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Cancer-induced Muscle Wasting is IKKβ-dependent and NF-kappaB-independent

ABSTRACT: 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-κB transcription factors that regulate wasting is far from complete. In muscles of C26 tumor bearing mice, overexpression of d.n. IKKβ blocked muscle wasting by 69%, the IκBα-super repressor blocked wasting by 41%. In contrast, overexpression of d.n. IKKα or d.n. NIK did not block C26-induced wasting. Surprisingly, overexpression of d.n. p65 or d.n. c-Rel did not significantly block 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-κB transcription factors, we compared genome-wide p65 or p50 binding to DNA in control and cachectic muscle using ChIP-sequencing. Bioinformatic analysis of ChIP-seq data from control and C26 muscles showed increased p65 and p50 binding to a few regulatory and structural genes but only two of these genes were upregulated with atrophy. The p65 and p50 ChIP-seq data are consistent with our finding of no significant change in protein binding to an NF-κB oligo in a gel shift assay. Taken together, these data support the idea that although inhibition of IκBα, and particularly IKKβ, blocks cancer-induced wasting, the alternative NF-κB signaling pathway is not required. In addition, the downstream NF-κB transcription factors do not regulate the transcriptional changes. These data are consistent with the growing body of literature showing that there are NF-κB-independent substrates of IKKβ and IκBα that regulate physiological processes.

ORGANISM(S): Mus musculus

PROVIDER: GSE48363 | GEO | 2013/11/15

SECONDARY ACCESSION(S): PRJNA209857

REPOSITORIES: GEO

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Project description: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-M-NM-:B transcription factors that regulate wasting is far from complete. In muscles of C26 tumor bearing mice, overexpression of d.n. IKKM-NM-2 blocked muscle wasting by 69%, the IM-NM-:BM-NM-1-super repressor blocked wasting by 41%. In contrast, overexpression of d.n. IKKM-NM-1 or d.n. NIK did not block C26-induced wasting. Surprisingly, overexpression of d.n. p65 or d.n. c-Rel did not significantly block 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-M-NM-:B transcription factors, we compared genome-wide p65 or p50 binding to DNA in control and cachectic muscle using ChIP-sequencing. Bioinformatic analysis of ChIP-seq data from control and C26 muscles showed increased p65 and p50 binding to a few regulatory and structural genes but only two of these genes were upregulated with atrophy. The p65 and p50 ChIP-seq data are consistent with our finding of no significant change in protein binding to an NF-M-NM-:B oligo in a gel shift assay. Taken together, these data support the idea that although inhibition of IM-NM-:BM-NM-1, and particularly IKKM-NM-2, blocks cancer-induced wasting, the alternative NF-M-NM-:B signaling pathway is not required. In addition, the downstream NF-M-NM-:B transcription factors do not regulate the transcriptional changes. These data are consistent with the growing body of literature showing that there are NF-M-NM-:B-independent substrates of IKKM-NM-2 and IM-NM-:BM-NM-1 that regulate physiological processes. To compare gene expression changes in atrophied muscles from C26 tumor bearing mice, gastrocnemius/plantaris muscles were harvested from 4 C26 tumor-bearing mice, and 3 control non tumor-bearing mice. Total RNA were isolated and pooled (2-3 muslces in the same group per RNA sample ) to make equal amount of total RNA per sample. Three pooled total RNA samples from healthy control muscles and 3 pooled total RNA from muscles of C26 tumor bearing mice were labelled and hybridized on 6 Mouse Affyemtrix Gene 1.0 ST arrays. Two-side t-tests and multiple test corrections were performed to identify differentially expressed genes due to C26 tumor bearing induced cachexia.

Project description:Skeletal muscle atrophy is a debilitating condition associated with weakness, fatigue, and reduced functional capacity. Nuclear factor-kappaB (NF-κB) transcription factors play a critical role in atrophy. Knockout of genes encoding p50 or the NF-κB co-transactivator, Bcl-3, abolish disuse atrophy and thus they are NF-κB factors required for disuse atrophy. We do not know however, the genes targeted by NF-κB that produce the atrophied phenotype. Here we identify the genes required to produce disuse atrophy using gene expression profiling in wild type compared to Nfkb1 (gene encodes p50) and Bcl-3 deficient mice. There were 185 and 240 genes upregulated in wild type mice due to unloading, that were not upregulated in Nfkb1-/- and Bcl-3-/- mice, respectively, and so these genes were considered direct or indirect targets of p50 and Bcl-3. All of the p50 gene targets were contained in the Bcl-3 gene target list. Most genes were involved with protein degradation, signaling, translation, transcription, and transport. To identify direct targets of p50 and Bcl-3 we performed chromatin immunoprecipitation of selected genes previously shown to have roles in atrophy. Trim63 (MuRF1), Fbxo32 (MAFbx), Ubc, Ctsl, Runx1, Tnfrsf12a (Tweak receptor), and Cxcl10 (IP-10) showed increased Bcl-3 binding to κB sites in unloaded muscle and thus were direct targets of Bcl-3. p50 binding to the same sites on these genes either did not change or increased, supporting the idea of p50:Bcl-3 binding complexes. p65 binding to κB sites showed decreased or no binding to these genes with unloading. Fbxo9, Psma6, Psmc4, Psmg4, Foxo3, Ankrd1 (CARP), and Eif4ebp1 did not show changes in p65, p50, or Bcl-3 binding to κB sites, and so were considered indirect targets of p50 and Bcl-3. This work represents the first study to use a global approach to identify genes required to produce the atrophied phenotype with disuse. 24 mice were used based on 4 mice per group, 3 mouse genotypes (wild type, Nfkb1-/-, Bcl3-/-) and 2 conditions (weight-bearing and unloaded).

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Cancer-induced Muscle Wasting is IKKβ-dependent and NF-kappaB-independent

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