A role for increased Fra-1 expression in cancer cachexia: Novel mechanisms of increased apoptosis and impaired myogenesis

TNFα is strongly associated with cachexia, promoting pleiotropic effects in skeletal muscle, including apoptosis, inhibition of myogenesis, and increased protein degradation. These effects are mediated in part by pro-inflammatory transcription factors, including AP-1. C2C12 muscle cells treated with TNFα exhibit altered expression of cJun, JunB, and Fra-1, components of the AP-1 transcription factor. Constitutive overexpression of Fra-1 in C2C12 cells under differentiation conditions results in increased numbers of satellite cells and increased apoptotic signaling. These morphological changes are accompanied by increased mRNA expression of caveolin-1 and Pax7, two satellite cell markers. Additional myogenesis inhibitors, including IGF-1, TGF-β1, and BMP4 are also up-regulated at the mRNA level. Furthermore, expression of pro-apoptotic caspases 1 and 8 are up-regulated at both the mRNA and protein level.;Our results indicate that Fra-1 is a viable therapeutic target for drug development in the treatment of cancer cachexia. Consequently, we investigated Fra-1 inhibitors potential therapeutics. This investigation identified LS474, a quinazolinone-based small molecule, as a potential therapeutic because it restores MyHC expression in TNFα-treated C2C12 cells, an indicator of restored balance between protein production and degradation in muscle cells. Further investigation of LS474 and other small molecules that block Fra-1 activity, could identify effective new therapeutic drugs with more direct efficacy on cachexia than present palliative measures.;Downstream of Fra-1 expression, we see increased caspase 3 activity and cleaved PARP, hallmarks of apoptosis. Moreover, inhibiting caspase 8 in both the TNFα-treated and the Fra-1 overexpressing cells reduces apoptosis. Inhibiting the ERK pathway in the TNFα-treated cells reduces the expression of p-Fra-1, while inhibiting the JNK pathway reduces the expression of p-Fra-1 and p-cJun. Under the same conditions, JNK inhibition greatly reduces apoptosis. These results suggest that TNFα signals through the ERK and JNK pathways, causing a switch to Jun:Fra-1 heterodimer composition, which, in turn, leads to increased caspase 3 activity, potentially through transcriptional up-regulation of caspase 8. The ERK pathway has also been implicated in the up-regulation of Pax7, caveolin-1, and TGF-β1, which supports a model in which Fra-1, an important ERK target, mediates the up-regulation of these genes.