Further evidence on the janus‐faced nature of the epidermal growth factor receptor: From liver regeneration to hepatocarcinogenesis
Authors: Carmen Berasain, Matias A. Avila
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. See Article on Page 604 After partial resection or parenchymal injury, the liver mounts a remarkable regenerative and self‐protective reaction. The mechanisms orchestrating this process have been the subject of intensive research for almost a century. Among the multiple mediators involved in liver regeneration after partial hepatectomy (PH), inflammatory cytokines and growth factors play a central role, promoting the synchronous entry into the cell cycle of the otherwise quiescent and differentiated hepatocytes.1 c‐Met and the epidermal growth factor (EGF) receptor (EGFR or ErbB1), both highly expressed in hepatocytes, are the two principal tyrosine kinase growth factor receptors (receptor tyrosine kinases; RTKs) involved in liver regeneration.1 Interestingly, their extensive crosstalk and funtional overlapping manifests in different cellular backgrounds, including hepatocytes.1 c‐Met is bound and activated by hepatocyte growth factor (HGF), which is induced after liver resection.1 However, besides EGF, several ligands activate EGFR in hepatocytes leading to DNA synthesis and cell survival4 (Fig. 1A). Interestingly, persistent activation of the inflammatory signals and growth factor pathways involved in liver regeneration, including c‐Met and the EGFR, is also believed to participate in hepatocarcinogenesis.4Figure 1: Role of EGFR in liver regeneration and hepatocarcinogenesis evaluated in ΔEGFR transgenic mice. (A) Left panel: EGFR/ErbB1 signaling in hepatocytes is triggered by EGFR ligands, including EGF, transforming growth factor alpha (TGFα), amphiregulin (AREG), epiregulin (EREG), and heparin‐binding EGF (HB‐EGF). Binding of EGFR results in receptor dimerization and activation of its tyrosine‐kinase domain (KD), inducing the activation of intracellular signaling pathways, such as MAPK and AKT, leading to cell proliferation and survival. Right panel: Transgenic ΔEGFR mice express a truncated EGFR devoid of its KD in hepatocytes. This mutant allows the binding of EGFR ligands through heterodimerization with WT EGFR, but blocks intracellular signaling behaving as a dominant‐negative effector. (B) Left panel: Liver regeneration after PH is delayed in ΔEGFR mice. Inhibition of EGFR activity results in enhanced TGF‐β signaling, leading to increased expression of cell‐cycle inhibitors, and in reduced lipogenic activity. Up‐regulation of Hgf and urokinase‐type plasminogen activator (uPa), resulting in increased c‐MET signaling, partially compensate for lack of EGFR signaling. Right panel: ΔEGFR mice show delayed DEN‐induced hepatocarcinogenesis. Inhibited EGFR signaling is associated with reduced inflammation and enhanced expression of the purported tumor suppressor, Nox4. As observed in liver regeneration, Hgf expression is also up‐regulated.ΔEGFR mice initially develop less tumoral lesions than WTs, but at later stages, tumor burden is similar in both genotypes. Abbreviations: Ccl2, chemokine (C‐C motif) ligand 2; Cxcl1, chemokine (C‐X‐C motif) ligand 1; Fasn, fatty acid synthase; G6pdh, glucose‐6‐phosphate dehydrogenase, Tnf; tumor necrosis factor.The contribution of EGFR to liver regeneration has been addressed using different experimental approaches. Given that early studies showed that EGFR‐null mice were not viable,4 alternative strategies to elucidate EGFR function in the adult animal were needed. Mice with hepatocyte‐specific perinatal deletion of EGFR were generated and after PH delayed cell‐cycle progression, reduced hepatocellular proliferation and increased mortality were appreciated.6 Interestingly, these mice were more susceptible to acute liver injury induced by the carcinogen, diethylnitrosamine (DEN).7 Partially agreeing with these findings, a recent study also using hepatocyte‐specific EGFR‐null mice found delayed cell‐cycle progression and DNA synthesis after PH, but not increased CCl4‐mediated acute liver injury.3 However, codeletion of EGFR and c‐Met in hepatocytes markedly exacerbated CCl4‐induced damage, further supporting the cooperation between these two receptors.3 Other investigators employed EGFR‐targeted RNA interference to examine the involvement of EGFR in rat liver regeneration after PH.2 Consistent with the phenotype of hepatocyte‐specific EGFR‐null mice, EGFR knockdown reduced hepatocellular proliferation and a potentially compensatory up‐regulation of c‐Met was also observed.2 Albeit altogether, these observations supported the implication of EGFR in the early phases of liver regeneration, many unsolved questions remained, including the cellular mechanisms triggered by EGFR that might regulate hepatocellular proliferation and survival in vivo. Moreover, all these studies relied on EGFR elimination or down‐regulation and lack of EGFR in hepatocytes might promote adaptive mechanisms, complicating the interpretation of results. With these issues in mind, López‐Luque et al. developed an elegant transgenic mouse model in which EGFR tyrosine kinase activity is specifically impaired in hepatocytes. Their study, reported in this issue of Hepatology confirms the role of EGFR in liver regeneration and addresses its involvement in hepatocarcinogenesis providing insightful mechanistic observations.8 This model, the ΔEGFR mice, consisted in hepatocyte‐specific expression of a truncated form of the EGFR devoid of most of the cytoplasmic domain, where the tyrosine kinase activity resides (Fig. 1A). This EGFR mutant had been previously shown to undergo EGF‐induced heterodimerization with wild‐type (WT) EGFR, behaving as a dominant negative receptor capable of reducing WT EGFR tyrosine autophosphorylation and biological signaling (Fig. 1A).8 The inhibitory activity of mutant EGFR was validated when two‐thirds PH was performed in ΔEGFR mice. EGFR phosphorylation and downstream protein kinase B (AKT) and ERK signaling was impaired, and ΔEGFR mice displayed less and delayed hepatocyte proliferation, but apparently not increased hepatocellular apoptosis. Overall, these findings were consistent with previous reports on the role of EGFR in the early stages of liver regeneration.2 However, this study also identifies mechanisms contributing to explain the importance of EGFR signaling in hepatocellular proliferation. Previous in vitro findings demonstrated that EGFR‐mediated signaling counteracts the cytostatic effects of transforming growth factor beta (TGF‐β),8 a cytokine also up‐regulated during liver regeneration.1 This contention is now validated in vivo, given that markedly increased small mothers against decapentaplegic homolog 3 (Smad3) phosphorylation was noticed in regenerating ΔEGFR mouse livers accompanied by induction of the cell‐cycle inhibitors and TGF‐β target genes, Cdkn1a and Cdkn2b (Fig. 1B). Intriguingly, ΔEGFR mice not only displayed increased TGF‐β signaling, but also markedly elevated TGF‐β levels, particularly before PH. In the normal and regenerating liver, TGF‐β can be produced by different cell types, including stellate and inflammatory cells, but not hepatocytes. It would be very interesting to examine the source of TGF‐β in ΔEGFR mouse livers and how lack of EGFR signaling in hepatocytes regulates TGF‐β expression. Another noteworthy finding was the disruption of hepatic adipogenesis in ΔEGFR mice during liver regeneration. Transient hepatic steatosis after PH is thought to be important for liver regeneration to proceed.9 López‐Luque et al. showed that EGFR signaling was required for fat accumulation and adipogenic gene expression during liver regeneration, suggesting a relevant role for EGFR in hepatic lipid metabolism (Fig. 1B). This finding is consistent with the phenotype of mutant mice expressing a constitutively active EGFR allele.10 The influence of EGFR signaling on normal (i.e., nontransformed) liver metabolism certainly deserves further exploration. As observed in models of hepatocyte‐specific EGFR knockout or knockdown, in spite of impaired hepatocellular proliferation, ΔEGFR mice were also able to fully regenerate their livers. Without downplaying the role of EGFR in liver regeneration, this response attests to the remarkable functional redundancy among the signals involved in the process. Coherent with the aforementioned intense crosstalk between EGFR and c‐Met, increased expression of HGF and urokinase‐type plasminogen activator, as well as c‐Met phosphorylation, were found in ΔEGFR livers after PH. Activation of c‐Met signaling may not only partially compensate the impaired hepatocellular proliferation, but also protect from apoptosis that could occur by overactivation of the TGF‐β pathway in ΔEGFR livers. Nevertheless, it is worth noting that ΔEGFR mice showed elevated expression of HGF and TGF‐β even under basal conditions, suggesting a role for EGFR signaling in the quiescent liver deserving additional investigation. The involvement of EGFR in chemically induced (DEN) hepatocarcinogenesis was also examined in ΔEGFR mice. Multiple clinical and experimental evidences suggest the implication of this RTK in hepatocarcinogenesis, including the inhibition of experimental hepatocarcinogenesis by EGFR inhibitory drugs.4 Consistently, ΔEGFR mice clearly showed delayed tumorigenesis, with markedly reduced cellular proliferation and tumoral lesions at early stages. However, by 12 months, liver tumor burden was similar in WT and ΔEGFR mice, suggesting that EGFR activity is important during the early phases of hepatocarcinogenesis, being dispensable when tumors have developed. These findings agree with the lack of clinical efficacy of EGFR inhibitors in human hepatocellular carcinoma (HCC) and suggest that EGFR targeting could be a chemopreventive, rather than a therapeutic, strategy.4 Mechanistically, López‐Luque et al. show that, by 9 months after DEN administration, ΔEGFR mice displayed higher levels of HGF, as well as reduced expression of the purported tumor suppressor, Nox4, and of inflammatory cytokines, many of which participate in hepatocarcinogenesis5 (Fig. 1B). Although these observations confirm the overall proinflammatory effects of EGFR activation, the underlying mechanisms merit further study. In ΔEGFR mice, EGFR signaling is impaired in hepatocytes, but not in nonparenchymal liver cells, including cytokine‐producing inflammatory cells also capable of expressing EGFR.7 In contrast to the findings of López‐Luque et al., hepatocyte‐specific EGFR‐null mice developed significantly larger tumors than WT controls. This phenotype was attributed to enhanced DEN‐induced hepatotoxicity owing to lack of EGFR, which would lead to a compensatory repair reaction ultimately favoring hepatocarcinogenesis.7 The differential response between these model mice in terms of HCC development may reside in the carcinogenic protocol used in each case.7 However, and as reasoned by the investigators, lack of EGFR in hepatocyte‐specific EGFR‐null mice would result in an increased availability of EGFR ligands to act on nonparenchymal cells and promote hepatocarcinogenesis. It has been over 50 years that the first RTK, the EGFR, was discovered. Still, its biological functions are not completely understood. The transgenic model presented here will surely further our knowledge on the hepatic functions of this fascinating receptor. Author names in bold designate shared co‐first authorship.