Approximately two million people die world-wide of the consequences of cancer cachexia annually. For skeletal muscle, the primary organ involved in cancer cachexia, there have been no major studies where high throughput approaches have been used to identify target genes that drive the wasting responsible for a poor prognosis. The overall goal is to use an entirely in vivo and unbiased approach with new frontier technology to identify the genes involved in producing muscle wasting due to cancer and due to disuse. The comparison is to decipher differences in muscle wasting with and without immune system triggers. This goal will entail measurement of global gene expression in conjunction with chromatin immunoprecipitation (ChIP) using transcription factor antibodies followed by massively parallel sequencing of the immunoprecipitated DNA fragments (ChIP-sequencing). For transcriptional regulation of muscle atrophy, the most evidence exists for involvement of nuclear factor kappaB (NF-?B) transcription factors so these will be studied first.
Aim 1 : Determine the upstream ?B signaling proteins required for muscle wasting due to cancer as has been done for disuse. This will confirm the requirement of NF-?B transcriptional regulation of muscle wasting due to cancer. It will be accomplished by overexpressing plasmids that encode dominant negative forms of NF-?B signaling proteins in muscles of tumor and non-tumor bearing mice. Inhibition of cancer induced muscle wasting will indicate the requirement of the ?B signaling protein being studied and the associated ?B mediated transcription.
Aim 2 : To identify genome-wide differentially expressed transcripts using Affymetrix high-density microarrays (39,000 transcripts) in muscle wasting due to cancer and due to disuse. These data will be used as a functional readout for comparison to the genome-wide determination of ?B transcription factor binding in Aim 3.
Aim 3 : On a genome-wide scale, discover the NF-?B transcription factor binding to gene regulatory sites in muscle due to disuse atrophy and due to cancer using ChIP-sequencing. At least three different ?B transcription factor libraries will be used for analysis of each type of atrophy. Identification of genome-wide binding sites of ?B transcription factors will represent a major advance in the understanding of muscle wasting because it will reveal novel genes at work during atrophy. Moreover, these datasets will allow modeling of the biological regulatory networks involved in the atrophy process and will open new avenues for research. By discovering gene targets and transcriptional networks involved in the development of muscular wasting, new foci for therapeutic drug development will be identified.
This work will identify key proteins required for the progression of muscle wasting due to cancer cachexia and to disuse atrophy. Understanding the molecular underpinnings of cancer-induced muscle wasting is essential to develop rational pharmacological and/or nutritional compounds that can be used to attenuate the loss of muscle strength and fatigue. Overall completion of this work will lead to development of therapies to reduce the morbidity and mortality associated with cancer cachexia and inactivity induced muscle wasting.
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