Hepatocellular carcinoma is an often lethal malignancy with limited treatment options. Here, we have uncovered a central role for the winged helix transcription factors Foxa1 and Foxa2 in controlling estrogen and androgen signaling through recruitment of ERα and AR to their relevant targets in the liver, thereby explaining the sexual dimorphism of liver cancer in mammals. Not only are the Foxa factors required for the sex hormone receptors to bind to many of their targets, importantly, tumor growth is also strongly dependent on Foxa1/2. Thus, tumor load is dramatically increased in the livers of female Foxa1/2 mutant mice, and decreased in the livers of male Foxa1/2 mutants exposed to hepatocarcinogens. Our current view of how the Foxa1/2 factors control multiple aspects of hepatocarcinogenesis is summarized in the schema provided in Supplementary Figure S7
The Foxa factors play a dominant role in determining the gender specificity of HCC development. Interestingly, without carcinogen treatment, Foxa1/2 mutant mice maintain much of the sexual dimorphic gene expression profile that is present in control livers (data not shown). This suggests that the loss of gender specificity in Foxa1/2-deficient mice occurs with the onset of carcinogen exposure. Given the fact that multiple physiological and pathophysiological processes are sexually dimorphic in the organs expressing Foxa proteins, we speculate that other stresses, like dietary deprivation and aging, might also invoke gender-specific responses from Foxa factors, which we aim to address in the future.
A previous study had attributed the disparity in liver cancer between the sexes to differences in IL-6 production by Kupffer cells in response to chemical carcinogens (Naugler et al., 2007
). In fact, DEN-induced tumor incidence was reduced in both male and female IL-6 null mice. However, while we confirmed higher IL-6 plasma levels in wild type male mice treated with DEN compared to female controls, IL-6 levels did not correlate with tumor load in Foxa1/2-deficient mice ( and Figure S5
). In addition, because Foxa1/2 were not ablated in Kupffer cells but in hepatocytes in our model, the effect of the sex hormones on tumor susceptibility shown here is exclusively due to direct action of the Foxa factors on the parenchymal cells in the liver.
Estrogen promotes and estrogen antagonists (such as tamoxifen) prevent the growth of breast cancer cells, at least in estrogen receptor-positive tumors (Hollingsworth et al., 1998
; Zumoff, 1998
). Androgen promotes and androgen deprivation prevents the growth of prostate cancer cells (Paulson, 1984
; Smolev et al., 1977
). As discussed above, in both cancers, binding of the sex hormone receptor occurs near FOXA1 binding sites. However, in liver cancer, a different scenario has been recognized for years, in that estrogen prevents cancer development, opposite to the situation in the mammary gland. As we have shown here, both the effects of androgens and estrogens are Foxa1/2-dependent in the liver. Thus, a puzzle emerges: how can it be that estrogen signaling, dependent on Foxa factors in both the mammary gland and the liver, is tumor-promoting in the former, and tumor-preventing in the latter? This paradox suggests the existence of tissue-specific targets or tissue-specific co-regulation of the Foxa/ERα axis in the two tissues, an issue that will be of great interest for future investigation.
Thus far, no mutations in the FOXA1 or FOXA2 gene have been linked to human HCC. This might not be too surprising given the redundant function of the two proteins, which suggests that mutations in one or the other might not be sufficient to affect tumor initiation or progression. However, strikingly, we found multiple examples of single nucleotide polymorphisms in FOXA binding sites that affect FOXA and ERα occupancy, which were significantly more frequent in HCC samples than in normal livers from women. Not only does this finding provide supporting evidence that the co-regulation of target genes by FOXA and ERα extends from mice to humans, but it also suggests that SNPs in FOXA binding sites could contribute to the risk of hepatocarcinogenesis in women. Future large-scale studies investigating HCC risk in women with respect to these SNPs appear warranted.
We previously found that glucocorticoid receptor (GR)-mediated transcriptional regulation also depends on Foxa1/2 (Li et al., 2009
; Zhang et al., 2005
). Full activation of certain genes in the liver in response to a prolonged fast, and engagement of their cis
-regulatory elements by GR requires Foxa2 (Zhang et al., 2005
). Likewise, repression of the IL-6 promoter by GR is dependent on the Foxa factors in hepatocytes (Li et al., 2009
). Why are Foxa factors required for steroid hormone signaling? Our current study suggests that Foxa factors serve as a scaffold for steroid hormone receptors to regulate gene transcription in the liver on a genome-wide scale, and extends to at least three nuclear hormone receptors. Our finding that two sets of sex hormone cis
-regulatory elements, AREs and EREs, are found close to the same Foxa binding site indicates that Foxa-dependent estrogen and androgen signaling regulates the same set of genes in the liver (Figure S7
). More importantly, our studies showed that ERα and AR had opposite effects on this gene set between females and males ( and Figure S7
), which provides a molecular explanation for previous findings that estrogen and androgen were able to reverse HCC incidence in the opposite genders (Naugler et al., 2007
; Shimizu et al., 1998
; Tsutsui et al., 1992
; Yamamoto et al., 1991
). In conclusion, we have identified a set of regulatory units in which juxtaposition of Foxa binding sites next to both EREs and AREs allows for the mediation of the gender-specific effects of the sex hormones in the liver.