Fxr/Shp double knockout (DKO) mouse model recapitulates sex-difference observed in HCC incidence.

(A-E) One-year-old DKO male mice developed hepatocellular carcinoma, which was not observed in age-matched wild type (WT) and DKO female mice. (B and E) Representative H&E stained liver sections from a (B) DKO male and (E) female. Inflammation and injury are evident at 1 year and the dotted line (B) separates the HCC with large nuclei on lower right. Sirius red staining shows increased collagen in a perisinusoidal distribution, which is greater in the DKO males (C and F). Liver-to-body weight ratio was significantly higher in DKO males (G). Compared to WT animals, serum markers of liver injury, (H) AST and ALT were higher in DKO mice. (I-J) Analysis of five different HCC clinical cohorts (n=1000) patients reveals a reduction in Fxr and Shp transcript levels in patients with liver tumors. n=5-10 mice /group; mean ± SEM; *p<0.01, **p<0.001 compared to genotype or gender controls. One-way ANOVA with Bonferroni post hoc analysis was performed.

Transcriptome analysis reveals striking sex differences in hepatic metabolism.

Microarray was performed on liver tissue from WT and DKO mice of both sexes (n = 6/group). (A-B) GO categories were determined using genes with >1.3 fold change in expression between DKO males and females. Enrichment of overlapping GO categories between males and females was determined by comparing – log p-values for each term. (C) GO categories unique to the set of genes upregulated >1.3 fold in DKO males and (D) DKO females.

Gene signatures obtained from the DKO mouse model correlate well with the clinical outcomes of HCC patient.

The survival probability based on WT and DKO transcriptome changes was evaluated using five different HCC clinical cohorts. (A-C) Analysis of OS (Overall Survival) and RFS (Recurrence Free Survival) in patients using the gene signatures representative of either male WT or DKO mice (A), female WT or DKO mice (B) and unique changes observed in female DKO mice (C).

Estrogen signaling protects against liver tumorigenesis and can regulate BA synthesis in DKO female mice.

(A-B) Ovariectomized female DKO mice were aged to a year and examined for liver tumorigenesis, where a dotted line demarcates the tumor margin. (C) Serum total bile acid concentrations. (D-H) Experimental design of chow and 1% cholic acid (CA) diet for 1 week with or without (OVX). (E) Expression of hepatic Era was induced with CA diet in DKO female mice and reduced in both WT and DKO females following ovariectomy. (F) CA-mediated suppression of Cyp7a1 and (G) Cyp8b1 in WT and DKO females was completely lost in DKO females after OVX. (H) Sult2a1 has greater baseline expression in DKO mice, induced to a lesser extent upon CA challenge compared to WT animals (n = 4-5/group). (I) ChIP-PCR was performed in WT and DKO male and female livers to test ERα recruitment to BA synthesis and metabolism genes, Cyp7a1, Cyp8b1, and Sult2a1.Mean ± SEM; Two-way ANOVA with Bonferroni post hoc analysis was performed. #p<0.05, *p<0.01, **p<0.001 compared to controls.

BA composition and metabolism are differentially regulated between the sexes of DKO mice.

(A-B) Hepatic mRNA expression of classical BA synthetic enzymes was elevated in DKO compared to WT mice. Alternative BA synthesis enzyme, Cyp27a1, was increased in females only. Expression of hepatic BA transporters (B) and BA sulfotransferase in WT and DKO mice (C). (D) Percentages of sulfated BAs in DKO male and female serum (one tailed t-test). (E-F) Serum BA composition is slightly varied, whereas it remains unchanged in the liver between DKO males and females. (G-H) BA composition in the urine was variable between the sexes, and BAs constitute a higher proportion of fecal metabolites in the DKO female compared to males (n = 5-7/group). Mean ± SEM; #p<0.05, *p<0.01, **p<0.001 compared to genotype or gender controls. One-way ANOVA with Bonferroni post hoc analysis was performed.

Treating with BA-binding resin reduces the tumor burden in DKO male mice.

(A) DKO male mice were fed a 2% cholestyramine (CHR)-enriched diet for 3 months until one year of age. (B-C) Serum BA levels and composition upon feeding DKO male mice CHR-enriched diet. (D) CHR dramatically reduced HCC burden in DKO males. (E) Histological analysis shows HCC, bland tumor cells and enlarged nuclei with irregular membranes in DKO male mice. CHR treatment results in smaller and fewer nodules but increases steatosis that is also reflected in the liver weight (D). (n = 6-7/group). Mean ± SEM; *p<0.01; **p<0.001 compared to DKO controls.