Sex differences in bile acid homeostasis and excretion underlie the disparity in liver cancer incidence between males and females
- Department of Molecular and Integrative Physiology, University of Illinois, Urbana-Champaign, United States
- Division of Nutritional Sciences, University of Illinois, Urbana-Champaign, United States
- Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, United States
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska, United States
- Department of Systems Biology, The University of Texas MD Anderson Cancer Center, United States
- CHA Bundang Medical Center, CHA University School of Medicine, Republic of Korea
- Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, United States
- Cancer center at Illinois, University of Illinois, Urbana-Champaign, United States
- Department of Pathology, Baylor College of Medicine, United States
Figures
Figure 1 with 1 supplement
Fxr/Shp double knockout (DKO) mouse model recapitulates sex difference observed in HCC incidence.
(A) One-year-old DKO male mice developed hepatocellular carcinoma, which was not observed in age-matched wild type (WT) and DKO female mice (D). (B and E) Representative H&E stained liver sections from a (B) DKO male and (E) DKO female. Inflammation and injury are evident at 1 year, and the dotted line (B) separates the HCC with large nuclei on the lower right. (C and F). Sirius red staining shows increased collagen in a perisinusoidal distribution, which is greater in the DKO males. (G) The liver-to-body weight ratio was significantly higher in DKO males. (H) Compared to WT animals, serum markers of liver injury AST and ALT were higher in DKO mice. (I–J) Analysis of five different HCC clinical cohorts (n=1000) reveals a reduction in NR1H4 and NR0B2 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.
Figure 1—figure supplement 1
DKO female mice exhibit reduced liver injury.
(A–B) Compared to DKO males, the female mice showed smaller liver size and liver-to-body weight ratio. (C) Sirius red staining revealed less collagen staining (red) in DKO females. (Six-month-old mice, n=6/group). Mean ± SEM. *p<0.01, **p<0.001 compared to genotype or gender controls. Unpaired t-test was used to analyze the data.
Figure 2
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.
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Figure 2—source data 1
List of genes in different DKO gene signature categories used for analysis.
See the Excel sheet.
- https://cdn.elifesciences.org/articles/96783/elife-96783-fig2-data1-v1.xlsx
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Figure 2—source data 2
Transcription motifs enriched in DKO male livers compared to DKO females.
- https://cdn.elifesciences.org/articles/96783/elife-96783-fig2-data2-v1.docx
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Figure 2—source data 3
Transcription motifs enriched in DKO female livers compared to DKO males.
- https://cdn.elifesciences.org/articles/96783/elife-96783-fig2-data3-v1.docx
Figure 3 with 4 supplements
Correlation of gene signatures obtained from WT and DKO mouse model with the survival data of HCC patients.
The Kaplan Meier Survival graphs were generated based on WT and DKO transcriptome changes using five different HCC clinical cohorts. (A–C) Analysis of OS (Overall Survival) and RFS (Recurrence Survival) in patients using the gene signatures representative of either (A) male WT or male DKO, (B) female WT or female DKO, and (C) unique changes observed in female DKO mice but not in male DKO mice.
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Figure 3—source data 1
Table of different HCC gene expression data sets used for analysis.
- https://cdn.elifesciences.org/articles/96783/elife-96783-fig3-data1-v1.docx
Figure 3—figure supplement 1
Schematic of the pipeline used to obtain gene signatures to predict outcomes in HCC patient cohorts.
(A) Workflow schematic of the gene expression analysis. (B–C) Different gene signatures were extracted from the microarray data by comparing wild-type (WT) male and female gene expression patterns with that of DKO male and DKO female, respectively. These gene lists were used to predict overall survival and recurrence-free survival using five human clinical cohorts. (D) Gene changes in these defined sets, DKO males (DKO_M), DKO females (DKO_F), or combined (DKO_ALL), or DKO_F vs M (DKO female gene signature that does not overlap with DKO males), DKO_Estrogen, DKO_BA, and DKO_Urea, are shown. These genes were selected by t-test and log2 Fold Change (P<0.001 and log2 FC >1 or <-1).
Figure 3—figure supplement 2
Association of DKO-derived gene signatures with clinical stages of HCC.
The clinical relevance of DKO gene signatures—DKO_Combined (shared male/female), DKO_Male, DKO_Female, and DKO_FvsM (female vs. male)—was assessed in HCC cohorts using a Bayesian Compound Covariate Predictor (BCCP). For each sample, prediction probabilities (range: 0=low correlation, 1=high correlation) were calculated and stratified by clinical stage using (A–D) CLIP (Cancer of the Liver Italian Program) scores and (E–H) TNM (Tumor-Node-Metastasis) stages. Each box represents the interquartile range (IQR)—the middle 50% of BCCP prediction probabilities—for a given clinical group. The bold horizontal line within each box indicates the median probability, while individual dots represent BCCP probabilities for individual samples. Vertical spread of the dots reflects variability within each stage group. These plots illustrate the relationship between DKO gene signatures and liver cancer stage.
Figure 3—figure supplement 3
Expression of the urea cycle genes in human liver cancer.
RNA-seq from TCGA-LIHC (The Cancer Genome Atlas- Liver Hepatocellular Carcinoma Collection) database was analyzed. Several genes that encode enzymes involved in ureagenesis exhibited a reduction in their transcript levels in tumors compared to non-tumor (NT) tissue. (Males: n=29 NT, n=245 tumors; Females: n=20 NT and n=114 tumors). Mean ± SEM; *p<0.01, **p<0.001, ***p<0.0001 compared to their respective sex-specific controls. One-way ANOVA with Bonferroni post hoc analysis was performed.
Figure 3—figure supplement 4
DKO female livers exhibit increased expression of urea cycle genes, and they correlate with better patient survival.
(A) Schematic of the urea cycle, with a representative heat map of urea cycle genes from the microarray showing higher expression in DKO female livers (n=6/group). (B) Survival curves from HCC patients who exhibit increased expression of urea cycle genes.
Figure 4 with 2 supplements
Estrogen signaling protects against liver tumorigenesis and may 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) 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 a 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 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 ERa recruitment to BA synthesis and metabolism genes, Cyp7a1, Cyp8b1, and Sult2a1. Rabbit IgG was used as a control for the ChIP-PCR experiments. Mean ± SEM; Two-way ANOVA with Bonferroni post hoc analysis was performed.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to controls.
Figure 4—figure supplement 1
Loss of endogenous estrogen lowers the expression of urea cycle transcripts in DKO females.
(A) Expression of urea cycle genes was reduced in DKO female mice following OVX (n=4–5/group). and (B) LC-MS quantification of urea cycle intermediates (n=4–5/group). Mean ± SEM; *p<0.01, **p<0.001 compared to genotype or gender controls. Unpaired t-test was used for analysis.
Figure 4—figure supplement 2
Estrogen receptor signaling positively correlates with better survival in HCC clinical samples.
(A) RNA-seq from TCGA-LIHC (The Cancer Genome Atlas- Liver Hepatocellular Carcinoma Collection) data was analyzed. ERα transcript levels were reduced in tumors compared to non-tumor (NT) tissue. (Males: n=29 NT, n=245 tumors; Females: n=20 NT and n=114 tumors). Mean ± SEM; *p<0.01, **p<0.001, ***p<0.0001 compared to their respective sex-specific controls. One-way ANOVA with Bonferroni post hoc analysis was performed. (B) Kaplan Meier survival graphs using Era targets altered in DKO females (DKO_Estrogen) showed better clinical outcomes for HCC patients.
Figure 5
BA composition and metabolism are differentially regulated between the sexes of DKO mice.
(A) Hepatic mRNA expression of classical BA synthetic enzymes was elevated in 6-month-old DKO compared to WT mice. While the alternative BA synthesis encoding gene, Cyp27a1, was increased in females only. (B) Expression of hepatic BA transporters and (C) BA sulfotransferase in WT and DKO mice. (D) Percentages of sulfated BAs in DKO male and female serum (one-tailed t-test, #Pp<0.05). (E–F) BA composition is slightly varied in serum, 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 year old DKO females compared to males (n=5–7/group). Mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to genotype or sex-specific controls. One-way ANOVA with Bonferroni post hoc analysis was performed.
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Figure 5—source data 1
Serum BA composition in DKO mice.
- https://cdn.elifesciences.org/articles/96783/elife-96783-fig5-data1-v1.docx
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Figure 5—source data 2
Urine BA composition in DKO mice.
- https://cdn.elifesciences.org/articles/96783/elife-96783-fig5-data2-v1.docx
Figure 6 with 2 supplements
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 1 year of age. (B–C) Serum BA levels and composition upon feeding DKO male mice a CHR-enriched diet. (D) CHR dramatically reduced the 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. (n=6–7/group). Mean ± SEM; ****p<0.0001 compared to DKO controls.
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Figure 6—source data 1
Hepatic and serum BA profiles after different diet regimens (Chow, DDC, or CHR) in WT and DKO mice.
See the Excel sheet.
- https://cdn.elifesciences.org/articles/96783/elife-96783-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
DKO females challenged with the DDC-enriched diet developed liver tumors.
(A–B) DKO females were fed a 0.1% DDC diet, as shown in the schematic, for 3 months, and this treatment led to visible tumors at one year. (C–D) DDC diet elevated the liver-to-body weight ratio and serum BAs in DKO females. (E) Histology revealed cholangitis, ductular reaction, and tumor nodules (marked by a dotted line) in DKO female livers + DDC treatment. (n=4–7/group). Mean ± SEM; *p<0.01; **p<0.001 compared to chow-fed DKO controls. Unpaired t-test was used for analysis.
Figure 6—figure supplement 2
BA composition and expression of BA synthesis genes in DKO mice after different treatments.
(A) Correlation analysis of serum and hepatic composition of BAs post-DDC diet in DKO female mice (n=5–8 mice/group) and post-CHR diet in DKO male mice (n=4–5 mice/group), along with their sex-specific chow controls. (B–C) Relative expression of classical and alternative BA synthesis genes after CHR and DDC treatments. Mean ± SEM; *p<0.01; **p<0.001, *** p<0.0001 compared to their chow-fed DKO controls. Unpaired t-test was used for analysis.
Author response image 1
Gross liver images of a year-old WT and DKO mice which show prominent hepatocarcinogenesis in DKO male mice.
Author response image 2
Correlation of gene signatures obtained from WT and DKO mouse model with the survival data of HCC patients segregated by age and sex.
The Kaplan Meier Survival graphs were generated based on WT and DKO transcriptome changes using five HCC clinical cohorts. Analysis of OS (Overall Survival) in patients ((A) Men and (B) Women) using the gene signatures representative of either male WT or male DKO, female WT or female DKO, and unique changes observed in female DKO mice but not in male DKO mice.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Farnesoid x receptor (FXR) and Small heterodimer partner (SHP) | GenBank | Nr1h4 Nr0b2 | Key regulators of bile acid homeostasis |
| Strain background (Mus musculus) | C57BL/6 | In house PMCID:PMC3007143 | FXR/SHP Ddouble knockout (DKO) mice; RRID:MGI:2159769 | Both sexes of DKO model and wild type mice were used for analysis in this paper |
| Chemical compound used in the diet - (0.1%) | 3,5-Di-ethoxycarbonyl-1,4-Dihydrocollidine (DDC) | Purchased from Sigma | Catalog# 137030 | Envigo – (chemical compound was mixed with base diet and pelleted) |
| Chemical compound used in the diet – (2%) | Cholestyramine (CHR) | Purchased from Sigma | Catalog # C4650 | Envigo –(chemical compound was mixed with base diet and pelleted) |
| Other | Hematoxylin & Eosin Sirius Red Staining | Epredia VWR-Avantor | Catalog # 71311, # 7211 Catalog # 10065–176 | Stains used to analyse liver histology |
| Sequence-based reagent | Several genes | This paper | PCR primers | See Supplementary file 1 |
| Antibody | Anti-rabbit polyclonal ERα-MC10 | sc-542 Santacruz | RRID:AB_631470 | Used for ERα ChIP |
| Commercial assay or kit | Bile acid analysis | Genway Biotech | Total BA concentration | |
| Software | EndNote Prism | Clarivate GraphPad | EndNote Prism | Reference Statistical analysis |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/96783/elife-96783-mdarchecklist1-v1.docx
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Supplementary file 1
The title is written in the word doc- Primer Sequences used.
- https://cdn.elifesciences.org/articles/96783/elife-96783-supp1-v1.docx
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Sex differences in bile acid homeostasis and excretion underlie the disparity in liver cancer incidence between males and females
eLife 13:RP96783.
https://doi.org/10.7554/eLife.96783.4
