Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 2007;317:121–124. (Reprinted with permission.)
Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males.
Hepatocellular carcinomas (HCCs) represent the fifth most common cancer worldwide and account for approximately 90% of primary liver cancers. A general characteristic of HCC (across almost all geographical areas) is the striking male predominance ranking from 2:1 up to 5:1.1 Earlier hypotheses suspected higher exposition of men to HCC risk factors due to a different socialization. However, animal experiments revealed a higher susceptibility to HCC in male rodents, which indicates that the prevalence for HCC in males has basic molecular reasons. However, the mechanisms responsible for this gender difference are largely unknown, although it suggests that sex hormones such as estrogen and testosterone might be involved in this phenomenon.2
Accordingly, studies in the widely used diethylnitrosamine (DEN) model of murine hepatocarcinogenesis revealed a positive effect of androgen receptors on tumor growth3 whereas estrogens seemed to be protective.4 DEN is a DNA-damaging agent inducing either acute cell death of damaged cells or accumulation of oncogenic mutations in surviving hepatocytes, eventually leading to HCC development in a time frame of 20-40 weeks after application. In this model, 100% of male but only 10%-30% of female mice develop HCC thereby reflecting the unequal distribution of HCC prevalence in human. Although the mechanism leading to DEN-induced HCC is clearly different from the pathogenesis in humans, comparative functional genomics revealed that the gene expression signatures of DEN-induced mouse HCCs were most similar to human HCCs with poor survival.5
Naugler et al.6 recently uncovered a molecular mechanism explaining the lower HCC susceptibility in females. In their study, they treated male and female mice with DEN and investigated the relationship between HCC development and gender-dependent expression of interleukin-6 (IL-6) which is a promising candidate molecule for at least 3 different reasons: First, HCC is always linked with liver inflammation and IL-6 is a typical proinflammatory cytokine playing important roles for the acute phase response and during chronic hepatitis.7 Second, IL-6 provides important functions for hepatocyte proliferation8 which is critical for HCC formation. Finally, the transcriptional control of IL-6 expression is clearly sex hormone regulated as it was shown earlier that estrogens inhibit IL-6 promoter activity through a physical interaction with IL-6 activators nuclear factor κB (NF-κB) and CCAAT-enhancer-binding protein beta (C/EBPβ),9 thereby defining IL-6 as a good candidate for affecting gender-dependent inflammatory processes.
Indeed, Naugler et al. found significantly higher IL-6 levels in male compared to female mice 48 hours after DEN application. Simultaneous application of estrogens in males was sufficient to reduce IL-6 to levels found in females. High IL-6 levels in male mice correlated with higher serum aminotransferases, apoptosis, and hepatocyte proliferation, indicating acute liver damage induced by DEN. All these effects in male animals could be completely blocked by cotreatment with the estrogen receptor-α agonist PPT, indicating a negative regulatory role of estrogens on IL-6 expression. However, IL-6 levels after partial hepatectomy are equally elevated in both genders, implicating that the induction of IL-6 through DEN occurs via different signaling cascades. Naugler et al. further show that male IL-6 knockout mice were protected from short-term effects of DEN, namely, increased serum aminotransferases, apoptosis, and hepatocyte proliferation, and showed a strongly decreased tumor incidence even lower than that found for female wild-type mice, demonstrating the central involvement of IL-6 in DEN-induced tumorgenesis.
The source of IL-6 production following DEN stimulation could be traced back to the Kupffer cells, and the stimulus turned out to be the cell debris of necrotic hepatocytes. Cellular debris stimulated Kupffer cells, which was dependent on the myeloid differentiation primary response gene 88 (MyD88). This demonstrates an involvement of Toll-like receptors (TLR), because MyD88 represents a crucial adaptor protein in TLR signaling which results in activation of the transcription factor NF-κB, leading to gene transcription of IL-6 and other targets. Consistently, Naugler et al. demonstrated that deletion of MyD88 blocks both the short-term effects of DEN treatment in males and, even more important, inhibits HCC formation with reduced tumor size in MyD88-deficient males compared to wild-type animals.
In summary, this study gives us an idea about the mechanism leading to higher incidence of DEN-induced HCCs in male mice, which is summarized in Fig. 1: DEN induces strong DNA damage in hepatocytes, leading either to immediate death of hepatocytes or leaves the surviving cells with accumulated mutations. The cell debris stimulates Kupffer cells via TLR-MyD88, eventually leading to NF-κB activation and subsequent induction of IL-6, which in turn then triggers proliferation of surviving hepatocytes carrying DEN-induced mutations. In females, this cascade leading to HCC development is interrupted simply because estrogens inhibit IL-6 promoter activity in Kupffer cells.
Although this study contributes greatly to our understanding of DEN-induced hepatocarcinogenesis, some questions regarding the role of IL-6 in this model still remain unanswered. The IL-6 response occurs during the first 48 hours after stimulation, whereas tumorigenesis requires 8 months. Therefore, one has to postulate that this short IL-6 response is able to trigger very strong and sustained hepatocyte proliferation. Compensatory hepatocyte proliferation after liver damage could also contribute in part to HCC development. However, it seems unlikely that both mechanisms alone are sufficient to induce tumor formation after 8 months. Therefore, very likely additional IL-6–dependent and sex-dependent mechanisms are important to explain malignant growth in the liver of these animals.