Characterization And Interaction Of EGFR Family And NGF/TrkA In Cholangiocarcinoma

Cholangiocarcinoma is commonly considered a rare cancer. However, if we consider the hepato-biliary system a single entity, CCA represent approximately30%of the total with incidence rates second to that of hepatocellular carcinoma,Cholangiocarcinoma is characterized by a very poor prognosis and virtually no response to chemotherapeutics; radical surgery, the only effective treatment, is not frequently applicable because late diagnosis. To the present, Biomarkers for screening programs and for follow-up of categories at risk be scarcely reached clinical application. Cancers of the cholangiocarcinoma should merit much more scientific attention and novel biomarkers are urgently needed for CCA management and treatment because a progressive increase in incidence and mortality for these cancers has been reported worldwide.The growth factor receptors are known to play a role in the development of several carcinomas. The EGFR family consists of four receptor tyrosine kinases, EGFR (ERBB1, HER1), ERBB2(HER2, neu), ERBB3(HER3), and ERBB4(HER4). Binding of the appropriate growth factor leads to dimerization and subsequent activation of the receptor and then the down-stream intracellular signaling pathways to regulate cell proliferation, differentiation, and migration. Most of the previous research has focused on investigating the expression of individual members in relation to different clinicopathological studies. However, in different tumor entity, EGFR family members showed discrepancy in quantity of expression and different style of co-expresion leading different biological behavior. Comprehensive investigation should carry out on base of regarding EGFR family members as a whole entity.To the present, a series of studies concerning EGFR family members’biological functions and clinicopatholgical association in cholangiocarcimoma have carried out which focus on two members of EGFR and HER2and revealed that EGFR is a prognostic factors and targets of novel biologic agent.HER2overexpression is uncommon event in cholangiocarcinoma unlike the cases in breast cancer and gastric cancer. By contrast, the data of HER3and HER4is very limited. The biological function and prognostic role of HER4have not been investigated in CCA so far.Chronic inflammation caused by various factors are risk factors for the development of cholangiocarcinoma, inflammatory cytokines, growth factors, chemokines around the cholangiocarcinoma cells form a complex tumor microenvironment. In this complex tumor microenvironment, activation of the EGFR family is not an isolated incident, in which the formations of homodimers or heterologous dimers,"trans-activation" between different molecular pathways are involved.NGF (nerve growth factor, NGF) belong to the family of neurotrophic factors, NGF can be used as an inflammatory mediators play an important role in inflammation; NGF binding to TrKA receptors cause cell proliferation, differentiation and cell migration. Previous studies showed during inflammatory processes existing "Crosstalk"between NGF-TrkA and EGFR pathway, and the detailed circumstances is not yet clear in cholangiocarcinoma.In this experiment, tissue microarrays were constructed containing175cases of specimens from patients of cholangiocarcinomas. Using immunohistochemistry and fluorescence in situ hybridization technology, we investigate the expressions of EGFR family members, the relationship between members in the group, and their associations with clinical pathological features.To further investigate the biological functions of HER4in proliferation, invasion and metastasis, we construct HER4 overexpression plasmid and siRNA-HER4interference fragments and relevant experiments were caried out.In the final section of this paper, we use immunohistochemistry to detect the expression of NGF and TrkA in cholangiocarcinoma and the associations with clinicopathological features. Furthermore the mechanisms affecting prognosis were discussed.Part1Characterization of EGFR family gene aberrations in cholangiocarcinomaObjectiveTo explore the expressions of EGFR family members in cholangiocarcinoma and their associations with clinicopathological characteristicsMethods1. Establishing database of cholangiocarcinoma patients and construct175cases of cholangiocarcinoma patients of QiLu hospital from2004-2008were adopted in this experiment. Clinical information concerning gender, age, clinical stage, tumor size, lymph node metastasis, surgical methods, calculus, and hepatitis infection were collected. Two tissue microarrays of cholangiocarcinomas was constructed.2. Using immunohistochemical method to detect EGFR members’ expressions in cholangiocarcinomas,Monoclonal antibody of EGFR, HER2, HER3and HER4were used to detect the expression of EGFR family members’ immunohistochemically on TMA.nalysis. Each EGFR member’s expression and inner link and its relationship clinicopathological features were analyzed accordingly.3. The prognostic significance of EGFR family members in cholangiocarcinomasIn the survival analysis, the starting point for the survival time was the date three months after surgery. Cumulative overall survival rates were calculated by Kaplan-Meier method and statistical significance for survival curves comparison was analyzed by log-rank test. Univariate and multivariate survival analyses were performed using the Cox multiple hazards model to estimate hazard ratio.differences for all the tests were regarded as statistically significant when the P-value from a two-tailed test as P＜0.05.4. Fluorescence in situ hybridization (FISH)The GLP EGFR/CSP7probe and GLP HER2/CSP17were utilized and slides were examined using an ImagingZl microscope.Result1. A detailed information database of175CCAs was built and follow-up information was obtained form165cases CCAs. In all two tissue microarrays was construct.2. In all, EGFR, HER3and HER4were overexpressed in20(30.8%),8(12.3%) and41(63.1%) of the65intrahepatic cholangiocarcinomas (IHCCs), and23(20.9%),13(11.5%) and62(56.4%) of the110extrahepatic cholangiocarcinomas (EHCCs), respectively. Overexpression of HER2was exclusively identified in EHCCs, among which the rate was4.5%(5/110).FISH analysis for EGFR and HER2was available for169and171cases, respectively. In all, amplification of EGFR was identified in1(1.6%) of the63IHCC cases, comparable with that of2.8%(3/106) in patients with EHCC. By contrast, HER2amplification was seen in8of the108(7.4%) EHCC, but absent in IHCC cases. Of note, a significant association was identified between EGFR amplification and EGFR overexpression (P=0.002). Similarly, HER2amplification was strongly associated with HER2overexpresssion (P<0.001).3. In IHCC, univariate analysis revealed that EGFR overexpression was a prognostic factor (P=0.016). Additionally, histological differentiation (P=0.004), pT stage (P=0.003), Tumor size (P=0.012), lylmph node metastasis (P＜0.001) were also significantly related to overall survival. Notably, in a multivariate analysis, EGFR overexpression remained an independent prognostic factor (HR (95%CI):3.689(1.253-10.587), P=0.018).In EHCC,4factors including EGFR overexperssion were identified as prognostic factors by univariate analysis. In multivariate analysis, only lymph node status was an independent prognostic factor (HR (95%CI):4.248(1.761-10.249), P=0.001). In contrast, EGFR expression lost its predictive value. In all, no statistical significance was identified between HER4expression and overall survival in CCA by univariate analysis. However, HER4expression was identified as a prognostic factor (P=0.023). ConclusionIn summary, our results revealed expression of the EGFR family members in CCA development and progression. CCAs differentially express HER2protein based on tumor location. For the first time, our study suggested that HER4is a prognostic factor in subset of EGFR-Chinese CCA patients with IHCC. Determination of HER4expression allows stratification of CCA patients into different survival categorie.Part2In vitro studies of HER4influence on cell proliferation, invasion and invasion abilities in cholangiocarcinoma cellsObjectiveThe first part of this study found that the expression of HER4with good prognosis, but the specific mechanism is unknown; this section intends to study HER4RBE in bile duct cancer cells and the proliferation of HUCCT-1, the influence of the invasion and metastasis ability, to explore the role of HER4in bile duct cancer mechanism.Methods1. Construction of HER4overexpression plasmid and siRNA-HER4interference fragmentsThe HER4cDNA fragment was cloned by PCR and digisted and ligased into the GV135vector. This vector was identified by squencing, and we successfully constructed the adenovirus expression vector containing HER4. The1x109virus were obtained by JiMa Biological Company. The siRNA targeting siRNA was designed and synthesised by Shanhai KaiJi Company.2. Investigation of the biological functions of HER4in proliferation, invasion and metastasis CCA cell line RBE were cultured and is divided into three groups:MOCK (Lipofectamine), NC (Lipofectamine+Scramble RNA) and siRNA-HER4(Lipofectamine+siRNA-HER4) group. After24hours incubation in each group,5x103cells were seeded in96-well plates, proliferation rate of each group were detected at time points of in24h,48h,72h respectively. Wound healing assay was performed in12-well plates in the three differently treated group, the span of scratches were observed at time point of6h,24h,48h and72h respectively. The ability of RBE and HUCCT-1migration was evaluated by transwell assay. RBE cells transfected with siRNA-HER4and HUCCT-1with GV135-HER4for24h,then Equal numbers of RBE and HUCCT-1cells were suspended and cultured in transwells for6hours,the invade cells were fixed with4%Formaldehyde and stained with0.05%crystal violet.The invaded cells on the lower surface were counted from3random fields.3. HER4detects the impact of cholangiocarcinoma cell apoptosis.RBE cells were cultured and divided MOCK, NC, and siRNA-HER4group, respectively, after treatment24h,48h,72h, apoptosis were detectd by flow cytometry. Changes of caspase-8,-9, and3were detected by ELIS A method. Apoptosis in empty vector and GV135-HER4treated HUCCT-1cells were detected by flow cytometry.ResultUsing the MTS assay, we found that siRNA knockdown of HER4in RBE cells significantly increased cell proliferation at24h and48h after treatment compared with negative controls(P<0.05). Wound healing assay indicated that siRNA-HER4-transfected RBE cells displayed a significant decrease in cell migration ability compared to control conditions (P＜0.05). To further examine the effect of HER4on cell invasion, siHER4-transfected RBE and PcDNA3.1-HER4-transfected Hucct-1cells were cultured on a transwell apparatus. The percentage of migrated cells was significantly less in siHER4-treated groups when compared to the negative control groups (for both P＜0.05). PcDNA3.1-HER4-transfected Hucct-1cell line showed decreased percentage of migrated cells (both P＜0.05).Conclusion HER4knockdown increased RBE cell proliferation, infiltration and invasion ability, in contrast, overexpressed HER4decreased percentage of migrated cells.Our result demonstrated that EGFR family member play an dual role in CCAs and HER4exert a protective influence in CCAs progression.Part3Cell signaling pathway of EGF and NGF in cholangiocarcinomaObjectiveTo explore the variation and connection of NGF and EGF activated signal transduction cascades.MethodsA.Detection of EGFR and TrkA pathway protein by westen blot. Cholangiocarcinoma cell line RBE was incubated by recombinant human EGF and recombinant human. MAPK signaling pathway protein ERK, p-ERK was detected at different time points.B. RBE cells were cultured in the medium were added to recombinant EGF, EGF+K252a and EGF+NGF monoclonal antibodies rspectivly. Changes of total and phosphorylation of TrkA and concentration of NGF in culture medium were detected.C. RBE cells were cultured in the medium were added to recombinant NGF+K252a, recombinant NGF+EGFR inhibitor AG1478respectively. Variations of total and phosphorylation of EGFR and concentration of EGF in culture medium were detected.ResultsAfter separately addition of recombinant human EGF and recombinant NGF respectivly, ERK and p-ERK protein levels increased in early phase, then the influence gradually abate with time cause. When EGF and NGF added in the same time, ERK protein levels increased than EGF or NGF alone.K252a, NGF monoclonal antibody reduced the level of phosphorylation of TrkA in EGF incubated cholangiocarcinoma cells; K252a, AG1478reduced levels of NGF induced phosphorylation of EGFR. ELISA results show that recombinant human EGF protein in RBE cells can stimulate the secretion of NGF; recombinant NGF protein can cause elevated levels of EGF.ConclusionIn cholangiocarcinoma, EGF (R) signaling pathway and NGF-TrkA receptors and signaling pathways influent bilateral, EGF and NGF has a synergistic role in the development of cholangiocarcinoma.Part4Clinicopathological study of NGF and TrkA expression in intrahepatic cholangiocarcinomaObjective To investigate the correlation between nerve growth factor-tropomyosin-receptor-kinase (NGF-TrkA) signaling pathway and prognosis in intrahepatic cholangiocarcinoma (IHCC)Method1. Expression of NGF and TrkA were detected by immunohistochemistory in cholangiocarcinoma.NGF and TrkA expression in83samples of IHCC was assessed by immunohistochemistry (IHC). Correlations between NGF-TrkA expression and clinicopathological features were analyzed.ResultsNGF and TrkA expression was significantly related with differentiation (P=0.024) and intraneural invasion (P=0.003), respectively. Additionally, double higher expression of NGF and TrkA was identified as an independent prognostic factor in IHCC (P=0.003). Moreover, NGF-TrkA signaling pathway can promote IHCC proliferation and invasion.ConclusionNGF-TrkA double higher expression is an independent prognostic factor in IHCC. NGF-TrkA signaling pathway can promote IHCC progression, indicating that NGF-TrkA may become a potential drug target.