The susceptibility of BR mice to hepatocarcinogenesis is primarily caused by two susceptibility loci, Hcf1 and Hcf2, on chromosomes 17 and 1, respectively. These loci were initially identified through a linkage analysis of crosses between the susceptible BR and resistant B6 strains and their existence has now been further validated through the analysis of congenic mice. Homozygosity for the full-length BR chromosome 1 region caused a 3-fold increase in liver tumor multiplicity in females and a 7-fold increase in males. The chromosome 17 region had a 4-fold effect in females and a 2- to 4.5-fold effect in males. Both of these regions appear to act at least semidominantly in both sexes. The high susceptibility seen in double heterozygotes, with one copy of each region, indicates that these two loci are responsible for the majority of the susceptibility in the BR strain. For the Hcf1 locus on chromosome 17, a set of 16 recombinant lines covering the entire congenic region was generated, each containing a portion of chromosome 17 from the BR strain on a B6 background. Susceptible heterozygous chromosome 17 recombinants demonstrated between a 3- and 7.5-fold effect in females and between a 1.5- and 2-fold effect in males, completely recapitulating the increase seen in heterozygous full-length congenics. The analysis of these recombinants indicates that Hcf1 is located in a single region from 30.05 to 35.83 Mb that is responsible for the increase in liver tumor sensitivity.
BR females are up to 50-fold more susceptible than females of all other inbred strains that have been evaluated (8
). This effect is partly due to the loss of protection against liver tumor development typically offered by ovarian hormones: ovariectomized and intact BR females do not show a significant difference in liver tumor multiplicity (11
). However, the fact that the two susceptibility loci are the major cause of susceptibility in BR females and males suggests that the same underlying pathway must be at work in both sexes. Work from other labs indicates that the protective effect of the ovaries is most probably mediated by estrogen. Chronic administration of estrogen has been shown to protect intact males (24
) and ovariectomized females (25
). Furthermore, progesterone was seen to have no effect on liver tumor incidence. As estrogen levels in females during diestrus have been shown to be only approximately twice that measured in males (26
), estrogen could be exerting a protective effect in both sexes that is overwhelmed in males by the promotion of liver tumors caused by androgens (7
). The increase in liver tumor multiplicity in BR males and females due to the chromosome 17 locus could therefore be caused by the loss of the same protective effect in both sexes. There is no difference in the levels of estrogen in B6 and BR females (M.H.Feld and N.D., unpublished data). The amounts and affinities of estrogen receptor in the livers of B6 and BR mice are similar as well (13
), suggesting that the chromosome 17 locus acts downstream of estrogen binding.
Estrogen may have either a direct or an indirect effect on hepatocarcinogenesis. When chimeras between the BR and B6 strains were treated with DEN, a majority of the tumors that developed originated from the BR strain (12
). This result indicates that the BR susceptibility genes collectively have a cell-autonomous effect. The chromosome 17 locus may act downstream of estrogen binding and so Hcf1
could cause cell-autonomous effects in the liver in response to estrogen. However, it is unknown whether only one locus or both Hcf
loci act cell autonomously. Chimeras between C3H and B6 have similarly demonstrated the inherent susceptibility of the C3H strain to both spontaneous (28
) and DEN-induced (29
) liver tumors. As the C3H and BR strains appear to share a susceptibility locus on chromosome 1, it is possible that the chromosome 1 locus acts cell autonomously in both of these strains, whereas the chromosome 17 locus acts non-cell autonomously.
There is explicit evidence that estrogen may have an indirect effect on hepatocarcinogenesis: subcutaneous implantation of estrogen pellets decreases liver tumor incidence, whereas implantation of estrogen pellets in the spleen, which drains directly into the liver, does not (25
). The change in expression of interleukin-6 (IL-6) in response to DEN is greater in males and ovariectomized females than in intact females (30
), and the differences among these groups are reduced by the injection of estrogen. Knocking out IL-6 confers on males a resistance to hepatocarcinogenesis that is comparable with that of wild type and IL-6 knockout females. Naugler et al.
) have hypothesized that the death of hepatocytes due to DEN causes Kupffer cells to release IL-6, which promotes compensatory hepatocyte regeneration, increasing the fixation of initiating mutations that result in liver tumors. Estrogen might protect livers from tumors indirectly by inhibiting the production of IL-6.
The multistage model of carcinogenesis, originally developed to describe tumor formation in the skin (31
), can also be applied to tumor formation in the liver (14
). This model divides tumor development into the three stages of initiation, promotion and progression. Ovarian hormones exert much of their protection during the promotion phase. Estrogen could act during the conversion stage of promotion (31
), when an initiated cell divides and begins to express its preneoplastic phenotype, by inhibiting production of IL-6 (30
). Estrogen may also act later during the propagation stage of promotion, by inhibiting the proliferation or enhancing apoptosis of preneoplastic cells. Preneoplastic lesions in the liver have similar growth rates in B6 and C3H females, but grow more rapidly in BR females (11
). These lesions grow more slowly in B6 and C3H females than in the corresponding males, but the growth rates of lesions in BR males and BR females are similar. Moreover, ovariectomy performed weeks after carcinogen treatment increases the growth rate of preneoplastic lesions in B6, C3H and B6C3F1
females, but not in BR females (7
). Further evidence that the modifiers act after initiation is provided by the observation that BR males haves a high spontaneous incidence of liver tumors (32
) and that both sexes are susceptible to liver tumors induced by the direct-acting carcinogen N
The liver tumor susceptibility locus Hcf1
has so far been mapped to a region of chromosome 17 from 30.05 to 35.83 Mb, which corresponds to a section of human chromosome 6p21. Amplification of this portion of the p arm of chromosome 6 has repeatedly been found in human liver tumors. The proportion of tumors with this amplification varied from 20 to 61% and was found in patients with hepatitis B or C virus infection (33
), as well as patients without hepatitis virus infection (37
). An increase in the copy number of this region may indicate the presence of a dominantly acting oncogene. Additionally, this amplification was not associated with liver tumor grade, indicating that it could be an early event (40
). Interestingly, amplification of chromosome arm 6p was also seen specifically in hepatic metastases from colorectal cancers (43
), which are the leading cause of colorectal cancer deaths. Genetic aberrations in primary tumors and metastases from Dukes’ stage C colorectal cancer patients with lymph node metastasis were compared with Dukes’ stage D colorectal cancer patients with liver metastasis (44
). Amplification of chromosome arm 6p was only significantly associated with Dukes’ stage D and liver metastases. Consequently, identification of the causative mutation in the Hcf1
locus could have wide-ranging effects on other diseases in addition to liver cancer.
The current minimal region contains ~215 genes. Several genes in this region are potential candidates for Hcf1
is a member of the cytochromes P450 4F subfamily. These enzymes are involved in arachidonic acid metabolism (45
) and metabolize leukotriene B4
into biologically less active metabolites (46
). Leukotriene B4
is a powerful promoter of inflammation, which can in turn lead to liver cancer (4
). The sequence of Cyp4F14
in mice has 95% sequence similarity with Cyp4F1
in rats and both are expressed in the liver (45
). The expression of this enzyme in rats is sex specific, with significantly higher expression in females (47
). Moreover, its expression was shown to decrease after ovariectomy (47
) and increase after exposure to aflatoxin B1
), a known risk factor for liver cancer (4
was also the first P450 found to be constitutively overexpressed in rat hepatomas (49
Another candidate for Hcf1
is a member of the nicotinamide adenine dinucleotide-dependent 17β-hydroxysteroid dehydrogenase family of enzymes. It is expressed in the ovaries, testes and liver, in addition to other tissues (50
). This family of enzymes efficiently catalyzes the oxidation of estradiol, testosterone and dihydrotestosterone as well as the reduction of estrone to form estradiol (51
). These enzymes carry out a key reaction in the synthesis and metabolism of sex hormones and regulate the last step required to form all androgens and estrogens in both gonadal and non-gonadal tissues (52
). Expression of H2-Ke6
has previously been linked to the development of cysts in the livers and kidneys of mice.
A final candidate is tumor necrosis factor (TNF)-α, a proinflammatory cytokine. TNF can trigger the acute phase response and a cascade of other cytokines, and it also has a crucial role in the balance of hepatocyte proliferation and death (53
). The transcription factor nuclear factor-kappa B is at least partly responsible for cell proliferation in response to TNF, and the activation of nuclear factor-kappa B in liver regeneration is primarily due to IL-6 induction. As previously stated, the production of IL-6 by non-parenchymal cells has been hypothesized to cause the gender disparity in DEN-induced liver tumors (30
). In addition, TNF and IL-6 also have inhibitory effects on each other (54
). IL-6 can inhibit TNF expression and TNF can block IL-6 induction of type II acute phase response genes and activation of signal transducer and activator of transcription signaling (55
All of the exons of Cyp4F14, H2-Ke6 and Tnfa and their splice sites have been sequenced in BR, B6 and C3H strains to look for unique mutations in the susceptible BR strain (S.Peychal and N.Drinkwater, unpublished data). This sequencing did not identify any unique mutations in either the exons or splice sites of these three candidate genes. In addition, the hepatic expression levels of these genes at 10 weeks of age were compared using microarrays. In B6, BR and BR chromosome 17 recombinant females, treated with DEN at 12 days and ovariectomized or sham operated at 6 weeks of age, the expression levels of the three genes were not significantly different among the strains. However, as there could be a causative difference in the expression of these genes at another time, or in another tissue, that could result in susceptibility to liver tumors, these genes cannot be excluded as the Hcf1 locus.
Men have a 2- to 5-fold higher risk of developing liver cancer than women (1
). At least some of this difference is due to the different hormonal environments, but it is not currently known what contribution, if any, is due to other complicating risk factors such as hepatitis virus infection or aflatoxin B1
exposure. Inbred mice offer a simpler system in which to study liver cancer and the effect of sex hormones. Work with these mice has shown the stimulatory effect of androgens as well as the protective effect of ovarian hormones (7
). The BR females are highly susceptible to liver cancer due, in part, to their unique lack of ovarian hormones’ protection (11
). Therefore, they offer a singular model to explore the suppressive pathways at work. Elucidation of how these pathways are disrupted in this unique strain may shed light on how these pathways function in all other strains and may uncover information of relevance to women and liver cancer development in humans as a whole.