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

Reviewer #1 (Public Review):

Summary:

Liver cancer shows a higher 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 the 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 levels and signaling may be beneficial in HCC treatment.

Strengths:

(1) Chronic liver diseases often preceed 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 develops 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.

Weaknesses:

(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.

(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.

(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.

(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.

Reviewer #2 (Public Review):

Summary:

The manuscript of Patton et al. shows that in mice in which both FXR and SHP are knocked out, the sex difference in liver cancer risk is recapitulated. Authors show that the protection against tumor development seen in female mice is dependent upon ovarian hormone secretion and higher fecal bile acid excretion in females compared to males. The female liver-specific gene signature correlates with low-grade tumors and better survival in human HCC patients.

The combination of the use of the double knockout mice together with ovariectomy in female mice and using a bile acid raisin in male mice to underscore their conclusion is strong. However, there are also some shortcomings, that should be addressed.

Strengths:

(1) Using computational modelling, Patton and colleagues correlate mouse DKO transcriptome data to the clinical outcomes of HCC patients using HCC transcriptome datasets.

(2) The dependence of female protection on ovarian hormones and increased fecal bile acid excretion is nicely shown by combining ovariectomy and bile acid raisin with the use of double knockout mice.

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.

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

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