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C26 cancer-induced muscle wasting is IKK?-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-?B transcription factors that regulate wasting is far from complete. In muscles of C26 tumor bearing mice, overexpression of dominant negative (d.n.) IKK? blocked muscle wasting by 69% and the I?B?-super repressor blocked wasting by 41%. In contrast, overexpression of d.n. IKK? 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-?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-?B oligonucleotide in a gel shift assay, no activation of a NF-?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?B?, and particularly IKK?, blocks cancer-induced wasting, the alternative NF-?B signaling pathway is not required. In addition, the downstream NF-?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-?B-independent substrates of IKK? and I?B? that regulate physiological processes.

SUBMITTER: Cornwell EW

PROVIDER: S-EPMC3906224 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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C26 cancer-induced muscle wasting is IKKβ-dependent and NF-kappaB-independent.

Cornwell Evangeline W EW Mirbod Azadeh A Wu Chia-Ling CL Kandarian Susan C SC Jackman Robert W RW

PloS one 20140129 1

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 dominant negative (d.n.) IKKβ blocked muscle wasting by 69% and the IκBα-super repressor blocked wasting by 41%. In contrast, overexpression of d.n. IKKα or d.n. NIK did not block C26- ...[more]

PMID: 24489962

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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:BackgroundRetinoids, through their cognate nuclear receptors, exert potent effects on cell growth, differentiation and apoptosis, and have significant promise for cancer therapy and chemoprevention. These ligands can determine the ultimate fate of target cells by stimulating or repressing gene expression directly, or indirectly through crosstalking with other signal transducers.ResultsUsing different breast cancer cell models, we show here that depending on the cellular context retinoids can signal either towards cell death or cell survival. Indeed, retinoids can induce the expression of pro-apoptotic (i.e. TRAIL, TNF-Related Apoptosis-Inducing Ligand, Apo2L/TNFSF10) and anti-apoptotic (i.e. cIAP2, inhibitor of apoptosis protein-2) genes. Promoter mapping, gel retardation and chromatin immunoprecipitation assays revealed that retinoids induce the expression of this gene mainly through crosstalk with NF-kappaB. Supporting this crosstalk, the activation of NF-kappaB by retinoids in T47D cells antagonizes the apoptosis triggered by the chemotherapeutic drugs etoposide, camptothecin or doxorubicin. Notably apoptosis induced by death ligands (i.e. TRAIL or antiFAS) is not antagonized by retinoids. That knockdown of cIAP2 expression by small interfering RNA does not alter the inhibition of etoposide-induced apoptosis by retinoids in T47D cells reveals that stimulation of cIAP2 expression is not the cause of their anti-apoptotic action. However, ectopic overexpression of a NF-kappaB repressor increases apoptosis by retinoids moderately and abrogates almost completely the retinoid-dependent inhibition of etoposide-induced apoptosis. Our data exclude cIAP2 and suggest that retinoids target other regulator(s) of the NF-kappaB signaling pathway to induce resistance to etoposide on certain breast cancer cells.ConclusionsThis study shows an important role for the NF-kappaB pathway in retinoic acid signaling and retinoic acid-mediated resistance to cancer therapy-mediated apoptosis in breast cancer cells, independently of cIAP2. Our data support the use of NF-kappaB pathway activation as a marker for screening that will help to develop novel retinoids, or retinoid-based combination therapies with improved efficacy.

Dataset Information

C26 cancer-induced muscle wasting is IKK?-dependent and NF-kappaB-independent.

Publications

C26 cancer-induced muscle wasting is IKKβ-dependent and NF-kappaB-independent.

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