Inflammation-induced cholestasis in cancer cachexia

Abstract

Background: Cancer cachexia is a debilitating metabolic syndrome contributing to cancer death. Organs other than the muscle may contribute to the pathogenesis of cancer cachexia. This work explores new mechanisms underlying hepatic alterations in cancer cachexia.

Methods: We used transcriptomics to reveal the hepatic gene expression profile in the colon carcinoma 26 cachectic mouse model. We performed bile acid, tissue mRNA, histological, biochemical, and western blot analyses. Two interventional studies were performed using a neutralizing interleukin 6 antibody and a bile acid sequestrant, cholestyramine. Our findings were evaluated in a cohort of 94 colorectal cancer patients with or without cachexia (43/51).

Results: In colon carcinoma 26 cachectic mice, we discovered alterations in five inflammatory pathways as well as in other pathways, including bile acid metabolism, fatty acid metabolism, and xenobiotic metabolism (normalized enrichment scores of -1.97, -2.16, and -1.34, respectively; all Padj < 0.05). The hepatobiliary transport system was deeply impaired in cachectic mice, leading to increased systemic and hepatic bile acid levels (+1512 ± 511.6 pmol/mg, P = 0.01) and increased hepatic inflammatory cytokines and neutrophil recruitment to the liver of cachectic mice (+43.36 ± 16.01 neutrophils per square millimetre, P = 0.001). Adaptive mechanisms were set up to counteract this bile acid accumulation by repressing bile acid synthesis and by enhancing alternative routes of basolateral bile acid efflux. Targeting bile acids using cholestyramine reduced hepatic inflammation, without affecting the hepatobiliary transporters (e.g. tumour necrosis factor α signalling via NFκB and inflammatory response pathways, normalized enrichment scores of -1.44 and -1.36, all Padj < 0.05). Reducing interleukin 6 levels counteracted the change in expression of genes involved in the hepatobiliary transport, bile acid synthesis, and inflammation. Serum bile acid levels were increased in cachectic vs. non-cachectic cancer patients (e.g. total bile acids, +5.409 ± 1.834 μM, P = 0.026) and were strongly correlated to systemic inflammation (taurochenodeoxycholic acid and C-reactive protein: ρ = 0.36, Padj = 0.017).

Conclusions: We show alterations in bile acid metabolism and hepatobiliary secretion in cancer cachexia. In this context, we demonstrate the contribution of systemic inflammation to the impairment of the hepatobiliary transport system and the role played by bile acids in the hepatic inflammation. This work paves the way to a better understanding of the role of the liver in cancer cachexia.

Keywords: Bile acids; Cholestyramine; Hepatobiliary transport system; IL-6; Liver.

Figure 1

Hepatic whole transcriptome analysis in cachectic mice. (A) Principal component analysis of 22 389 expressed genes in sham‐injected mice (CT) and in colon carcinoma 26 ‐transplanted mice (C26). (B) Volcano plot of genes differentially expressed in the liver of CT mice as compared with C26 mice [Padj < 0.01, absolute log₂(fold‐change) >1]. (C) Table of the most significantly modified gene pathways between CT and C26 mice using gene set enrichment analysis. N = 8 mice per group.

Figure 2

Bile acid pathways are altered in cachectic mice. (A) Bile acid profile in the systemic serum of colon carcinoma 26 ‐transplanted mice (C26) as compared with sham‐injected mice (CT). (B) Total bile acid levels in the liver of C26 mice as compared with CT mice. (C–E) Hepatic mRNA expression levels. Baat, bile acid‐CoA:amino acid N‐acyltransferase; Bacs, Solute Carrier Family 27 Member 5; Csad, cysteine sulfinic acid decarboxylase; Cyp27a1, cytochrome P450 family 27 sub‐family A member 1; Cyp7a1, cytochrome P450 family 7 sub‐family A member 1; Cyp7b1, cytochrome P450 family 7 sub‐family B member 1; Cyp8b1, cytochrome P450 family 8 sub‐family B member 1; Slc6a6, solute carrier family 6 member 6. N = 7–8 mice per group; data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Impairment of the hepatobiliary transport system in cachectic mice. (A) Schematic illustration of alterations in the hepatobiliary transport system in cachectic mice revealed by hepatic whole transcriptome analysis of cachectic mice. (B) Hepatic mRNA expression levels of genes involved in the hepatobiliary transport system in the liver of colon carcinoma 26‐transplanted mice (C26) as compared with sham‐injected mice (CT). (C) Hepatic function parameters reflected by total bilirubin levels and alanine aminotransferase levels (ALAT) in the serum of C26 mice as compared with CT mice. Data from (A) and (B) come from independent experiments. Abcg5, ATP‐binding cassette sub‐family G member 5; Abcg8, ATP‐binding cassette sub‐family G member 8; Bsep, bile salt export pump; Mdr2, multidrug resistance protein 2; Mrp2, multidrug resistance‐associated protein 2; Ntcp, Na(+)/taurocholate transport protein; Oatp1b2, organic anion transporter family member 1B2; Ostβ, organic solute transporter subunit beta; BA, bile acids; chol, cholesterol; CL‐, chloride ion; GSH, glutathione; HC03‐, ion bicarbonate; OA, organic anion; OC, organic cation; PL, phospholipids; ster, steroids. N = 7–8 mice per group; data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Hepatic inflammation in cachectic mice. (A) Western blot analysis of NFκB‐p65 in nuclear fraction extracts from the liver of colon carcinoma 26‐transplanted mice (C26) as compared with sham‐injected mice (CT). (B) Immunohistochemistry of Ly6G, a specific marker of neutrophils, in the liver of C26 mice and CT mice. N = 6–8 mice per group; data are presented as mean ± SEM, *P < 0.05, **P < 0.01.

Figure 5

Strong association between hepatobiliary alterations and the progression of cachexia in colon carcinoma 26 (C26) mice. (A) Pearson correlations between cachectic parameters and hepatic gene expression levels in C26 mice euthanized at 8, 9 and 10 days after injection. (B) Pearson correlations between cachectic parameters and bile acid profiles in C26 mice euthanized at 8, 9, and 10 days after injection. BAT, brown adipose tissue; GAS, gastrocnemius; SAT, subcutaneous adipose tissue; Bsep, bile salt export pump; Cxcl2, C‐X‐C motif chemokine ligand 2; Cyp27a1, cytochrome P450 family 27 sub‐family A member 1; Cyp7a1, cytochrome P450 family 7 sub‐family A member 1; Cyp7b1, cytochrome P450 family 7 sub‐family B member 1; Cyp8b1, cytochrome P450 family 8 sub‐family B member 1; Fbxo32, F‐box protein 32 (also known as Atrogin1); Icam1, intercellular adhesion molecule 1; Mdr2, multidrug resistance protein 2; Mmp8, matrix metallopeptidase 8; Mrp2, multidrug resistance‐associated protein 2; Ntcp, Na(+)/taurocholate transport protein; Oatp1b2, organic anion transporter family member 1B2; Ostβ, organic solute transporter subunit beta; Trim63, tripartite motif containing 63 (also known as Murf1). N = 8 mice per group, *P < 0.05, **Padj < 0.05.

Figure 6

Cholestyramine treatment counteracts alterations in hepatic bile acid profile and reduces hepatic inflammation in cachectic mice. (A) Hepatic bile acids profile in sham‐injected mice (CT), in untreated colon carcinoma 26‐transplanted mice (C26) and in C26‐transplanted mice receiving cholestyramine in their diet (C26‐CHO). (B) Volcano plot of genes differentially expressed in the liver of C26‐CHO mice as compared with C26 mice [Padj < 0.05, absolute log₂(fold‐change) >1]. (C) Table of the most significantly modified gene pathways between C26 and C26‐CHO mice using gene set enrichment analysis. (D) Hepatic mRNA expression levels of genes involved in inflammation in CT, C26 and C26‐CHO. Ccl2, C‐C motif chemokine ligand 2; Cd68, CD68 molecule; Cxcl1, C‐X‐C motif chemokine ligand 1; Cxcl2, C‐X‐C motif chemokine ligand 2; Icam1, intercellular adhesion molecule 1; Il6, interleukin‐6; Mmp8, matrix metallopeptidase 8; Tnfα, tumour necrosis factor. N = 7–8 mice per group; data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs. C26.

Figure 7

IL‐6 is the main driver of hepatic alterations in cachectic mice. (A) Hepatic mRNA expression levels of genes involved in the hepatobiliary transport system in the liver of colon carcinoma 26‐transplanted mice treated with phosphate‐buffered saline (C26), a neutralizing antibody targeting IL‐6 (anti‐IL‐6) or an isotope control (IgG) and control mice injected with phosphate‐buffered saline (CT). (B) Hepatic mRNA expression levels of genes involved in bile acid synthesis in the liver of CT, C26, anti‐IL‐6 and IgG mice. (C) Hepatic mRNA expression levels of genes involved in inflammatory response in the liver of CT, C26, anti‐IL‐6 and IgG mice. Abcg5, ATP‐binding cassette sub‐family G member 5; Abcg8, ATP‐binding cassette sub‐family G member 8; Bsep, bile salt export pump; Cxcl1, C‐X‐C motif chemokine ligand 1; Cxcl2, C‐X‐C motif chemokine ligand 2; Cyp7a1, cytochrome P450 family 7 sub‐family A member 1; Cyp8b1, cytochrome P450 family 8 sub‐family B member 1; Il1b, interleukin‐1β; Icam1, intercellular adhesion molecule 1; Ntcp, Na(+)/taurocholate transport protein; Oatp1b2, organic anion transporter family member 1B2; Ostβ, organic solute transporter subunit beta. N = 7–8 mice per group; data are presented as mean ± SEM. One‐way ANOVA with Tukey's post‐tests. *P < 0.05 vs. CT, #P < 0.05 vs. C26, $P < 0.05 vs. anti‐interleukin 6 (IL‐6).

Figure 8

Similar hepatic alterations were found in cachectic Lewis lung carcinoma‐injected mice. (A) Hepatic mRNA expression levels of genes involved in the hepatobiliary transport system in Lewis lung carcinoma ‐injected mice (LLC) as compared with sham‐injected mice (CT). (B) Hepatic mRNA expression levels of genes involved in bile acid synthesis in the liver of LLC mice as compared with CT mice. (C) Hepatic mRNA expression levels of genes involved in inflammation in the liver of LLC mice as compared with CT mice. Bsep, bile salt export pump; Ccl2, C‐C motif chemokine ligand 2; Cxcl1, C‐X‐C motif chemokine ligand 1; Cxcl2, C‐X‐C motif chemokine ligand 2; Cyp27a1, cytochrome P450 family 27 sub‐family A member 1; Cyp7b1, cytochrome P450 family 7 sub‐family B member 1; Cyp7a1, cytochrome P450 family 7 sub‐family A member 1; Cyp8b1, cytochrome P450 family 8 sub‐family B member 1; Icam1, intercellular adhesion molecule 1; Il6, interleukin‐6; Il1b, interleukin‐1β; Mmp8, matrix metallopeptidase 8; Mrp2, multidrug resistance‐associated protein 2; Ntcp, Na(+)/taurocholate transport protein; Ostβ, organic solute transporter subunit beta; Tnfα, tumour necrosis factor. N = 5–6 mice per group; data are presented as mean ± SEM, *P < 0.05, **P < 0.01.

Figure 9

Alteration of serum bile acids correlates with inflammatory markers in cachectic and non‐cachectic colorectal cancer patients. (A) Bile acid profile in the serum of cachectic and non‐cachectic colorectal cancer patients. (B) Serum total bile acid levels in cachectic and non‐cachectic colorectal cancer patients. (C) Partial Spearman rank‐based correlations between serum bile acids and clinical parameters (corrected for age and sex). N = 51 non‐cachectic patients, 43 cachectic patients, mean ± SEM, *P < 0.05, **Padj < 0.05. BMI, body mass index; CRP, C‐reactive protein.