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.