Hepatocyte-specific HDAC3 ablation promotes hepatocellular carcinoma in females by suppressing Foxa1/2

Abstract

Background: Hepatocellular carcinoma (HCC), the most common primary liver cancer, prevails mainly in males and has long been attributed to androgens and higher circumstantial levels of interleukin-6 (IL-6) produced by resident hepatic macrophages.

Methods: Constitutively hepatocyte-specific histone deacetylase 3 (HDAC3)-deficient (HDAC3^LCKO) mice and constitutively hepatocyte-specific HDAC3 knockout and systemic IL-6 simultaneously ablated (HDAC3^LCKO& IL-6^-/-) mice were used in our study to explore the causes of sex differences in HCC. Additionally, we performed human HCC tissues with an IHC score. Correlation analysis and linear regression plots were constructed to reveal the association between HDAC3 and its candidate genes. To further elucidate that HDAC3 controls the expression of Foxa1/2, we knocked down HDAC3 in HUH7 liver cancer cells.

Results: We observed a contrary sex disparity, with an earlier onset and higher incidence of HCC in female mice when HDAC3 was selectively ablated in the liver. Loss of HDAC3 led to constant liver injury and the spontaneous development of HCC. Unlike the significant elevation of IL-6 in male mice at a very early age, female mice exhibit stable IL-6 levels, and IL-6 ablation did not eliminate the sex disparity in hepatocarcinogenesis in HDAC3-deficient mice. Oestrogen often protects the liver when combined with oestrogen receptor alpha (ERα); however, ovariectomy in HDAC3-ablated female mice significantly delayed tumourigenesis. The oestrogen-ERα axis can also play a role in tumour promotion in the absence of Foxa1 and Foxa2 in the receptor complex. Loss of HDAC3 profoundly reduced the expression of both Foxa1 and Foxa2 and impaired the binding between Foxa1/2 and ERα. Furthermore, a more frequent HDAC3 decrease accompanied by the simultaneous Foxa1/2 decline was found in female HCC compared to that in male HCC.

Conclusion: In summary, we reported that loss of HDAC3 reduces Foxa1/2 and thus promotes HCC development in females in an oestrogen-dependent manner.

Keywords: Foxa1; Foxa2; HCC; HDAC3; IL-6; Oestrogen receptor; Sex difference.

Fig. 1

Significant hepatocyte damage in 2-month-old HDAC3^LCKO mice. (A) Western blot detection of HDAC3 protein in livers from HDAC3^LCKO mice. GAPDH and histone H3 were used as the loading controls. (B) Immunohistochemistry staining shows the negative expression of HDAC3 in HDAC3^LCKO hepatocytes. Scale bar, 100 μm. (C) Gross morphology of HDAC3^loxP/loxP and HDAC3^LCKO livers. (D) The ratio of liver weight (l.w) to body weight (b.w) between HDAC3^loxP/loxP mice and HDAC3^LCKO mice. (E-F) Serum levels of ALT and AST. (G) H&E staining and immunohistochemistry staining of glutamine synthetase (GS), cytokeratin 19 (CK19), and Ki67 in livers from 2-month-old HDAC3^LCKO mice. Scale bar, 100 μm. * p ＜ 0.05, **** p ＜ 0.0001, and ns, not significant

Fig. 2

HDAC3 deletion results in earlier HCC onset in female mice. (A) Gross morphology of HDAC3^LCKO livers at 9 and 12 months old. (B) The number of female HDAC3^LCKO mice and male HDAC3^LCKO mice with liver nodules. (C) The ratio of liver weight (l.w) to body weight (b.w) in HDAC3^LCKO mice at 9 and 12 months old. (D) The number of tumours in female and male HDAC3^LCKO mice at the indicated times. (E) The number of tumours larger than 5 mm in diameter in female and male HDAC3^LCKO mice. (F) Histological detection of the livers of 9-month-old female HDAC3^LCKO mice. T, tumour; NT, not tumour. Scale bar, 100 μm. * p ＜ 0.05, ** p ＜ 0.01, *** p ＜ 0.001, and ns, not significant

Fig. 3

HDAC3 deletion resulted in higher IL-6 levels in female mice and more severe liver damage was observed in HDAC3^LCKO&IL-6^−/− mice at 2 months of age. (A) Serum IL-6 levels in mice of different genotypes and sexes at 2, 6, and 9 months. (B) Western blot detection of HDAC3 protein in the liver. (C) Serum IL-6 levels in HDAC3^LCKO and HDAC3^LCKO&IL-6^−/− mice. (D) Gross morphology of HDAC3^LCKO and HDAC3^LCKO&IL-6^−/− livers. (E) The ratio of liver weight (l.w) to body weight (b.w) between HDAC3^LCKO and HDAC3^LCKO&IL-6^−/− mice. (F-G) Serum AST and ALT levels. (H) H&E staining and immunohistochemistry staining of GS, CK19, and Ki67 in livers from 2-month-old HDAC3^LCKO mice. Scale bar, 100 μm. * p ＜ 0.05, ** p ＜ 0.01, *** p ＜ 0.001, **** p＜ 0.0001, and ns, not significant

Fig. 4

HDAC3^LCKO&IL-6^-/- female mice developed HCC earlier than males. (A) Gross liver morphology in HDAC3^LCKO&IL-6^−/− mice at 7 and 9 months old. (B) The number of female HDAC3^LCKO&IL-6^−/− mice and male HDAC3^LCKO&IL-6^−/− mice with liver nodules. (C) The ratio of liver weight to body weight in HDAC3^LCKO&IL-6^−/− mice at 7 and 9 months old. (D) The number of tumours in female HDAC3^LCKO&IL-6^−/− mice and male HDAC3^LCKO&IL-6^−/− mice. (E) The number of tumours larger than 5 mm in diameter in female HDAC3^LCKO&IL-6^−/− mice and male HDAC3^LCKO&IL-6^−/− mice. (F) Histological detection of the liver in 7-month-old female HDAC3^LCKO&IL-6^−/− mice. T, tumour; NT, not tumour. Scale bar, 100 μm. * p ＜ 0.05, ** p ＜ 0.01, and *** p ＜ 0.001

Fig. 5

Ovariectomy significantly delayed tumorigenesis. (A) Gross morphology of HDAC3^LCKO-OVX livers at 9 and 12 months old. (B) The number of HDAC3^LCKO and HDAC3^LCKO-OVX mice with liver nodules at the indicated times. (C) The ratio of liver weight (l.w) to body weight (b.w) in HDAC3^LCKO and HDAC3^LCKO-OVX mice. ** p ＜ 0.01, *** p ＜ 0.001, and **** p ＜ 0.0001

Fig. 6

HDAC3 ablation suppresses Foxa1 and Foxa2 expression and their binding to ERα and AR. (A) Immunohistochemical detection of Foxa1 and Foxa2 in the livers of HDAC3^LCKO mice. Scale bar, 100 μm. (B) Western blot detection of Foxa1 and Foxa2 protein expression in the livers of HDAC3^LCKO mice. (C) Western blotting indicating that knockdown of HDAC3 reduced Foxa1 and Foxa2 in HUH7 cells. (D) The mRNA level of HDAC3, Foxa1, and Foxa2 in HDCA3^loxP/loxP and HDAC3^LCKO mice. (E) The mRNA level of Foxa1 and Foxa2 in HUH7 cells. (F) The interactions between ERα and Foxa1/2 were analyzed by co-immunoprecipitation (co-IP). (G) The interactions between AR and Foxa1/2 were analyzed by co-immunoprecipitation (co-IP).

Fig. 7

Human HCCs display sex differences in HDAC3 levels. (A) staining intensity of HDAC3 in HCC. Scale bar, 100 μm. (B-C) Staining intensity of Foxa1 and Foxa2 in HCC. Scale bar, 100 μm. (D) The chi-square test revealed HDAC3 expression was presumably lower in female HCC. (E-F) Correlation analysis revealed that Foxa1 (r = 0.5265; p < 0.0001) and Foxa2 (r = 0.5550; p < 0.0001) were positively associated with HDAC3 expression in female HCC patients. (G) Diagram of the hypothetical roles of HDAC3 in female HCC development by regulating the Foxa1/2 signalling pathway. * p ＜ 0.05 and **** p ＜ 0.0001