Growth Factors and Regeneration Dig Dis 2012;30:524–531 DOI: 10.1159/000341705
Epidermal Growth Factor Receptor Signaling in Hepatocellular Carcinoma: Inflammatory Activation and a New Intracellular Regulatory Mechanism Carmen Berasain a Alexandra Nicou a Oihane Garcia-Irigoyen a M. Ujue Latasa a Raquel Urtasun a Maria Elizalde a Fabiana Salis a María J. Perugorría a Jesús Prieto a, b Juan A. Recio c Fernando J. Corrales a Matías A. Avila a a
Division of Hepatology and Gene Therapy, CIMA, and b CIBERehd, University Clinic, University of Navarra, Pamplona, and c Vall d’Hebron Research Institute, Barcelona, Spain
Key Words Epidermal growth factor receptor ⴢ ADAM17 ⴢ Amphiregulin ⴢ Protein methylation ⴢ Mitogen-activated protein kinases
Abstract Background/Aims: Hepatocellular carcinoma (HCC) is a chemoresistant tumor strongly associated with chronic hepatitis. Identification of molecular links connecting inflammation with cell growth/survival, and characterization of pro-tumorigenic intracellular pathways is therefore of therapeutic interest. The epidermal growth factor receptor (EGFR) signaling system stands at a crossroad between inflammatory signals and intracellular pathways associated with hepatocarcinogenesis. We investigated the regulation and activity of different components of the EGFR system, including the EGFR ligand amphiregulin (AR) and its sheddase ADAM17, and the modulation of intracellular EGFR signaling by a novel mechanism involving protein methylation. Methods: ADAM17 protein expression was examined in models of liver injury and carcinogenesis. Crosstalk between tumor necrosis factor (TNF)- ␣, AR and EGFR signaling was evaluated in human HCC cells and mouse hepatocytes. Modulation of EGFR signaling and biological responses by methylation
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reactions was evaluated in AML12 mouse hepatocytes. Results: ADAM17 was upregulated in liver injury and hepatocarcinogenesis. TNF- ␣ triggered AR shedding and EGFR transactivation in HCC cells. AR was necessary for TNF- ␣ activation of ERK1/2 and Akt signaling in hepatocytes. Inhibition of methylation reactions increased the ERK1/2 signal amplitude triggered by AR/EGFR and reduced DNA synthesis in AML12 cells. Conclusions: Increased ADAM17 in preneoplastic liver injury further supports its implication in hepatocarcinogenesis. AR release and EGFR transactivation by TNF- ␣ constitutes a novel link between inflammatory signals and pro-tumorigenic mechanisms in liver cells. Finally, the identification of a new mechanism controlling growth factor signaling, and biological responses, involving methylation reactions within the RAS/RAF/MEK/ERK pathway, exposes a new target for antineoplastic intervention. Copyright © 2012 S. Karger AG, Basel
Introduction
Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the fifth most prevalent human malignancy worldwide [1]. Most HCCs develop on a background of persistent injury and inflammation, Matías A. Avila Division of Hepatology and Gene Therapy, CIMA University of Navarra, Avda. Pio XII, n55 ES–31008 Pamplona (Spain) Tel. +34 948 194 700, E-Mail maavila @ unav.es
caused by chronic alcohol consumption, viral infections, and metabolic alterations associated with obesity and type 2 diabetes [1]. From a molecular point of view, HCCs are a very heterogeneous type of tumor, and thus mutations in key tumor suppressors or oncogenes, such as TP53 and CTNNB1, are observed in no more than 50% of cases [reviewed in 2]. Remarkable efforts have been made to classify HCCs according to their gene expression profiles captured by high-throughput analyses. A common finding in these studies and in others focusing on specific pathways is the frequent dysregulation of signaling systems involving growth factors and their intracellular signaling routes [2, 3]. Some studies have correlated specific gene expression profiles with disease outcome, finding that a signature characteristic of increased growth factor signaling and cell proliferation was associated with the worst prognosis [4]. Moreover, a groundbreaking report demonstrated that the gene expression profiles of the cirrhotic tissues surrounding the tumors could be also correlated with disease recurrence and patients’ survival [5]. Interestingly, the poor-prognosis signature that was validated in these cirrhotic tissues also included the upregulation of growth factors, such as the epidermal growth factor (EGF), and genes involved in signaling mediated by inflammatory cytokines like tumor necrosis factor (TNF)- ␣ and interleukin-6 (IL-6) [5]. These observations underscore the mechanistic relevance of chronic inflammation and growth factor signaling in hepatocarcinogenesis and have paved the way for the development of molecularly targeted agents like sorafenib, which have shown therapeutic activity [6]. However, the clear antitumoral effects displayed by these targeted agents in preclinical models have not translated into the expected clinical benefits. Among other factors, this limited response could be due to the extensive crosstalk between extracellular and intracellular signaling pathways that are established in HCC [3]. Characterization of these complex interactions may lead to the development of more efficacious therapies, based on combination of drugs or the use of other multitargeted agents. Perhaps one of the most promiscuous signaling systems activated from the early stages of hepatocarcinogenesis is that mediated by the EGF receptor (EGFR). The EGFR is highly expressed in hepatocytes, is necessary for liver regeneration, and is part of the endogenous defence mechanisms triggered in acute liver injury [7]. In HCC cells and tissues the EGFR is found to be activated, and interference with its downstream signaling reduces tumor growth [8]. The EGFR can be bound and activated Signaling Crosstalks in Inflammation and Hepatocarcinogenesis
Inflammatory mediators and cytokines (e.g. TNF-␣)
TNFR
ADAM17
Soluble EGFR ligands
Pro-EGFR ligands
EGFR
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Raf
Akt
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ERK Cell proliferation
Fig. 1. Crosstalk between inflammatory signals and the EGFR system. Cytokines may signal through the EGFR receptor by stimulating the EGFR ligand sheddase ADAM17.
by a family of ligands that, in addition to EGF, include amphiregulin (AR), transforming growth factor (TGF)␣, betacellulin, epiregulin, and heparin-binding EGF (HB-EGF) [7, 8]. Although most of these factors are induced during liver injury and regeneration, as well as in HCC, AR seems to play a nonredundant biological role stimulating survival and proliferation and maintaining the neoplastic properties of HCC cells [8]. Of note, these ligands are synthesized as membrane-anchored precursors that need to be proteolytically cleaved by a metalloprotease to be released as soluble factors. Members of the ‘A disintegrin and metalloprotease’ (ADAM) family, in particular ADAM17, also known as the TNF-␣ converting enzyme (TACE), play a central role in EGFR ligand shedding [7–9]. Interestingly, ADAM17 can in turn be activated by a number of signals, including cytokines and inflammatory mediators, leading to the release of EGFR ligands and activation of the EGFR in a process known as ‘receptor transactivation’ (fig. 1) [8]. These interactions make the EGFR system a key ‘signaling hub’ in the hepatocyte that conveys and amplifies a variety of biological cues relevant to the carcinogenic process [10]. Downstream of the activated EGFR a number of intracellular pathways, of which RAF/MEK/ERK, PI3K/AKT, and Dig Dis 2012;30:524–531
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mTOR are central, lead to the activation of survival and proliferation mechanisms [3, 6], including upregulation of other growth factors such as the connective tissue growth factor (CTGF) [11]. In this study we address the role of the ADAM17/AR/EGFR axis in inflammatory signaling and hepatocarcinogenesis. We also introduce a novel mechanism that modulates intracellular signaling triggered by the EGFR/RAF/MEK/ERK phosphorylation cascade based on protein arginine methylation, which may constitute a new target for intervention in the molecular therapy of HCC.
24 h
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a
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CCl4 Pro-ADAM17 ADAM17
Results
New Insights into the ADAM17/AR/EGFR Axis in Liver Injury, Hepatocarcinogenesis, and Inflammatory Signaling ADAM17 plays a key role in defending the liver parenchyma from injury elicited by Fas activation or acetaminophen overdosing. This protective effect was mainly attributed to ADAM17-mediated shedding of the EGFR ligands TGF- ␣, HB-EGF, and AR [12]. In line with these findings, we had previously shown that AR–/– mice were more sensitive to lethal doses of Fas ligand [13]. The expression of AR is readily induced in acute liver injury, including Fas activation, and remains elevated in liver cirrhosis and HCC [8]. Several reports have described that ADAM17 activity can be triggered by different inflammatory molecules and cytokines, having a significant impact on cellular responses through EGFR transactivation [10, 12] (fig. 1). However, the expression levels of ADAM17, which are known to be elevated in human HCC tissues [14], have not been examined in relevant experimental models of liver injury, regeneration, and carcinogenesis. Here we show that liver ADAM17 protein levels are significantly increased in mice treated with the Fas agonist antibody Jo-2 or the hepatotoxin CCl4 (fig. 2a, b). Interestingly, ADAM17 protein was also upregulated in mice undergoing neoplastic liver transformation upon single injection of the carcinogen diethylnitrosamine (DEN). ADAM17 was increased not only in DEN-induced tumors but also already at pre-neoplastic stages, i.e. 5– 7 months after DEN injection (fig. 2c). Transactivation of the EGFR by the key inflammatory cytokine TNF-␣ has been reported in different cellular backgrounds, including mouse hepatocytes [8, 15]. Here we show that in human HCC cells TNF-␣ treatment also leads to EGFR phosphorylation (Tyr1148), and that in the 526
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-Actin
b
C
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HCC
DEN Pro-ADAM17 ADAM17 Glypican 3 -Actin
c Fig. 2. Expression of ADAM17 in mouse liver during injury and
carcinogenesis. Western blot analyses of ADAM17 in liver extracts from mice treated with the Fas agonistic antibody Jo2 (a) or the hepatotoxin CCl4 (b) as described previously [13], or after a single injection of DEN to 15-day-old mice (15 mg/kg body weight) (c). ADAM17 was assessed with an antibody raised against the C-terminal region of the protein, which recognizes ADAM17 precursor and processed species (AB19027; Chemicon Int.) [27]. Expression of the HCC marker glypican 3 is also shown. Western blots were performed as described before [13]. C = Control; FasL = Fas ligand.
presence of the ADAM17 inhibitor GM6001 this effect is completely abolished (fig. 3a). Interestingly, we could demonstrate that TNF-␣ elicited a dose-dependent release of AR to the cells’ culture medium, which was also abrogated by GM6001 (fig. 3b). TNF-␣ has been shown to stimulate the shedding of different EGFR ligands [15, 16]. In order to evaluate the specific contribution of AR to TNF-␣ signaling in liver cells, hepatocytes isolated from AR+/+ and AR–/– mice were treated with this Berasain et al.
cytokine and intracellular signaling was examined. As shown in figure 3c, activation of ERK and PI3K/Akt signaling by TNF-␣ was significantly attenuated in hepatocytes from AR-null mice, while signaling through the NF-B pathway was not significantly affected, as evidenced by similar levels of P-IB in both types of hepatocytes.
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TNF-␣ + GM p-EGFR
-Actin
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5.0 4.0 AR (pg/ml)
Modulation of Growth Factor Signaling through the RAS-ERK Pathway by Methylation Reactions: A Novel Mechanism and Target for Therapeutic Intervention Signaling through the RAS-ERK cascade is essential for cell proliferation, differentiation and survival, and this pathway is known to play an important role in hepatocarcinogenesis [3, 6, 8]. From an oversimplified point of view, phosphorylation and dephosphorylation reactions determine whether kinases are active or inactive; however, there are other mechanisms, including subcellular distribution and presumably posttranslational modifications in addition to phosphorylation, that may influence the functional outcome of the growth factor signal [17]. These additional mechanisms may control different aspects of the kinase signal’s dynamics, including its duration, amplitude, and integral strength [18]. Modulation of these parameters has profound effects on the biological responses elicited by a particular signaling pathway, including growth factor-triggered ERK phosphorylation. Recently we could demonstrate that specific arginine methylation of RAF proteins mediated by protein arginine methyltransferase 5 (PRMT5) limited ERK signaling activated by growth factors in different cell lines, including hepatocytes [19]. Treatment of cells with the methylation inhibitor 5ⴕmethylthioadenosine (MTA) significantly enhanced the amplitude of growth factor-mediated ERK1/2 phosphorylation. This was also observed with other methylation inhibitors, such as 3-deazaadenosine, and upon depletion of intracellular levels of the methyl donor Sadenosylmethionine by incubation with cycloleucine [19]. In the case of PC12 pheochromocytoma cells, manipulation (increase) of the ERK1/2 signal amplitude with MTA (or by PRMT5 knockdown) shifted the biological response to EGF from proliferation to growth arrest and differentiation into neuron-like cells [19]. Interestingly, MTA has been tested in different experimental models of liver inflammatory injury, fibrosis, and carcinogenesis, showing notorious hepatoprotective, antifibrotic, and antitumoral effects [20–24]. One of the most prominent cellular effects of MTA was its ability to limit growth factor-induced cell proliferation;
TNF-␣
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TNF-␣ + TNF-␣ + TNF-␣ + TNF-␣ + GM GM GM GM (1 μM) (3 μM) (10 μM) (40 μM)
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c Fig. 3. TNF- ␣ transactivates the EGFR and elicits functional AR shedding in HCC cells and hepatocytes. Human HCC cells PLC/ PRF5 were treated with TNF- ␣ (30 ng/ml, 15 min) in the absence or presence of the metalloprotease inhibitor GM6001 (40 M), and the levels of pEGFRTyr1148 were evaluated by Western blotting (a). AR concentrations in conditioned media from PLC/PRF5 cells treated with TNF- ␣ (30 ng/ml, 5 h) in the presence of the indicated concentrations of GM6001. AR was measured by ELISA as described [14] (b). Isolated and cultured hepatocytes from AR+/+ and AR–/– mice were treated with TNF-␣ (30 ng/ml, 15 min), and intracellular signaling was evaluated as described (c) [11, 13, 14]. C = Control.
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DNA synthesis (fold-change vs. control)
3 C MTA
C
MTA
AR
AR + MTA
*
2
*
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1
ERK1/2 0 C
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FCS
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b Fig. 4. Effect of the methylation inhibitor MTA on DNA synthesis
and MEK/ERK signaling. AML12 mouse hepatocytes were incubated for 24 h in serum-free medium (control), with AR (50 ng/ ml) or with fetal calf serum (FCS; 10%) in the absence or presence of MTA (500 M). 3H-thymidine was added for the last 8 h of the
however, the molecular mechanisms responsible for this antiproliferative effect were not completely understood [22, 24]. As indicated above, the intensity of the RAFERK signal can dictate the ultimate response of the cell to a growth factor, i.e. proliferation versus cell cycle arrest at a high signal intensity, and this has been observed in different cell types, including human HCC cells [25, 26]. Here we now show that MTA treatment inhibited DNA synthesis in AML12 immortalized mouse hepatocytes [15] when stimulated with fetal calf serum, or with the EGFR ligand AR (fig. 4a). MTA did not show cytotoxic effects towards AML12 cells (not shown). In line with the aforementioned modulation of the RAF-ERK pathway by RAF arginine methylation, we found that in the presence of MTA the phospho-ERK1/2 signal elicited by growth factor (AR) treatment was significantly increased (fig. 4b). These findings support the involvement of methylation reactions in AR-elicited mitogenic signaling in hepatocytes. Interference with these methylation reactions, likely occurring at the level of RAF proteins as we have previously shown [19], may constitute the basis of novel targeted interventions to modulate cell proliferation (fig. 5).
Discussion
The implication of growth factors and their intracellular signaling pathways in hepatocarcinogenesis is a widely accepted phenomenon, and is the basis for the de528
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incubation and its incorporation into newly synthesized DNA was measured as described [24]. * p ! 0.05 vs. control (a). AML12 cells were pretreated or not with MTA (500 M, 30 min) and then stimulated with AR (50 ng/ml, 15 min). p-ERK1/2 levels were evaluated by Western blotting (b). C = Control.
velopment of most of the targeted therapies so far implemented [6]. Unfortunately, up to now these new therapeutic approaches have shown limited clinical efficacy. The reasons for this are not completely known, but the complexity and plasticity of the mechanisms governing growth factor availability and intracellular signaling are likely involved. One important event in the activation of the EGFR is proteolytic processing of the EGFR ligands at the cell’s surface, which is carried out mainly by the metalloproteinase ADAM17/TACE [9, 27]. Importantly, ADAM17 activity and expression is induced in different types of cancers, including HCCs, and has been recently shown to contribute to cytotoxic drug resistance through the enhanced availability of EGFR ligands [9, 14, 27–29]. It is known that HCC slowly unfolds on a background of chronic injury and inflammation. Here we have shown that ADAM17 expression is increased in different models of acute liver injury (i.e. CCl4 and Fas ligation). Interestingly, we also found enhanced ADAM17 levels in mice undergoing neoplastic transformation after single-dose DEN administration. These findings are consistent with the recent report of increased ADAM17 protein levels in the livers of mice fed a pro-inflammatory high-fat diet [30]. While upregulation of ADAM17 during acute injury may be part of the endogenous protective mechanisms of the liver due to EGFR ligand shedding [7, 12, 13], its persistent overexpression could indeed contribute to the tumorigenic process. Moreover, the impact of ADAM17 upregulation in the liver tissue may extend beyond the EGFR system, given the variety of ADAM17 Berasain et al.
GF EGFR
RAS
RAS
RAF
RAF PRMT5
RAF
CH3
MEK
MEK
p-ERK1/2
Degradaon
MTA
p-ERK1/2
RAF
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Time
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Fig. 5. Modulation of the EGFR-triggered RAS/RAF/MEK/ERK signaling pathway and downstream biological responses by methylation reactions.
substrates which include key inflammatory cytokines such as TNF-␣ and the soluble IL-6 receptor (sIL-6R) [9]. Therefore, ADAM17 sits at a crossroad between inflammatory and growth factor signaling pathways, a critical interface in hepatocarcinogenesis [8, 9]. Mechanistic links between inflammation and cancer have been identified in many experimental models and clinical studies [reviewed in 8]. Here we further illustrate this crosstalk by showing the ability of the inflammatory cytokine TNF-␣ to activate the EGFR in HCC cells. This effect was likely due to the rapid release of AR upon TNF-␣ treatment, which was abrogated by the ADAM17 inhibitor GM6001. Interestingly, activation of the pro-mitogenic (MEK/ERK) and survival (PI3K/Akt) pathways by TNF-␣ in mouse hepatocytes was dependent on AR expression. These findings lend further support to the involvement of the EGFR system in TNF-␣-induced hepatocellular proliferation [15] and highlight the unique role of AR among other EGFR ligands in growth regulation and carcinogenesis [31]. In view of all of these findings, targeting ADAM17 with novel oral agents might in-
crease the efficacy of EGFR inhibitors, and perhaps also that of sorafenib by enhancing the inhibition of the RAS/ RAF pathway [28]. As mentioned above, downstream of the EGFR, and of other tyrosine kinase receptors involved in hepatocarcinogenesis, is the RAS/RAF/MEK/ERK pathway. Oncogenic mutations in RAS and RAF genes are rarely found in HCCs, and increased activation of this pathway results from dysregulated expression of growth factors and their receptors [3]. Targeting of this pathway is therefore an attractive strategy to quell HCC growth. Control of RAS/RAF/MEK/ERK signaling is highly complex and not so well understood [18]. There are compelling reports showing that while moderate activation of the pathway leads to cell cycle progression, strong ERK activation above a certain threshold results in a blockade of cell proliferation and even in the induction of differentiation [25, 26]. We have identified a previously unknown regulatory mechanism controlling ERK signal amplitude that is triggered by growth factors concomitantly with the activation of the RAS/RAF/MEK/
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ERK pathway [19]. This mechanism is based on the methylation of RAF proteins at specific arginine residues, for instance Arg563 for CRAF, catalyzed by PRMT5 [19]. This posttranslational modification reduces the half-life of activated RAF proteins, and therefore modulates signal amplitude and the biological output (fig. 5). Methylation reactions can be pharmacologically targeted, and we show here that treatment of hepatocytes with the methylation inhibitor MTA significantly enhanced the level of ERK phosphorylation triggered by growth factors. Strong activation of the RAS/ RAF/MEK/ERK pathway has been associated with the induction of cell cycle arrest [25, 26]; consistent with this we also found that MTA inhibited DNA synthesis in parenchymal liver cells. These findings identify a new regulatory mechanism amenable to therapeutic intervention that may be exploited in HCC targeted therapy.
Acknowledgements This work was supported by the agreement between FIMA and the ‘UTE project CIMA’, and by RTICC-RD06 00200061 (C.B., F.J.C., and M.A.A.), CiberEhd (J.P.), FIS PI10/02642 and PI10/00038 (C.B. and M.A.A.), SAF2011-29312 (F.J.C.), the Ramón y Cajal Program (M.U.L.), the Torres Quevedo Program (R.U.), a ‘Gobierno de Navarra’ fellowship (M.E.), and the Erasmus program (F.S.), and a fellowship from Ministerio de Educación, FPV program, supported OG-I.
Disclosure Statement The authors declare that no financial or other conflict of interest exists in relation to the content of the article.
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