ErbB4/HER4 Acts As A Tumor Suppressor In Hepatocellular Carcinoma

BACKGROUND&OBJECTIVEHepatocellular carcinoma(HCC)is the third leading cause of cancer death in man worldwide.The overall 5-year survival rate for HCC patients is less than 5%.Because of the lack of understanding of liver carcinogenesis and the failure to develop interventions that are targeted at blocking or reversing the steps of malignant transformation,the prognosis of HCC patients remain poor.Thus,it is necessary to elucidate the molecular mechanisms underlying HCC and identify novel therapeutic targets and biomarkers for the early detection of HCC.The ErbB4 gene encodes one of the four members of the mammalian ErbB family of transmembrane tyrosine kinases.The expression of ErbB4 gene can appear in many tissues in our body,especial in brain and heart.The ErbB4 protein plays a role as a receptor for the neuregulins,a large group of structurally related molecules and a few other epidermal growth factors(EGF)-related polypeptides,such as heparin-binding EGF,betacellulin and epiregulin.The importance of this receptor tyrosine kinase in tumors has been demonstrated by the generation of mice with a targeted inactivation of the ErbB4 gene.However studies to investigate the relationship between ErbB4 gene and HCC are fewer,and the result is contradictory.The present study is designed to investigate the possible function of ErbB4 in liver.Methods1.Correlation analysis of expression levels of ErbB4 in tissue of liver diseases and patients with hepatocellular carcinomaExpressions of ErbB4 in 104 cases of clinical samples of normal liver and patients with hepatitis,cirrhosis and hepatoma were detected by immunohistochemical staining.The staining intensity was scored on a scale of 0 to 3 as negative(0),weak(1),medium(2),or strong(3).The extent of the staining,defined as the percentage of positive staining areas of cancer cells in relation to the whole tumor area,was scored on a scale of 0 to 4:0(none),1(≤25%),2(26%-50%),3(51%-75%),4(>75%).An overall protein expression score(overall score range,0-12)was calculated by multiplying the intensity and positivity scores.The relationship between clinicopathological features of tissues and ErbB4 scores was analyzed.Moreover,the relationship between clinicopathological features of tissues and ErbB4 scores and survival in hepatocellular carcinoma patients according to ErbB4 expression were analyzed.2.Estabalishment of acute liver injury models induced by CCL4 and LPSC57 mice were received an intraperitoneal injection of 2.5 ul/g olive oil and CCL4 in olive oil(1:4)respectively(model 1).After injections(12,24,48 and 72 hours,respectively),mice were sacrificed and marine serum and liver tissues were collected.To define hepatic inflammatory responses in ErbB4 deletion,we assessed induction of liver damage by lipopolysaccharides(LPS)challenge.ErbB4-/-HER4heart and ErbB4+/+ mice were received an intraperitoneal injection of 0.5 ml/25g LPS respectively(model 2).After injections(1,3 and 13 hours,respectively),mice were sacrificed and marine serum and liver tissues were collected.Marine serum ALT/AST was detected by microplate method.The structure and degree of injury in the liver were detected by H&E.Expression of ErbB4 and cytokines mRNA was detected by Western-Blotting and real-time PCR.3.Estabalishment of liver tumor models induced by DEN and CCL4To further determine the action of ErbB4 in liver malignancy,we evaluated the effect of ErbB4 removal on liver tumor in mice induced by chemical carcinogen.Control and ErbB4-/-HER4heart mice were injected with a single dose of diethylnitrosamine(DEN)on postnatal day 15,and animals were dissected after 8 months to examine liver tumor incidences(model 1).Mice were sacrificed and marine serum and liver tissues were collected.ErbB4-/-HER4heart and ErbB4+/+ mice were received an intraperitoneal injection of 25mg/kg DEN on postnatal day 15 respectively.On postnatal 8 weeks mice were received an intraperitoneal injection 2.5 ul/g CCL4 in olive oil(1:4),2 times a week(model 2).On postnatal 20 weeks,mice were sacrificed and marine serum and liver tissues were collected.Marine serum ALT/AST was detected by microplate method.The structure and degree of injury in the liver were detected by H&E.4.The roles of ErbB4 in HCCAnalyze disparity expression spectra in liver tumor models induced by DEN using Affymetrix GeneChip MouseGene 2.0_Array.The expression of differential expresssion gene were detected and tested by Real-Time PCR in mice liver of tumor models and hepatoma cell lines of ErbB4 knockdown.Then the NR4A1/TR3 was futher determinded as key related to ErbB4 how to restrain HCC.Result:1.Correlation analysis of expression levels of ErbB4 in tissue of normal liver and patients with hepatitis,cirrhosis and hepatomaThe expression levels of ErbB4 in the carcinoma are significantly lower than adjacent tissue and in tissue of normal liver and patients with hepatitis and cirrhosis by immunohistochemical staining(P<0.01).No significant associations were found between ErbB4 expression and age,gender,tumor stage of HCC patients(P>0.05).Interestingly,ErbB4 expression was correlated with tumor differentiation(P<0.01).We further examined whether the ErbB4 expression level correlated with the outcome of HCC patients after hepatectomy.Kaplan-Meier survival curves were used to compare the low(n=23)and high(n=67)ErbB4 subgroups.Remarkably,the patients with lower ErbB4 expression level had poorer overall survival(P<0.01).2.Estabalishment of acute liver injury models induced by CCL4 and LPS2.1 Mice were sacrificed and marine serum and liver tissues were collected after being injected OIL and 20%CCL4.Serum levels of alanine aminotransferase(ALT,P<0.01))and aspartate aminotransferase(AST,P<0.01)were significantly higher in mice injiected CCL4 than those in controls.A large proportion of CCL4 groups contained areas with infiltrate of inflammatory cells,which appeared to concentrate around the portal triads with extension into the parenchyma.Inflammatory cells were visible outside,around,or inside the necrotic areas.We then examined local expression of ErbB4 in the liver by RT-PCR and Western Blotting.Hepatic expression of ErbB4 mRNA was descended in CCL4 group.2.2 At first,we identify different genotypes for ErbB4 by genomic polymerase chain reaction.The ErbB4 wild-type allele yields～150bp,whereas the mutant allele yields-320bp.Then ErbB4-/-HER4heart and ErbB4+/+ mice were sacrificed and marine serum and liver tissues were collected after being injected LPS.Serum levels of ALT/ASTwere significantly higher in ErbB4-/-HER4heart mice than those in controls(P<0.01).In ErbB4-/-HER4heart mice liver inflammatory cells concentrated around the portal triads with extension into the parenchyma were more than control mice.At last,we measured hepatic expression levels of inflammatory cytokines at different time points following LPS injection,and detected similarly increased levels for IFN-y,TNF-a,RANTES,IL-12,IL-6,and IL-10.However,higher levels of IFN-γ,TNF-α,IL-6,IL-10 were detected in ErbB4-/-HER4heart than in control mice.3.Estabalishment of liver tumor models induced by DEN and CCL42.1 Control and ErbB4-/-HER4heart mice were injected with a single dose of diethylnitrosamine(DEN)on postnatal day 15,and animals were dissected after 8 months to examine liver tumor incidences,as described previously.All male animals in ErbB4-/-HER4heart groups developed visible hepatic tumor foci.Moreover,hepatocytespecific deletion of ErbB4 increased the number of liver tumors(P<0.05).We also used serum levels of ALT/AST to determine the tumor burden in DEN-treated animals.The ALT levels increased almost 2.5-fold in ErbB4-/-HER4heart animals,compared to controls(P<0.01).2.2 Control and ErbB4-/-HER4heart mice were injected with diethylnitrosamine(DEN)and CCL4,and animals were dissected after 5 months to examine liver tumor incidences,as described previously.All the male animals in both ErbB4-/-HER4heart and control groups developed visible hepatic tumor foci.However,hepatocytespecific deletion of ErbB4 increased the size(>2mm)of liver tumors(P=0.012).Moreover the AST levels increased almost 2-fold in ErbB4-/-HER4heart animals,compared to controls(P<0.01).4.A model for ErbB4 regulation of STAT5A-stimulated NR4A1/TR3 expression and the TR3-Bcl2 apoptotic pathwayGrowth factor-stimulated ERBB4 undergoes sequential proteolytic processing at the cell membrane by TACE and presenilin-dependent r-secretase to release intracellular domain(ICD).ICD accumulates in the perinuclear region where a perinuclear/nuclear equilibrium is established favoring perinuclear over nuclear ICD.Cytosolic STAT5A associates with activated ERBB4/ICD in an SH2 domain-dependent manner and STAT5A is phosphorylated at multiple residues.The two nuclear proteins bind to STAT5A target promoters stimulating expression of STAT5A regulated genes including NR4A1/TR3,IL1B and so on.TR3 accumulates in the nuclear.Then it migrates from the nucleus to the cytoplasm as TR3/RXR aheterodimer,where it targets mitochondria by binding to Bcl2.Interaction with Bcl2 induces a Bcl2 conformational change,triggering cytochromec release and apoptosis.Conclusions:1.ErbB4 was down-regulated in the tumor tissues of liver.Analysising the tissues with tumor,the expression levels of ErbB4 in the carcinoma are significantly lower than adjacent tissue.In addition,ErbB4 expression was positively correlated with tumor differentiation.And,the patients with lower ErbB4 expression level had poorer overall survival.2.ErbB4 ablation leads to development of acute liver injury.3.ErbB4 ablation leads to development of hepatocellular tumor in two liver tumor models.4.The ErbB4-SATA5A-TR3-Bcl2 apoptotic pathways.Growth factor-stimulated ERBB4 undergoes sequential proteolytic processing to release ICD.ICD accumulates in the perinuclear region and conbines cytosolic STAT5A.Then they migrate from the cytoplasm to the nucleus and bind to STAT5A target promoters stimulating expression of STAT5A regulated genes including NR4A1/TR3 and IL1B.TR3 accumulates in the nuclear.Then it migrates from the nucleus to the cytoplasm as TR3/RXR aheterodimer,where it targets mitochondria by binding to Bcl2.Interaction with Bcl2 induces a Bcl2 conformational change,triggering cytochromec release and apoptosis.