Whole body wasting, or cachexia, is an adverse effect of many cancers and it is a prognosticator of morbidity and mortality. Catabolic cytokines, such as tumor necrosis factor (TNF) are key triggers of skeletal muscle atrophy and weakness in cancer cachexia. TNFalpha is a robust activator of NF-kappaB mediated transcription in muscle and NF- kappaB has been shown to be involved in cancer-induced muscle wasting. We do not know the genes that are targeted by NF-kappaB in muscle in response to TNF or with cancer, presenting a roadblock to the discovery of proteins involved in the progression of muscle wasting. The overall objective of this work is to identify NF-kappaB target genes in myotubes due to TNF treatment and in whole muscle due to cancer cachexia, and to make possible new therapies for muscle cachexia.
The specific aims are: (1) To identify if selected candidate genes are bona fide NF-kappaB DNA binding targets in TNF treated cultured myotubes and in muscle from mice carrying Lewis Lung Carcinoma (LLC). The candidate genes were selected from a list of genes that were upregulated in cancer cachexia and/or with TNF treatment, and that contain NF-kappaB consensus sites in their promoters. The initial 7 candidate genes will be studied in myotubes and whole muscle using chromatin immunoprecipitation (ChIP) assay with a p65 antibody to select for NF-kappaB specific DNA binding sites. Comparison of NF-kappaB binding targets in response to TNF vs. cancer may help determine the contribution of TNF to cancer cachexia. (2) To identify bona fide NF- kappaB binding targets in wasting muscle of cachectic mice (LLC) on a genome-wide basis. """"""""ChIP on chip"""""""" assays will be used where chromatin immunoprecipitation of cachectic muscle is performed using a p65 antibody followed by hybridization of the enriched DNA fragments to promoter DNA sequence tiling microarrays. The mRNA expression of identified targets will also be measured for comparison. Once the genes targeted by NF-kappaB have been identified, their function in muscle wasting can be determined by additional focused study. This work will identify key genes involved in the progression of muscle wasting due to cancer. Understanding the molecular underpinnings of muscle wasting due to cancer is essential to develop rational pharmaceutical compounds that can be used to block muscle weakness and fatigue that are symptoms of cancer. ? ?
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