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A Key Role for Leukemia Inhibitory Factor in C26 Cancer Cachexia.

ABSTRACT: Cachexia is an exacerbating event in many types of cancer that is strongly associated with a poor prognosis. We have identified cytokine, signaling, and transcription factors that are required for cachexia in the mouse C26 colon carcinoma model of cancer. C2C12 myotubes treated with conditioned medium from C26 cancer cells induced atrophy and activated a STAT-dependent reporter gene but not reporter genes dependent on SMAD, FOXO, C/EBP, NF-?B, or AP-1. Of the gp130 family members IL-11, IL-6, oncostatin M (OSM), and leukemia inhibitory factor (LIF), only OSM and LIF were sufficient to activate the STAT reporter in myotubes. LIF was elevated in C26 conditioned medium (CM), but IL-6, OSM, TNF?, and myostatin were not. A LIF-blocking antibody abolished C26 CM-induced STAT reporter activation, STAT3 phosphorylation, and myotube atrophy but blocking antibodies to IL-6 or OSM did not. JAK2 inhibitors also blocked C26 CM-induced STAT reporter activation, STAT3 phosphorylation, and atrophy in myotubes. LIF at levels found in the C26 CM was sufficient for STAT reporter activation and atrophy in myotubes. In vivo, an increase in serum LIF preceded the increase in IL-6 in mice with C26 tumors. Overexpression of a dominant negative Stat3C?-EGFP gene in myotubes and in mouse muscle blocked the atrophy caused by C26 CM or C26 tumors, respectively. Taken together, these data support an important role of LIF-JAK2-STAT3 in C26 cachexia and point to a therapeutic approach for at least some types of cancer cachexia.

SUBMITTER: Seto DN

PROVIDER: S-EPMC4528156 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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A Key Role for Leukemia Inhibitory Factor in C26 Cancer Cachexia.

Seto Danielle N DN Kandarian Susan C SC Jackman Robert W RW

The Journal of biological chemistry 20150619 32

Cachexia is an exacerbating event in many types of cancer that is strongly associated with a poor prognosis. We have identified cytokine, signaling, and transcription factors that are required for cachexia in the mouse C26 colon carcinoma model of cancer. C2C12 myotubes treated with conditioned medium from C26 cancer cells induced atrophy and activated a STAT-dependent reporter gene but not reporter genes dependent on SMAD, FOXO, C/EBP, NF-κB, or AP-1. Of the gp130 family members IL-11, IL-6, on ...[more]

PMID: 26092726

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Project description:BACKGROUND:Cancer cachexia is a metabolic wasting syndrome that is strongly associated with a poor prognosis. The initiating factors causing fat and muscle loss are largely unknown. Previously, we found that leukaemia inhibitory factor (LIF) secreted by C26 colon carcinoma cells was responsible for atrophy in treated myotubes. In the present study, we tested whether C26 tumour-derived LIF is required for cancer cachexia in mice by knockout of Lif in C26 cells. METHODS:A C26 Lif null tumour cell line was made using CRISPR-Cas9. Measurements of cachexia were compared in mice inoculated with C26 vs. C26Lif-/- tumour cells, and atrophy was compared in myotubes treated with medium from C26 vs. C26Lif-/- tumour cells. Levels of 25 cytokines/chemokines were compared in serum of mice bearing C26 vs. C26Lif-/- tumours and in the medium from these tumour cell lines. RESULTS:At study endpoint, C26 mice showed outward signs of sickness while mice with C26Lif-/- tumours appeared healthy. Mice with C26Lif-/- tumours showed a 55-75% amelioration of body weight loss, muscle loss, fat loss, and splenomegaly compared with mice with C26 tumours (P < 0.05). The heart was not affected by LIF levels because the loss of cardiac mass was the same in C26 and C26Lif-/- tumour-bearing mice. LIF levels in mouse serum was entirely dependent on secretion from the tumour cells. Serum levels of interleukin-6 and G-CSF were increased by 79-fold and 68-fold, respectively, in C26 mice but only by five-fold and two-fold, respectively, in C26Lif-/- mice, suggesting that interleukin-6 and G-CSF increases are dependent on tumour-derived LIF. CONCLUSIONS:This study shows the first use of CRISPR-Cas9 knockout of a candidate cachexia factor in tumour cells. The results provide direct evidence for LIF as a major cachexia initiating factor for the C26 tumour in vivo. Tumour-derived LIF was also a regulator of multiple cytokines in C26 tumour cells and in C26 tumour-bearing mice. The identification of tumour-derived factors such as LIF that initiate the cachectic process is immediately applicable to the development of therapeutics to treat cachexia. This is a proof of principle for studies that when carried out in human cells, will make possible an understanding of the factors causing cachexia in a patient-specific manner.

Project description: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.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.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/EBP? 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.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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A Key Role for Leukemia Inhibitory Factor in C26 Cancer Cachexia.

Publications

A Key Role for Leukemia Inhibitory Factor in C26 Cancer Cachexia.

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