Sex differences in bile acid homeostasis and excretion underlie the disparity in liver cancer incidence between males and females

Reviewer #1 (Public review):

Liver cancer shows a high incidence in males than females with incompletely understood causes. This study utilized a mouse model that lacks the bile acid feedback mechanisms (FXR/SHP DKO mice) to study how dysregulation of bile acid homeostasis and a high circulating bile acid may underlie the gender-dependent prevalence and prognosis of HCC. By transcriptomics analysis comparing male and female mice, unique sets of gene signatures were identified and correlated with HCC outcomes in human patients. The study showed that ovariectomy procedure increased HCC incidence in female FXR/SHP DKO mice that were otherwise resistant to age-dependent HCC development, and that removing bile acids by blocking intestine bile acid absorption reduced HCC progression in FXR/SHP DKO mice. Based on these findings, the authors suggest that gender-dependent bile acid metabolism may play a role in the male-dominant HCC incidence, and that reducing bile acid level and signaling may be beneficial in HCC treatment. This study include many strengths: 1. Chronic liver diseases often proceed the development of liver and bile duct cancer. Advanced chronic liver diseases are often associated with dysregulation of bile acid homeostasis and cholestasis. This study takes advantage of a unique FXR/SHP DKO model that develop high organ bile acid exposure and spontaneous age-dependent HCC development in males but not females to identify unique HCC-associated gene signatures. The study showed that the unique gene signature in female DKO mice that had lower HCC incidence also correlated with lower grade HCC and better survival in human HCC patients. 2. The study also suggests that differentially regulated bile acid signaling or gender-dependent response to altered bile acids may contribute to gender-dependent susceptibility to HCC development and/or progression. 3. The sex-dependent differences in bile acid-mediated pathology clearly exist but are still not fully understood at the mechanistic level. Female mice have been shown to be more sensitive to bile acid toxicity in a few cholestasis models, while this study showed a male dominance of bile acid promotion of HCC. This study used ovariectomy to demonstrate that female hormones are possible underlying factors. Future studies are needed to understand the interaction of sex hormones, bile acids, and chronic liver diseases and cancer.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Comments:

(1) HCC shows heterogeneity, and it is unclear what tissues (tumor or normal) were used from the DKO mice and human HCC gene expression dataset to obtain the gene signature, and how the authors reconcile these gene signatures with HCC prognosis.

Mice studies: Aged DKO mice develop aggressive tumors (major and minor nodules, See Figure 1), and the entire liver is burdened with multiple tumor nodules. It is technically challenging to demarcate the tumor boundaries as most of the surrounding tissues do not display normal tissue architecture. Therefore, livers from age- and sex-matched wild-type C57/BL6 mice were used as control tissue. All the mice were inbred in our facility. Spatial transcriptomics and longitudinal studies are ongoing to collect tumors at earlier time points wherein we can differentiate tumor and non-tumor tissue.

Human Studies: We mined five separate clinical data sets. The human HCC gene expression comprised of samples from the (i) National Cancer Institute (NCI) cohort (GEO accession numbers, GSE1898 and GSE4024) and (ii) Korea, (iii) Samsung, (iv) Modena, and (v) Fudan cohorts as previously described (GEO accession numbers, GSE14520, GSE16757, GSE43619, GSE36376, and GSE54236). We have added a new supplemental table 4, giving details of these datasets. Depending on the cohort, they are primarily HCC samples- surgical resections of HCC, control samples, with some tumors and paired non-tumor tissues.

(2) The authors identified a unique set of gene expression signatures that are linked to HCC patient outcomes, but analysis of these gene sets to understand the causes of cancer promotion is still lacking. The studies of urea cycle metabolism and estrogen signaling were preliminary and inconclusive. These mechanistic aspects may be followed up in revision or future studies.

We agree. Experiments to elicit HCC causality and promotion are complex, given the heterogeneous nature of liver cancer. Moreover, the length of time (12 months) needed to spontaneously develop cancer in this DKO mouse model makes it challenging. As mentioned by the reviewer, mechanistic studies are ongoing, and longitudinal time course experiments are actively being pursued to delineate causality. Having said that, we mined the TCGA LIHC (The Cancer Genome Atlas Liver Hepatocellular Carcinoma) database to examine the expression of the individual urea cycle genes and found them suppressed in liver tumorigenesis (new Supplementary Figure 4). We also evaluated if estrogen receptor a (Era) targets altered in DKO females (DKO_Estrogen) correlate with overall survival in HCC (new Supplementary Figure 6). We note that Era expression per se is reduced in males and females upon liver tumorigenesis. Also, DKO_Estrogen signature positively corroborated with better overall survival (new Supplementary Figure 6). These findings further bolster the relevance of urea cycle metabolism and estrogen signaling during HCC.

(3) While high levels of bile acids are convincingly shown to promote HCC progression, their role in HCC initiation is not established. The DKO model may be limited to conditions of extremely high levels of organ bile acid exposure. The DKO mice do not model the human population of HCC patients with various etiology and shared liver pathology (i.e. cirrhosis). Therefore, high circulating bile acids may not fully explain the male prevalence of HCC incidence.

We agree with this comment that our studies do not show bile acids can initiate HCC and may act as one of the many factors that contribute to the high male prevalence of HCC. This is exactly the reason why throughout the manuscript we do not write about HCC initiation. To clarify further, in the revised discussion of the manuscript, we have added a sentence to highlight this aspect, “while this study demonstrates bile acids promote HCC progression it does not investigate or provide evidence if excess bile acids are sufficient for HCC initiation.”

(4) The authors showed lower circulating bile acids and increased fecal bile acid excretion in female mice and hypothesized that this may be a mechanism underlying the lower bile acid exposure that contributed to lower HCC incidence in female DKO mice. Additional analysis of organ bile acids within the enterohepatic circulation may be performed because a more accurate interpretation of the circulating bile acids and fecal bile acids can be made in reference to organ bile acids and total bile acid pool changes in these mice.

As shown in this manuscript- we provide BA compositional analyses from the liver, serum, urine, and feces (Figures 5 and 6, new Supplementary Figure 8, Supplementary Tables 4 and 5). Unfortunately, we did not collect the intestinal tissue or gallbladders for BA analysis in this study. Separate cohorts of mice are being aged for future BA analyses from different organs within the enterohepatic loop. We thank you for this suggestion. Nevertheless, we have previously measured and reported BA values to be elevated in the intestines and the gall bladder of young DKO mice (PMC3007143).

Reviewer #2 (Public review)

Weaknesses:

(1) The translational value to human HCC is not so strong yet. Authors show that there is a correlation between the female-selective gene signature and low-grade tumors and better survival in HCC patients overall. However, these data do not show whether this signature is more highly correlated with female tumor burden and survival. In other words, whether the mechanisms of female protection may be similar between humans and mice. In that respect, it would also be good to elaborate on whether women have higher fecal BA excretion and lower serum BA concentration.

The reviewer poses an interesting question to test if the DKO female-specific signatures are altered differently in male vs. female HCC samples. As we found the urea cycle and estrogen signaling to be protective and enriched in our mouse model, we tested their expression pattern using the TCGA-LIHC RNA-seq data. We found urea cycle genes and Era transcripts broadly reduced in tumor samples irrespective of the sex (new Supplementary Figure 4 and Supplementary Figure 6), indicating that these pathways are compromised upon tumorigenesis even in the female livers.

While prior studies have shown (i) a smaller BA pool w synthesis in men than women (PMID: 22003820), we did not find a study that systematically investigated BA excretion between the sexes in HCC context. The reviewer is spot on in suggesting BA analysis from HCC and unaffected human fecal samples from both sexes. Designing and performing such studies in the future will provide concrete proof of whether BA excretion protects female livers from developing liver cancer. We thank you for these suggestions.

(2) The authors should perform a thorough spelling and grammar check.

We apologize for the typos, which have been fixed, and as suggested by the reviewer, we have performed a grammar check.

(3) There are quite some errors and inaccuracies in the result section, figures, and legends. The authors should correct this.

We apologize for the inadvertent errors in the manuscript, and we have clarified these inaccuracies in the revised version. Thank you.