Hepatocellular carcinoma (HCC) is the most rapidly increasing cause of cancer-related mortality in the United States. Because of the lack of viable treatment options for HCC, prevention in high-risk patients has been proposed as an alternative strategy. The main risk factor for HCC is cirrhosis and several lines of evidence implicate epidermal growth factor (EGF) in the progression of cirrhosis and development of HCC. We therefore examined the effects of the EGF receptor (EGFR) inhibitor erlotinib on liver fibrogenesis and hepatocellular transformation in three different animal models of progressive cirrhosis: a rat model induced by repeated, low-dose injections of diethylnitrosamine (DEN), a mouse model induced by carbon tetrachloride (CCl4), and a rat model induced by bile duct ligation (BDL). Erlotinib reduced EGFR phosphorylation in hepatic stellate cells (HSC) and reduced the total number of activated HSC. Erlotinib also decreased hepatocyte proliferation and liver injury. Consistent with all these findings, pharmacological inhibition of EGFR signaling effectively prevented the progression of cirrhosis and regressed fibrosis in some animals. Moreover, by alleviating the underlying liver disease, erlotinib blocked the development of HCC and its therapeutic efficacy could be monitored with a previously reported gene expression signature predictive of HCC risk in human cirrhosis patients. Conclusion: These data suggest that EGFR inhibition using Food and Drug Administration-approved inhibitors provides a promising therapeutic approach for reduction of fibrogenesis and prevention of HCC in high-risk cirrhosis patients who can be identified and monitored by gene expression signatures. (Hepatology 2014;59:1577-1590)
Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, and due to its poor prognosis it is the third leading cause of cancer-related death. In the United States, HCC is the most rapidly increasing cause of cancer-related mortality. While the cause of HCC is multifactorial, the common pathway for the vast majority of cases is cirrhosis. Cirrhosis is estimated to affect 1-2% of the world's population. Nearly one million people die from cirrhosis worldwide each year, and the annual cost for caring for complications of cirrhosis in the United States alone is estimated to be $4 billion. The major clinical consequences of cirrhosis are impaired liver function, portal hypertension, impaired cognitive function, and development of HCC, all of which increase the risk of death. Given the lack of successful treatment options for HCC, new strategies for the prevention of HCC by slowing the natural history of liver fibrosis and cirrhosis are urgently needed.
Epidermal growth factor (EGF) plays a role in both cirrhosis and HCC. EGF expression in the liver increases during cirrhosis. EGF is also a key member of a 186-gene signature predictive of progressive cirrhosis, HCC development, and death in patients with cirrhosis.[6, 7] In addition, a polymorphism in the human EGF gene that leads to increased EGF expression is associated with increased fibrosis and cirrhosis progression[8, 9] and elevated risk of developing HCC in patients with cirrhosis. Finally, transgenic mice with liver-specific overexpression of EGF rapidly develop HCC.
We report here that the small-molecule EGF receptor (EGFR) inhibitor erlotinib inhibits the activation of myofibroblastic hepatic stellate cells (HSC), prevents the progression of cirrhosis, regresses fibrosis in some animals, and blocks subsequent development of HCC in rodent models.
Materials and Methods
For details, please see the Supporting Information.
Animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” of the National Academy of Sciences. All animals were maintained in accordance with the guidelines of the Massachusetts General Hospital Subcommittee on Research Animal Care. Animals were treated as described in the Supporting Information.
Primary Rat HSC Isolation
HSC were isolated and cultured as described in the Supporting Information.
Histology, Immunohistochemistry, Immunofluorescence, and Hydroxyproline Analysis
Formalin-fixed samples were embedded in paraffin, cut into 5-μm-thick sections, stained, and analyzed as described in the Supporting Information.
Liver Function Tests
A cardiac terminal blood withdrawal was performed at the time of sacrifice and serum was isolated and analyzed as described in the Supporting Information.
Hydroxyproline Analysis and Western Blotting
Hydroxyproline analysis and western blot analysis are described in the Supporting Information.
Genome-wide gene expression profiling for the rats and mice was performed using RatRef-12 and Mouse Ref-8 Expression BeadChip microarrays, respectively (Illumina, San Diego, CA) as described in the Supporting Information.
An unpaired two-tailed t test was used to compare differences in body weights, liver weights, liver function tests, number of tumors, hydroxyproline levels, western blot densitometry, and quantifications of Sirius red and Ki67 stainings. Differences in Ishak scores were assessed by a Kruskal-Wallis test followed by post-hoc Dunn-Holland-Wolfe in the diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) studies and by a Mann-Whitney test in the bile duct ligation (BDL) study. Fisher's exact test was used to assess differences in tumor size.
The results of our investigation tie together several important observations. The first is that gene expression analyses have demonstrated that the EGF pathway is associated with progression of cirrhosis to mortality.[6, 7] Likewise, in cirrhosis patients the level of EGF mRNA expression in the cirrhotic tissues is associated with poor survival, whereas tumoral EGF expression in these same patients is not associated with survival (Supporting Fig. 11). Second, HSC play a pivotal role in hepatic fibrogenesis, and EGFR signaling has been shown to activate these cells.[26, 27] Third, polymorphism studies and transgenic mouse models have implicated EGF in hepatocellular transformation to HCC. Nonetheless, a common pathway to HCC is by way of progressive cirrhosis, and thus, effective strategies that limit or even regress hepatic fibrogenesis are expected to reduce the frequency of HCC.
Given the strong evidence implicating EGF and EGFR in these processes, there is a strong rationale to test an EGFR inhibitor for its ability to inhibit hepatic fibrogenesis and hepatocellular transformation. We observed that the FDA-approved EGFR inhibitor erlotinib, used at doses equivalent to or less than those used in humans, significantly reduced fibrogenesis in three separate animal models. Our results suggest that these models are similar with respect to fibrosis resolution but clearly differences do exist with respect to liver injury. Liver injury is more severe in the CCl4 model and this may be attributable to species differences, the different chemicals themselves, or the three times per week dosing with CCl4 as opposed to once a week dosing with DEN. To examine this further, we used gene set enrichment analysis (GSEA) to evaluate in the DEN-injured rats and CCl4-injured mice the effect of erlotinib on genes associated with lipopolysaccharide (LPS)-induced liver injury of HSC. Interestingly, genes suppressed by LPS were reexpressed in response to erlotinib in CCl4 mice (normalized enrichment score [NES] = −1.42, FDR = 0.066), whereas no such enrichment was observed in DEN rats (NES = 0.86, FDR = 0.67). This further supports our data indicating that erlotinib suppresses liver injury more significantly in CCl4 mice.
Compared to what has been reported previously in models of regeneration, we see a similar decrease in hepatocyte proliferation after EGFR inhibition, but we did not observe that EGFR conveyed antiapoptotic signals to hepatocytes. Instead, we observed less liver injury, suggesting that EGFR inhibition might have a role in protecting hepatocytes and this could be one mechanism by which erlotinib reduces fibrosis development. This difference will need to be examined further, but it is interesting to note that EGFR can promote both proliferation and apoptosis of HSC.
A further rationale for clinical evaluation of EGFR inhibition comes from studies demonstrating that EGFR is a cofactor important for hepatitis C virus (HCV) entry into cells. EGF accelerates HCV entry, and EGFR tyrosine kinase inhibitors—including erlotinib—have substantial antiviral activity. Given the prevalence of chronic HCV infection as a source of hepatic fibrosis and cirrhosis, these observations suggest that EGFR inhibition could be a new approach to simultaneously reduce fibrotic damage previously caused by the virus and treat HCV infection.
We observed that several EGFR ligands were increased in the DEN, CCl4, and BDL models and that treatment with erlotinib generally reduced their expression. Interestingly, EGFR ligands could have conflicting roles in liver fibrogenesis, as amphiregulin (AREG) has been shown to promote liver fibrosis, whereas heparin-binding EGF-like growth factor (HB-EGF) suppresses liver fibrosis. The relative importance of each of these ligands in liver disease will need to be elucidated in future studies especially given recent findings that EGFR ligands also play a role in HCC acquired resistance to sorafenib.
Another small-molecule EGFR inhibitor, gefitinib, has been previously shown to reduce the number of HCC nodules, but that effect was attributed to the antineoplastic effect of EGFR inhibition on the tumors themselves. No investigation was reported on the effect of gefitinib on liver injury, fibrogenesis, or synthetic function. In contrast, we observed a marked impact in the surrounding nontumoral liver tissue, but no effect of erlotinib within HCC tumors. Our analyses indicate that the effect of EGFR inhibition with erlotinib is purely on the surrounding liver, thereby reducing the risk of malignant transformation (the “field effect”) rather than a direct antineoplastic effect on tumors. The observed reduction in small tumors after erlotinib treatment rather than an effect on the growth of existing tumors is consistent with the ability of erlotinib to suppress the initiation of HCC tumors.
Indeed, our recent studies in predicting HCC survival suggest that nontumoral liver gene expression profiles are more predictive of clinical outcome than the profiles of the tumors themselves.[6, 7] We also note that the therapeutic benefit of EGFR inhibition in the treatment of established HCC is modest at best; only a minority of patients treated with erlotinib exhibited disease control.[33, 34] These clinical results are consistent with our observation that the most dramatic effects of EGFR blockade are on the prevention of fibrosis and cirrhosis, the principal risk factors for the development of HCC.
One potential problem with the design of antifibrotic and/or HCC prevention clinical trials is the lack of a sensitive way to assess treatment efficacy, as changes in liver biopsy histology might only occur after long periods and are also prone to considerable sampling error. We observed that the poor-prognosis cirrhosis gene signature was completely induced in DEN-injured livers before there were any notable changes in liver function tests or liver histology, and that it was reversed in response to erlotinib. Therefore, this poor-prognosis cirrhosis signature may be useful not only for the early detection of liver fibrosis and hepatocellular transformation-associated events but also for monitoring therapeutic efficacy of chemoprevention agents.
The details of the mechanisms by which erlotinib reduces liver injury, fibrogenesis, and HCC development remain to be worked out. Several different cell populations in the liver express EGFR and may each play contributory and interactive roles. It could be that erlotinib reduces the proliferation of hepatocytes, as indicated by Ki67 staining, and this directly prevents neoplastic transformation and indirectly prevents HSC activation through paracrine signaling. Consistent with this, erlotinib decreased the expression of several profibrogenic factors. However, our data also demonstrates that EGFR is activated in HSC and therefore erlotinib could directly inhibit HSC activation while at the same time reducing paracrine signals that stimulate hepatocyte proliferation. We suspect that both of these mechanisms are operant, and plan to examine the relative importance of hepatocytes and HSC on the efficacy of erlotinib in follow-up studies using cell-specific targeting and genetic models. Regardless, our results in three different preclinical models of liver fibrosis suggest that EGFR is an important mediator of disease progression.
To our knowledge, this is the first demonstration that EGFR inhibition regresses liver fibrosis. The results reported here have immediate and important clinical translational implications for both hepatic fibrosis and HCC. Cirrhosis exerts an enormous toll on human health worldwide, and there is great need for interventions to slow or even regress disease progression. As for HCC, identification of high-risk populations suitable for screening and chemoprevention has been proposed as the most efficient strategy to abrogate HCC-related mortality. And such high-risk populations within patients with early-stage cirrhosis may be more effectively identified with EGF genotype and/or liver gene expression profiles[6, 7] in combination with clinical and pathologic parameters. The present studies support the evaluation of EGFR inhibitors in clinical trials of cirrhosis patients at high risk of progression and HCC development.
The authors thank Scott Friedman and Young-Min Lee (Mount Sinai School of Medicine, New York, NY) for helpful advice on staining of HSC.