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Cited by 111Muscle Physiology and DisordersFOXO transcription factor regulationNutrition and Health in Aging

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Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia

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Judge, Sarah M, Wu, Chia-Ling, Beharry, Adam W, Roberts, Brandon M, Ferreira, Leonardo F, Kandarian, Susan C, Judge, Andrew R·2014·BMC Cancer
DOI 10.1186/1471-2407-14-997PMC PMC4391468
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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.

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Figure 1 Transduction of locomotor muscles and the diaphragm with AAV9-d.n.FoxO. (A) Alignment of the dominant negative (d.n.) FoxO protein sequence, which includes amino acids 141–265 of mouse FoxO3a, with the corresponding mouse FoxO1 and FoxO4 amino acid sequences. FoxO1 shares ~85% amino acid sequence identity and FoxO4 75% sequence identity with the d.n.FoxO protein (shared amino acids denoted in green), all of which share >90% sequence conservation within this region. The 6 amino acid residues involved in DNA binding of the Forkhead Domain are highlighted in red and are denoted by hash marks (#) above the aligned sequences. (B and C) The AAV9 vectors driving expression of d.n.FoxO (or empty vector), which also contain an IRES driving the expression of GFP, were injected directly into the anterior hind limb compartment of mice to transduce the TA and EDL muscles, or injected directly into the intrathoracic cavity of mice to transduce the diaphragm. (B) Representative muscle cross-sections showing AAV9 transfection efficiency in the TA, EDL and diaphragm ~26 days post-injection as visualized via direct GFP fluorescence. (C) Confirmation that the d.n.FoxO protein was also expressed in muscles transduced with AAV9-d.n.FoxO was confirmed through western blot using an antibody against DsRed, which is fused to the d.n.FoxO protein.
Figure 1 Transduction of locomotor muscles and the diaphragm with AAV9-d.n.FoxO. (A) Alignment of the dominant negative (d.n.) FoxO protein sequence, which includes amino acids 141–265 of mouse FoxO3a, with the corresponding mouse FoxO1 and FoxO4 amino acid sequences. FoxO1 shares ~85% amino acid sequence identity and FoxO4 75% sequence identity with the d.n.FoxO protein (shared amino acids denoted in green), all of which share >90% sequence conservation within this region. The 6 amino acid residues involved in DNA binding of the Forkhead Domain are highlighted in red and are denoted by hash marks (#) above the aligned sequences. (B and C) The AAV9 vectors driving expression of d.n.FoxO (or empty vector), which also contain an IRES driving the expression of GFP, were injected directly into the anterior hind limb compartment of mice to transduce the TA and EDL muscles, or injected directly into the intrathoracic cavity of mice to transduce the diaphragm. (B) Representative muscle cross-sections showing AAV9 transfection efficiency in the TA, EDL and diaphragm ~26 days post-injection as visualized via direct GFP fluorescence. (C) Confirmation that the d.n.FoxO protein was also expressed in muscles transduced with AAV9-d.n.FoxO was confirmed through western blot using an antibody against DsRed, which is fused to the d.n.FoxO protein.CC BY · PubMed Central
Figure 2 Inhibition of FoxO impedes C26-induced muscle fiber atrophy and weakness. (A-D) The average cross-sectional area of muscle fibers in the TA (A), EDL (B) and diaphragm (C) of control or cachectic C26 mice transduced with AAV9-ev or AAV9-d.n.FoxO was calculated following incubation of muscle cross-sections with Alexa Fluor-conjugated wheat germ agglutinin to label muscle fiber membranes. (D) Representative diaphragm muscle cross-sections from each group. Data represent mean ± SE, n = at least 6 animals per group. (E-H) Maximum absolute tetanic force (E), specific force (F), time to peak tension (G) and half-relaxation time (H) was calculated in EDL muscles from control or cachectic C26 mice transduced with AAV9-ev or AAV9-d.n.FoxO. Data represent mean ± SE, n = 4 animals/group. *p < 0.05 vs AAV9-ev control group. †p < 0.05 vs. AAV9-ev C26 group.
Figure 2 Inhibition of FoxO impedes C26-induced muscle fiber atrophy and weakness. (A-D) The average cross-sectional area of muscle fibers in the TA (A), EDL (B) and diaphragm (C) of control or cachectic C26 mice transduced with AAV9-ev or AAV9-d.n.FoxO was calculated following incubation of muscle cross-sections with Alexa Fluor-conjugated wheat germ agglutinin to label muscle fiber membranes. (D) Representative diaphragm muscle cross-sections from each group. Data represent mean ± SE, n = at least 6 animals per group. (E-H) Maximum absolute tetanic force (E), specific force (F), time to peak tension (G) and half-relaxation time (H) was calculated in EDL muscles from control or cachectic C26 mice transduced with AAV9-ev or AAV9-d.n.FoxO. Data represent mean ± SE, n = 4 animals/group. *p < 0.05 vs AAV9-ev control group. †p < 0.05 vs. AAV9-ev C26 group.CC BY · PubMed Central
Figure 3 Genome-wide identification of gene networks regulated by FoxO in muscles of cachectic C26 tumor-bearing mice. ( A ) Microarray analyses were performed on TA muscles from control and cachectic C26 mice transduced with either AAV9-ev or AAV9-d.n.FoxO. Comparison of control and C26 AAV9-ev groups revealed 2,194 gene transcripts which were differentially expressed in response to C26 (FDR q-value < 0.01 and fold-change ≥ 1.5-fold). Of these genes, 543 genes were differentially regulated in muscles from C26 mice transduced with d.n.FoxO (AAV9-ev C26 vs. AAV9-d.n.FoxO C26, FDR q-value < 0.01 and fold-change ≥ 1.5-fold) and were thus considered as downstream targets (direct or indirect) of FoxO. ( B and C ) Gene transcripts upregulated ( B ) or downregulated ( C ) in response to C26 which were identified as FoxO targets were analyzed using the DAVID functional annotation clustering module and Broad’s Molecular Signature Database to identify enriched biological processes and Canonical Pathways. The top 10 most highly enriched DAVID annotation clusters and MSigDB Canonical Pathways from each gene set are ranked in order of significance and are plotted against the -log of the p-value. ( D and E ) Gene expression changes of select transcripts identified via microarray as downstream targets of FoxO were validated using qRT-PCR analyses. Data represent mean ± SE, n = at least 3 animals/group. *p < 0.05 vs AAV9-ev control group. †p < 0.05 vs. AAV9-ev C26 group.
Figure 3 Genome-wide identification of gene networks regulated by FoxO in muscles of cachectic C26 tumor-bearing mice. ( A ) Microarray analyses were performed on TA muscles from control and cachectic C26 mice transduced with either AAV9-ev or AAV9-d.n.FoxO. Comparison of control and C26 AAV9-ev groups revealed 2,194 gene transcripts which were differentially expressed in response to C26 (FDR q-value < 0.01 and fold-change ≥ 1.5-fold). Of these genes, 543 genes were differentially regulated in muscles from C26 mice transduced with d.n.FoxO (AAV9-ev C26 vs. AAV9-d.n.FoxO C26, FDR q-value < 0.01 and fold-change ≥ 1.5-fold) and were thus considered as downstream targets (direct or indirect) of FoxO. ( B and C ) Gene transcripts upregulated ( B ) or downregulated ( C ) in response to C26 which were identified as FoxO targets were analyzed using the DAVID functional annotation clustering module and Broad’s Molecular Signature Database to identify enriched biological processes and Canonical Pathways. The top 10 most highly enriched DAVID annotation clusters and MSigDB Canonical Pathways from each gene set are ranked in order of significance and are plotted against the -log of the p-value. ( D and E ) Gene expression changes of select transcripts identified via microarray as downstream targets of FoxO were validated using qRT-PCR analyses. Data represent mean ± SE, n = at least 3 animals/group. *p < 0.05 vs AAV9-ev control group. †p < 0.05 vs. AAV9-ev C26 group.CC BY · PubMed Central
Figure 4 FoxO1 and FoxO3a regulate Cebpb , Fos and Stat3 gene expression. (A-C) Select transcripts identified via microarray as downstream targets of FoxO in locomotor muscles during C26 cancer cachexia were validated as targets in the diaphragm via qRT-PCR analyses. Data represent mean ± SE, n = 3–5 animals/group. *p < 0.05 vs AAV9-ev control group. †p < 0.05 vs. AAV9-ev C26 group. (D) FoxO1 TM or FoxO3a TM expression plasmids were injected and electroporated into TA muscles of mice and harvested 4 days later for qRT-PCR analyses. Data represent mean ± SE, n = 5 animals/group. *p < 0.05 vs empty vector (EV). (E) C2C12 myoblasts were transfected with a pRL-TK-Renilla reporter plus a WT Cebpb promoter reporter construct or a Cebpb reporter construct which is mutated at two putative Forkhead binding elements, which prevents FoxO DNA binding. Following 3 days of differentiation, myotubes were treated with IL-6 (10 ng/mL) for 3 hours and harvested for measurement of firefly/renilla luciferase activity. Data represent mean ± SE, n = at least 9 wells/group. *p < 0.05 vs control. †p < 0.05 vs. WT Cebpb promoter.
Figure 4 FoxO1 and FoxO3a regulate Cebpb , Fos and Stat3 gene expression. (A-C) Select transcripts identified via microarray as downstream targets of FoxO in locomotor muscles during C26 cancer cachexia were validated as targets in the diaphragm via qRT-PCR analyses. Data represent mean ± SE, n = 3–5 animals/group. *p < 0.05 vs AAV9-ev control group. †p < 0.05 vs. AAV9-ev C26 group. (D) FoxO1 TM or FoxO3a TM expression plasmids were injected and electroporated into TA muscles of mice and harvested 4 days later for qRT-PCR analyses. Data represent mean ± SE, n = 5 animals/group. *p < 0.05 vs empty vector (EV). (E) C2C12 myoblasts were transfected with a pRL-TK-Renilla reporter plus a WT Cebpb promoter reporter construct or a Cebpb reporter construct which is mutated at two putative Forkhead binding elements, which prevents FoxO DNA binding. Following 3 days of differentiation, myotubes were treated with IL-6 (10 ng/mL) for 3 hours and harvested for measurement of firefly/renilla luciferase activity. Data represent mean ± SE, n = at least 9 wells/group. *p < 0.05 vs control. †p < 0.05 vs. WT Cebpb promoter.CC BY · PubMed Central

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Metadata

Authors
Judge, S. M., Wu, C.-L., Beharry, A. W., Roberts, B. M., Ferreira, L. F., Kandarian, S. C., & Judge, A. R.
Affiliations
  • University of Florida Health (US)
  • Boston University (US)
  • University of Florida (US)
Corresponding author
Judge, Andrew R
ORCID
Venue
BMC Cancer
Publisher
Springer Science and Business Media LLC
Published
2014-12
Volume
14
Issue
1
Pages
997-997
ISSN
1471-2407 (ISSN-L)
DOI
10.1186/1471-2407-14-997
PMC
PMC4391468
OpenAlex
W1564746483
Cited by
111
References
59
Citations by year
2026: 7 · 2025: 8 · 2024: 8 · 2023: 14 · 2022: 2 · 2021: 14 · 2020: 20 · 2019: 12 · 2018: 8 · 2017: 5 · 2016: 9 · 2015: 3
FWCI
2.32
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Cancer (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • University of Florida Digital Collections (University of Florida) (submittedVersion)
  • OpenBU (Boston University) (submittedVersion, cc-by)
Funders
  • National Heart, Lung, and Blood Institute (R00HL098453)
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR060217, R01AR060209)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (T32HD043730)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › FOXO transcription factor regulation
  • Health Sciences › Medicine › Physiology (medical) › Nutrition and Health in Aging
Keywords
FOXO transcription factors, cancer cachexia, skeletal muscle atrophy, C26 colon carcinoma, FOXO1, FOXO3, gene regulatory networks, muscle fiber atrophy, diaphragm, limb muscles, STAT3, FOS, CEBPB, UBR2, IL-6 signaling, proteasome pathway, extracellular matrix genes, sarcomere proteins
MeSH terms
Dependovirus, Amino Acid Sequence, Animals, Cachexia, Colonic Neoplasms, Disease Models, Animal, Extracellular Matrix, Gene Expression Regulation, Genetic Vectors, Humans, Male, Molecular Sequence Data, Transcription Factors, Transduction, Genetic, Reproducibility of Results, Sequence Alignment, Muscle, Skeletal, Gene Expression Profiling, Proteasome Endopeptidase Complex, Mice, Forkhead Transcription Factors, Gene Regulatory Networks, Genome-Wide Association Study, Heterografts

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