This is the first study to directly examine the role of ERα in DEN-induced liver tumorigenesis. In the context of earlier literature indicating that ovarian estrogen inhibited tumorigenesis and male androgen enhanced it (8
), the lowered tumorigenic response in male ERαKO and the ERα- and ovary-independent protective effects in the female were unexpected findings.
Our results point to a role for ERα in mediating enhanced tumorigenesis in males. The lower tumorigenesis in the ERαKO males was not due to a decrease in androgen nor to a disruption in the male pattern of GH secretion. It has been shown that ERαKO males have serum testosterone concentrations that are several folds higher than those of WT males (33
). Although it is difficult to determine the GH secretion rates in mice, expression of sex-specific genes in the liver reflects male or female GH secretory patterns (34
). Our results on liver gene expression extend and confirm those reported by Sueyoshi et al.
), indicating that imprinting of the male GH secretion does not require ERα, whereas female ERαKO mice exhibited male patterns of gene expression, indicating that pubertal estrogen acts through this receptor to override the male pattern. Since tumorigenesis was decreased in male ERαKO mice in the face of continued male GH secretion and since there was no increase in tumorigenesis in females exhibiting the male pattern of GH secretion, these results suggest that the sexually dimorphic pattern of GH secretion is not responsible for sex differences in liver tumorigenesis.
It may be that the interplay between GH secretory patterns and activated ERα within the liver determine whether estrogen promotes or retards progression of preneoplastic foci to tumors. It has been suggested that the small decrement in body mass of male ERαKO mice is due to attenuation of the GH/IGF-I axis (39
). However, it is not known which part of that axis, GH secretion or IGF-I expression, is affected. It has been shown that estrogen can stimulate IGF-I expression in the liver and the decreased serum IGF-I concentrations seen in ERαKO mice may simply be due to the decrement in that stimulus (40
). Furthermore, the reduced levels of IGF-I in ERαKO males are comparable with levels seen in WT females (39
). It may be that both GH and estrogen are required for the male level of liver IGF-I expression and that the characteristic male tumorigenic response is related to this high level of IGF-I. Alternatively, even in the absence of estrogen ERα may play a role in IGF-I action (41
The cytokine, interleukin (IL)-6, is another growth factor implicated in liver tumorigenesis and this too may be modified by estrogen. In acute toxicological analyses performed in adult mice, it has been shown that DEN causes hepatocyte cell death, the debris from which induces production of IL-6 from Kupffer cells; the cytokine in turn stimulates a compensatory cell proliferation in the surviving hepatocytes (42
). In IL-6-null mice, the male bias for DEN-induced liver tumorigenesis is eliminated (44
). It was proposed that ERα mediates estrogen protection against hepatocarcinogenesis by reducing the production of IL-6 by Kupffer cells (44
). This hypothesis has been enthusiastically received for its clinical implications (45
). However, it is well established that the gender differences in the DEN-induced tumorigenesis model occur at the promotion stage, that is, preneoplastic foci develop equally in males and females over the course of 10–12 weeks after DEN administration, whereas fully developed tumor nodules exhibit sex bias only after several months (10
). Furthermore, the acute responses to DEN in the juvenile mouse tumorigenesis model would occur at a time when there is no ovarian production of estrogen, that is, in the prepubescent mouse. Thus, the very transient IL-6 response demonstrated following a single injection of DEN to adult mice may not be related to the gender effect that is evident several months after carcinogenic insult in neonates. Our data further question the relevance of the acute protective effect of estrogen as demonstrated by acute responses to high doses of DEN treatment in adult mice. Although full protection by estrogen against DEN-induced cell death required ERα in male mice (44
), we found that lack of ERα did not result in greater tumorigenesis in females and it actually reduced tumorigenesis in males. Thus, our results do not support the hypothesis that the protective effect of female factors is related to estrogen-induced reduction in IL-6 expression.
Estrogens are formed from androgens through the action of Cyp19a1, steroid aromatase. Cyp19a1
is expressed in the testis, brain and peripheral fat (48
). It is controversial whether Cyp19a1
is expressed in the normal adult human liver but it has been clearly demonstrated in diseased liver and in liver cancer cell lines (49
). Although circulating levels of estrogens are expected to be lower in males than females, it is not known whether localized liver tissue concentrations of estrogen differ according to sex. Thus, it is possible that androgens affect liver tumorigenesis through aromatization and subsequent activation of ERα to directly affect hepatocytes. Using the testicular feminization mutant (Tfm) mouse Kemp et al.
) found that the high rate of tumorigenesis in male mice was AR-dependent; in addition, observations on mice that were mosaic for AR in the liver suggested that the effect of androgen was mediated by secondary paracrine or endocrine factors. Furthermore, although the absolute numbers were lower in the animals lacking AR, testosterone treatment did increase tumor incidence and multiplicity in the AR-null males (11
). Notably, the aromatizable androgen, testosterone, was used in that experiment, thereby allowing for the possibility that the tumor promoting effect of testosterone in the absence of its cognate receptor might be mediated by estrogen metabolites. Our own data indicate that ERα is required for ~47% of the male effect, suggesting that estrogen derived from aromatization of testicular androgen plays a major role. Thus, estrogen may be the secondary secreted growth factor hypothesized to mediate the androgen effect in Tfm, AR- mosaic mice.
The role of ERα in male liver tumorigenesis demonstrated in the mouse may have bearing on the course of HCC in humans. Liver disease associated with hepatitis virus is aggravated by alcohol, indeed, alcohol and viral infection synergize to increase the risk of HCC (1
). Alcohol has also been shown to increase expression of aromatase in the diseased liver (53
). In light of our results, it might be hypothesized that this increase in aromatase is at least partially responsible for the increased risk in males due to alcohol intake.
ERα does mediate a protective effect of exogenously delivered estrogen as evidenced by the reduction in tumor counts in WT males treated with E2 but not in E2-treated ERαKO males. Our E2 treatment capsules produce a constant high physiological level of hormone (55
). Thus, ERα can mediate two opposing effects: a tumor-enhancing effect of low levels of endogenous estrogen and a tumor-inhibiting effect of high estrogen levels produced by exogenously delivered hormone. This type of biphasic dose–response is not uncommon for estrogen (56
On the other hand, in WT C57Bl/6J females, we did not observe an effect of ovariectomy nor was there an effect of ERα deficiency. The lack of effect of ovariectomy compared with the increase in tumorigenesis reported by others is probably due to two aspects of the study design: the strain of mice used and the dose of DEN administered. It has been shown earlier that the tumorigenic response to DEN and the effects of gonadal hormones is strain dependent, with C57Bl/6J female mice exhibiting a much lower tumor incidence and multiplicity compared with other strains (10
). In those earlier studies, the dose of DEN was ~5 mg/kg (0.05 μmol/g body wt) compared with the 20 mg/kg dose administered in our study; the higher dose produced a nearly 100% incidence in both intact and ovariectomized animals, compared with incidences of 7 and 45% for intact and ovariectomized female C57Bl/6J mice receiving the low dose of DEN (15
). Thus, in those earlier studies, the low multiplicity reported for intact females was mainly due to a low incidence of mice with tumors, whereas in our study, multiplicity more closely reflected the number of tumors per animal. Thus, when the dose of DEN is sufficient to overcome the resistance to tumor induction in C57Bl/6J mice, the protective effect of the female factor(s) is still evident but it is not dependent on ovarian hormone(s) or ERα.
Comparing the effects of ovariectomy in the ERαKO and the PRLR studies also points to a dramatic influence of genetic background on the role of ovarian factors in liver tumorigenesis. The PRLR-knockout mice are in a mixed genetic background (129Ola-X-C57Bl/6J); ovariectomy did result in heightened tumorigenesis in these animals. On the other hand, ovariectomy had no effect on tumorigenesis in the C57Bl/6J inbred animals. Thus, genetic components of the female C57Bl/6J mouse provide protection even in the estrogen-deficient state. Genetic studies have been performed to identify genes that are responsible for heightened sensitivity of female C57BR/cdJ to DEN-induced liver tumorigenesis compared with C57Bl/6J females (61
), however, it may be equally important to identify the genes that confer ovary-independent resistance to tumorigenesis in female C57Bl/6J mice.
This is also the first study to directly examine the role of PRLR in DEN-induced liver tumorigenesis. Since manipulation of endogenous PRL secretion indicates that it protects against tumorigenesis in the liver (13
) and since estrogen does increase PRL secretion by the pituitary (64
), it is an appealing candidate as a mediator of the protective action of the ovaries. However, the results of this study indicate that the PRLR does not mediate the protective effects of endogenous female factor(s).
In summary, exogenous E2 protects against carcinogen-induced liver tumorigenesis in an ERα-dependent manner, but ERα is not required for the low tumorigenic response in the female. In the male, the full tumorigenic response is partially dependent on ERα, implicating either a role for aromatization of endogenous androgens in the process or for a role of unliganded ERα. Although PRL may be a candidate as a mediator of protective effects of exogenously delivered estrogen, the unaltered response in PRLR-KO mouse to DEN indicates that it is not responsible for the protective effects of the ovarian factor(s).