C26 Cancer-Induced Muscle Wasting Is IKKβ-Dependent and NF-kappaB-Independent

Existing data suggest that NF-kappaB signaling is a key regulator of cancer-induced skeletal muscle wasting. However, identification of the components of this signaling pathway and of the NF-kappa B transcription factors that regulate wasting is far from complete. In muscles of C26 tumor bearing mice, overexpression of dominant negative (d.n.) IKK beta blocked muscle wasting by 69% and the I kappa B beta-super repressor blocked wasting by 41%. In contrast, overexpression of d.n. IKK alpha or d.n. NIK did not block C26-induced wasting. Surprisingly, overexpression of d.n. p65 or d.n. c-Rel did not significantly affect muscle wasting. Genome-wide mRNA expression arrays showed upregulation of many genes previously implicated in muscle atrophy. To test if these upregulated genes were direct targets of NF-kappa B transcription factors, we compared genome-wide p65 binding to DNA in control and cachectic muscle using ChIP-sequencing. Bioinformatic analysis of ChIP-sequencing data from control and C26 muscles showed very little p65 binding to genes in cachexia and little to suggest that upregulated p65 binding influences the gene expression associated with muscle based cachexia. The p65 ChIP-seq data are consistent with our finding of no significant change in protein binding to an NF-kappa B oligonucleotide in a gel shift assay, no activation of a NF-kappa B-dependent reporter, and no effect of d.n. p65 overexpression in muscles of tumor bearing mice. Taken together, these data support the idea that although inhibition of I kappa B alpha, and particularly IKK beta, blocks cancer-induced wasting, the alternative NF-kappa B signaling pathway is not required. In addition, the downstream NF-kappa B transcription factors, p65 and c-Rel do not appear to regulate the transcriptional changes induced by the C26 tumor. These data are consistent with the growing body of literature showing that there are NF-kappa B-independent substrates of IKK beta and I kappa B alpha that regulate physiological processes.