Leukemia inhibitory factor suppresses hepatic de novo lipogenesis and induces cachexia in mice

Abstract

Cancer cachexia is a systemic metabolic syndrome characterized by involuntary weight loss, and muscle and adipose tissue wasting. Mechanisms underlying cachexia remain poorly understood. Leukemia inhibitory factor (LIF), a multi-functional cytokine, has been suggested as a cachexia-inducing factor. In a transgenic mouse model with conditional LIF expression, systemic elevation of LIF induces cachexia. LIF overexpression decreases de novo lipogenesis and disrupts lipid homeostasis in the liver. Liver-specific LIF receptor knockout attenuates LIF-induced cachexia, suggesting that LIF-induced functional changes in the liver contribute to cachexia. Mechanistically, LIF overexpression activates STAT3 to downregulate PPARα, a master regulator of lipid metabolism, leading to the downregulation of a group of PPARα target genes involved in lipogenesis and decreased lipogenesis in the liver. Activating PPARα by fenofibrate, a PPARα agonist, restores lipid homeostasis in the liver and inhibits LIF-induced cachexia. These results provide valuable insights into cachexia, which may help develop strategies to treat cancer cachexia.

Fig. 1. LIF overexpression induces cachexia in the TgLC mice.

A The strategy to generate LIF-tg^flox/+ (TgL) mice. The mouse LIF gene, preceded by the CAG promoter and a transcriptional STOP cassette, was knocked into the Rosa26 locus (CAG-STOP-LIF-eGFP-Rosa26TV) using the CRISPR/Cas9 system. B The generation of TgL/R26-Cre^ERT2 (TgLC) mice. Right panel: the genotyping analysis of TgL and TgLC mice by PCR. All mice with the same genotype have similar results. C Serum LIF levels in TgL (n = 6) and TgLC mice (n = 10) with TAM injection measured by the ELISA assay. D Mouse body weight post TAM injection in TgL mice (n = 6) and TgLC mice (n = 14). E Fat and lean mass loss post TAM injection in TgL (n = 10) and TgLC (n = 8) mice. Body composition was measured by EchoMRI. F Representative H&E images of muscle and WAT tissues from TgL and TgLC mice with TAM injection. At least three independent biological replicates were performed. G–M Mice were housed in Promethion metabolic cages. Mice were injected with TAM at the first light cycle (n = 4–10/group). Shaded regions represent the dark cycle from 19:00 pm to 7:00 am. Values are hourly means. Energy balance (G), food intake (H), total energy expenditure (TEE) (I), oxygen consumption (VO₂) (J), carbon dioxide production (VCO₂) (K), Respiratory exchange ratio (RER) (L) and locomotor activity (M) of TgL and TgLC mice post TAM injection were measured. N The serum levels of albumin, blood urea nitrogen (BUN), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) reflecting kidney and liver functions in TgL and TgLC mice at 3 days after TAM injection for albumin and 9 days after TAM injection for other parameters (n = 3/group). O Kaplan-Meier survival curves of mice. The day of TAM injection was denoted as D0. Data are presented as mean ± SEM for (D, E), and as mean ± SD for (C, G–N). N.D. non-detectable. Each dot represents an individual mouse. Both female and male mice were used. For G–N: Two-tailed Student’s t-test; for D, E two-way ANOVA followed by Sidak’s multiple comparison test; and for O two-tailed Kaplan-Meier survival analysis. Source data are provided as Source Data file.

Fig. 2. LIF overexpression disrupts lipid homeostasis and decreases hepatic de novo lipogenesis.

A Heatmap of serum TG levels under fed and fasted conditions in TgL and TgLC mice with small LC-TGs (C ≤ 54) and larger LC-TGs (C > 54) clustered together (n = 4/group). B Representative serum TG levels in TgL (upper panels) and TgLC (bottom panels) mice injected with TAM under fed and fasted conditions (n = 4/group). The TG levels under the fed condition are designated as 1. C Representative serum TG levels under the fed condition (n = 4/group). The TG levels in TgL mice under the fed condition are designated as 1. D Heatmap showing the average TG levels in the livers of TgL and TgLC mice injected with TAM under fed and fasted conditions (n = 8/group). E Heatmap of the average TG levels in the livers from Balb/c mice with or without C26 or C26-LIF KO tumors (n = 6 for control (Con) mice without tumors, n = 10 for C26 tumor-bearing mice and n = 6 for C26-LIF KO tumor-bearing mice). F, G Hepatic de novo lipogenesis in TgL and TgLC mice with TAM injection (F; n = 6/group) and in non-tumor bearing Balb/c mice (n = 8) and Balb/c mice bearing C26 (n = 12) or C26-LIF KO tumors (n = 8) (G). Mice drank water containing 20% D₂O for 7 days before tissue collection. Levels of C16:0 and C18:0 in each group were shown. Data are presented as mean ± SD. Each dot represents an individual biological repeat. Both female and male mice were used. For B, C, F Two-tailed Student’s t-test was applied for comparison between two groups; for G One-way ANOVA followed by t-test with Tukey’s multiple comparison adjustment was applied for comparison among multiple groups. Source data are provided as Source Data file.

Fig. 3. Liver-specific LIFR blockade attenuates cachexia induced by LIF overexpression.

A The generation of TgL/LIFR^flox/flox mice. Liver-specific LIF expression and LIFR knockout was induced in TgL/LIFR^flox/flox mice by hydrodynamic tail vein injection of Ad5CMVCre-eGFP (Ad-Cre). B The genotyping analysis of TgL and TgL/LIFR^flox/flox mice with or without Ad-Cre injection by PCR. All mice with the same genotype have similar results. C Serum LIF levels in TgL and TgL/LIFR^flox/flox mice with or without Ad-Cre injection (n = 7–9/group). D Relative LIFR mRNA levels in TgL and TgL/LIFR^flox/flox mice with or without Ad-Cre injection (n = 4–8/group). E The levels of Tyr 705 phosphorylated STAT3 (pSTAT3) and total STAT3 protein in the liver of TgL and TgL/LIFR^flox/flox mice with or without Ad-Cre injection determined by Western-blot assays. At least three independent biological replicates were performed. F Relative body weight of TgL (n = 5) and TgL/LIFR^flox/flox (n = 10) mice post Ad-Cre injection. G Kaplan-Meier survival curves of TgL and TgL/LIFR^flox/flox mice post Ad-Cre injection. The day of Ad-Cre injection was denoted as D0. Data are presented as mean ± SD for (C, D), and as mean ± SEM for (F). N.D. non-detectable. Each dot represents an individual biological repeat. Both female and male mice were used. For D: one-way ANOVA followed by t-test with Tukey’s multiple comparison adjustment; for F: two-way ANOVA followed by Sidak’s multiple comparison test; for G: two-tailed Kaplan-Meier survival analysis. Source data are provided as Source Data file.

Fig. 4. LIF overexpression downregulates the PPARα signaling pathway.

A RNA-seq results showing gene expression levels in the liver from TAM-injected TgLC (n = 4) and TAM-injected TgL mice (n = 3). The number of identified genes and DEGs were shown (left). The DEGs were shown in the Heatmap (right). B KEGG analysis of DEGs by the DAVID database. C GSEA enrichment plots for lipid metabolism (left) and PPAR signaling pathway (right). D RNA-seq results showing the expression levels of genes (PPARa, PPARd, PPARg, PPARGC1a and PPARGC1b) encoding for five PPAR family members (n = 3 for TgL group, n = 4 for TgLC group). E Heatmap of PPARα target genes among the DEGs in the liver from TgLC and TgL mice with TAM injection. F KEGG map of PPARα signaling pathway. DEGs were mapped to the “PPARα signaling pathway”, according to the “PPARs signaling pathway” map in KEGG with some modifications. DEGs are colored in blue. G, H Validation of expression levels of PPARa and some of its target genes after LIF overexpression by qPCR assays (G; n = 4/group) and Western-blot assays (H). At least three independent biological replicates were performed. All data are presented as mean ± SD. Each dot represents an individual mouse. Both female and male mice were used. For D, G: two-tailed Student’s t-test. Source data are provided as Source Data file.

Fig. 5. LIF downregulates the expression of PPARa via the activation of the STAT3 signaling in hepatic cells.

A LIF overexpression increased the levels of pSTAT3 but not total STAT3 protein or other LIF downstream pathways, including STAT1, STAT4, AKT, ERK, and MAPK in the liver of TgLC mice post TAM injection as determined by Western-blot assays. At least three independent biological replicates were performed. B rLIF treatment (100 ng/ml for 30 min) of primary mouse hepatic cells increased the levels of pSTAT3 but not total STAT3 protein as determined by Western-blot assays. Three replicates were presented in each group. At least three independent biological replicates were performed. C rLIF treatment decreased the mRNA levels of PPARa in primary cultured hepatic cells (n = 4/group). D The sequence and location of a putative STAT3 binding site in the mouse PPARa promoter region. TSS: transcription start site. E rLIF increased the binding of STAT3 to a putative STAT3 binding site in the promoter of PPARa as determined by ChIP assays in primary mouse hepatic cells. A chromatin region without STAT3 binding site was included as a negative control (n = 4/group). F Blocking STAT3 by STAT3 inhibitors, Stattic (2 μM) or Galiellalactone (5 μM), largely abolished the inhibitory effect of rLIF on the expression of PPARa in primary mouse hepatic cells. The mRNA levels of PPARa were determined by qPCR assays and normalized to β-actin (n = 4–8/group). G STAT3 siRNAs largely abolished the inhibitory effect of rLIF on PPARa expression in primary mouse hepatic cells. Left: relative PPARa mRNA levels; right: relative STAT3 mRNA levels in primary cultured hepatic cells (n = 4/group). All data are presented as mean ± SD. Both female and male mice were used. For C, E: two-tailed Student’s t test; for F, G: one-way ANOVA followed by t-test with Tukey’s multiple comparison adjustment. Source data are provided as Source Data file.

Fig. 6. Fenofibrate restores hepatic lipid homeostasis and inhibits cachexia induced by LIF overexpression.

A, B mRNA levels (A; n = 4/group) and protein levels (B) of PPARα target genes in the livers of TgLC mice fed with regular chow or fenofibrate (Feno) diet (0.2% w/w). C, D mRNA levels (C; n = 4/group) and protein levels (D) in the liver of Balb/c mice bearing with or without C26 tumors fed with regular chow or fenofibrate diet. E Fenofibrate diet increased the levels of majority of small LC-TGs (C ≤ 54) in the liver of TgLC mice and C26 tumor-bearing mice. Heatmap showing the TG levels in the liver of TgLC mice (n = 8/group) and C26 tumor-bearing mice (n = 6/group) fed with regular chow or fenofibrate diet. F Body weight of TgLC mice fed with regular chow or fenofibrate diet (n = 5/group). G Lean mass (left) and fat mass (right) of TgLC mice fed with regular chow or fenofibrate diet post TAM injection. n = 6–8/group. H Kaplan-Meier survival curves of TgLC mice fed with regular chow or fenofibrate diet. I Body weight of C26 tumor-bearing mice fed with regular chow or fenofibrate diet. The day of C26 cells inoculation was denoted as D0. n = 5/group. J Kaplan-Meier survival curves of C26 tumor-bearing mice. K The diagram depicting the mechanism by which LIF induces cachexia. The diagram was created with BioRender.com. Data are presented as mean ± SD for (A, C), and as mean ± SEM for (F, G, I). Each dot represents an individual mouse. Both female and male mice were used. ns: non-significant. For A, C: one-way ANOVA followed by t-test with Tukey’s multiple comparison adjustment; for F, G, I: two-way ANOVA followed by Sidak’s multiple comparison test; for H, J: two-tailed Kaplan-Meier survival analysis. Source data are provided as Source Data file.