title: Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia.
abstract: BACKGROUND
  Evidence from cachectic cancer patients and animal models of cancer cachexia supports the involvement of Forkhead box O (FoxO) transcription factors in driving cancer-induced skeletal muscle wasting. However, the genome-wide gene networks and associated biological processes regulated by FoxO during cancer cachexia are unknown. We hypothesize that FoxO is a central upstream regulator of diverse gene networks in skeletal muscle during cancer that may act coordinately to promote the wasting phenotype.
  
  
  METHODS
  To inhibit endogenous FoxO DNA-binding, we transduced limb and diaphragm muscles of mice with AAV9 containing the cDNA for a dominant negative (d.n.) FoxO protein (or GFP control). The d.n.FoxO construct consists of only the FoxO3a DNA-binding domain that is highly homologous to that of FoxO1 and FoxO4, and which outcompetes and blocks endogenous FoxO DNA binding. Mice were subsequently inoculated with Colon-26 (C26) cells and muscles harvested 26 days later.
  
  
  RESULTS
  Blocking FoxO prevented C26-induced muscle fiber atrophy of both locomotor muscles and the diaphragm and significantly spared force deficits. This sparing of muscle size and function was associated with the differential regulation of 543 transcripts (out of 2,093) which changed in response to C26. Bioinformatics analysis of upregulated gene transcripts that required FoxO revealed enrichment of the proteasome, AP-1 and IL-6 pathways, and included several atrophy-related transcription factors, including Stat3, Fos, and Cebpb. FoxO was also necessary for the cancer-induced downregulation of several gene transcripts that were enriched for extracellular matrix and sarcomere protein-encoding genes. We validated these findings in limb muscles and the diaphragm through qRT-PCR, and further demonstrate that FoxO1 and/or FoxO3a are sufficient to increase Stat3, Fos, Cebpb, and the C/EBPβ target gene, Ubr2. Analysis of the Cebpb proximal promoter revealed two bona fide FoxO binding elements, which we further establish are necessary for Cebpb promoter activation in response to IL-6, a predominant cytokine in the C26 cancer model.
  
  
  CONCLUSIONS
  These findings provide new evidence that FoxO-dependent transcription is a central node controlling diverse gene networks in skeletal muscle during cancer cachexia, and identifies novel candidate genes and networks for further investigation as causative factors in cancer-induced wasting.
authors.affiliation: Department of Physical Therapy, University of Florida, 1225 Center Drive, HPNP Building 1142, Gainesville, Florida, USA. smsenf@ufl.edu.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Judge
authors.affiliation: Department of Health Sciences, Boston University, Boston, Massachusetts, USA. clwu@bu.edu.
authors.forename: Chia-Ling
authors.initials: CL
authors.lastname: Wu
authors.affiliation: Department of Physical Therapy, University of Florida, 1225 Center Drive, HPNP Building 1142, Gainesville, Florida, USA. beharrya1@phhp.ufl.edu.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Beharry
authors.affiliation: Department of Physical Therapy, University of Florida, 1225 Center Drive, HPNP Building 1142, Gainesville, Florida, USA. brob21@ufl.edu.
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Roberts
authors.affiliation: Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida, USA. ferreira@hhp.ufl.edu.
authors.forename: Leonardo F
authors.initials: LF
authors.lastname: Ferreira
authors.affiliation: Department of Health Sciences, Boston University, Boston, Massachusetts, USA. skandar@bu.edu.
authors.forename: Susan C
authors.initials: SC
authors.lastname: Kandarian
authors.affiliation: Department of Physical Therapy, University of Florida, 1225 Center Drive, HPNP Building 1142, Gainesville, Florida, USA. arjudge@phhp.ufl.edu.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Judge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002100:Cachexia / Q000209:etiology*; D003110:Colonic Neoplasms / Q000150:complications* / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D005109:Extracellular Matrix / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D005822:Genetic Vectors / Q000235:genetics; D055106:Genome-Wide Association Study; D064593:Heterografts; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D015203:Reproducibility of Results; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D051858:Forkhead Transcription Factors; D014157:Transcription Factors; D046988:Proteasome Endopeptidase Complex