT3 interaction plays a crucial role in the
pathological development of cachectic muscle wasting.