Functions Of FGF19-FGFR4and NGF-TrkA Signaling Pathway In Progression And Prognosis In Cholangiocarcinoma

Background and significanceCholangiocarcinoma(CCA) is a highly malignant tumor that arises from biliary tract. The CCAs can be further divided into intrahepatic cholangiocarcinomas (IHCCs), perihilar cholangiocarcinomas (PHCCs) and distal cholangiocarcinomas (DCCs) according to the7th edition of AJCC/UICC TNM staging classification. The IHCCs, PHCCs and DCCs originate from the intrahepatic, perihilar, or distal biliary tree respectively and are identified to have different biological and neoplastic features. All the IHCC,PHCC and DCC can be divided into4TNM stages according to the extent of tumor size, filtration, lymphoid and distal metastasis in AJCC/UICC TNM system. PHCC is the most common type of cholangiocarcinomas, followed by the DCC and then IHCC. Incidence of CCA just accounts for3%of gastrointestinal cancer, but the morbidity and mortality of cholangiocarcinoma, especially IHCC, are increasing worldwide. Morbidity of BTCs is low with approximately9,760new cases diagnosed annually in the United States, whereas the mortality of BTCs is very high. Currently, radical resection of the tumor and involved liver is the only curative treatment for CCA. However, when patients are hospitalized because of jaundice of pain, it is usually too late for surgery because of the invasiveness and metastasis, which make the resectability rate quite low and variable (18-70%). In conclusion, most patients lose surgical indications when diagnosed because of silent clinical features, difficult early diagnosis, high metastatic rate and low response to adjuvant therapy. Consequently, the5-year survival rates of BTCs are poor, ranging from5%to15%(10,11).Overall, CCAs are difficult to be treated successfully and have a poor prognosis as a result of clinically silent features, difficulty of prompt diagnosis, early metastasis and easy recurrence and low response to adjuvant therapy (8,12). Hence, there is an urgent need for the predictive, prognostic and therapeutic markers. Unfortunately, few studies aimed at identifying new prognostic factors of BTCs have been reported. Although several big breakthroughs on molecular analysis were made recently, researches on new biomarkers still make progress slowly because that the majority of patients present are at an unresectable stage, which makes a large randomized trials difficult. Hence, the understanding of genetic mechanisms, prognostic markers and potential inhibitor remains inadequate in CCA. In breast cancer, the exciting discovery of ErbB2association with prognosis leaded to the drug Herceptin, which markedly improved survival time of patients with breast cancer. It is obvious that the finding of new prognostic biomarker is very essential to new cancer drugs and therapies. We hope our screen of CCA biomarker can help introduce a new insight to drug development of BTCs and help find an attractive therapy.There are great breakthroughs in the field of IHCC biomarkers and molecular classficication recently. Large-scale screen of gene signatures and integrative molecular analysis were used to reveal transcriptional profile and classify molecular classes of IHCC. Daniela et al identified2classes of IHCC by genomic analysis and molecular signature, which are proliferation class and inflammation class. The former class has specific copy number alterations, activation of oncogenic pathways, and is associated with worse outcome. Different classes of ICC, based on molecular features, therefore might require different treatment approaches.Based on previous finding of CCA molecular and clinical study, we systematically read articles on CCA biomarker prediction, especially on the high throughout experiments on comparison of cancer and non-cancer tissue, including microarray, fast scan mass spectrometry, high-throughput gene sequencing studies etc.al. Combined with previous signaling study, we selected a serial of candidate tumor markers on purpose and perform protein screen by immunohistochemistry(IHC) on tissue samples firstly. The candidate markers included known CCA biomarker and suspected biomarker. The former ones included endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR), the latter ones included insulin-like growth factor1(IGF-1), insulin-like growth factor1receptor (IGF1R), fibroblast growth factor receptor1(FGFR1), FGFR2, FGFR3, FGFR4, fibroblast growth factor1(FGF1), fibroblast growth factor19(FGF19), Interleukin6(IL-6), IL-10, signal transducer and activator of transcription3(STAT3), p-Stat3,nerve growth factor(NGF), neurotrophic tyrosine kinase receptor type1(TrkA), ErbB2, ErbB3,CREB,ETS-1,FBOX9,WDFY-1et al. After first screen, we chose high-expressed and suspected proteins to analyze their relationship with clinicopathologic parameters. Subsequently we analyzed the correlation between these markers and overall survival rate by Kaplan-Meier method, which helped we found that FGFR4and NGF/TrkA were significantly related to prognosis. Then we identified these markers as independent prognostic factor by multiunivariate analysis with Cox-regression model. To elucidate why these markers correlated with poor prognosis and reveal the underlying molecular mechanism, we performed assay in vitro to study the importance of these markers and their involved signaling pathway on tumor progression in CCA cell lines, including proliferation, invasion, epithelial-mesenchymal transition(EMT) and vessel tube formation.The whole article can be divided into three parts. In the first part, we collected280CCA cases and divided it into primary cohort and validation cohort, and got the post-operation survival time by follow-up. After the approval of patients or their family, we got tissue samples and made them into TMA. Finally, we performed IHC and calculated the positive expression rate of all the candidate proteins, subsequently evaluated their correlation with overall survival rate. In the second part, we focused on FGFR4influence on CCA. We identified FGFR4as an independent prognostic factor in IHCC and PHCC, and proved that FGFR4can promote CCA cell line proliferation, invasion and EMT. Moreover, we used FGFR4inhibitor AP24354can suppress proliferation, invasion and induce apoptosis of CCA. In the third part, we indentified NGF/TrkA double positivity was an independent prognostic factor and can promote CCA progression. First part Screen of candidate prognostic markers of CCAObjectiveTo screen the predictive biomarkers of previous study by IHC, analyze the associations between biomarkers and overall survival rate by univariate analysis.MethodsFirst of all, we collected all the samples of CCA from the pathology department of Qilu Hospital and Yishui Central Hosptial. Samples were got from tumor resection during the period from2002to2010with prior patient consents and the approval of the Institutional Clinical Ethics Review Board. The diagnosis was confirmed by the routine pathology when resected and reconfirmed by a senior pathologist before experiments, In our study,280patients were identified to have CCA by routine pathology and199patients (83patients with IHCC,75patients with PHCC,41patients with DCC) were included in the validation cohort. After the approval of patients or their families, we made TMA from cancer tissues. We selected candidate biomarkers including FGFR1, FGFR2, FGFR3, FGFR4, FGF1, FGF19, ErbB2, ErbB3, IGF, IGF-1R, NGF, TrkA, Stat3, p-Stat3, IL-6, IL-10, CREB, Foxol, ETS-1, FBOX9, WDFY1and detected their expressions in CCA by IHC. Furthermore, we selected the candidate proteins whose positive rate was more than20%and calculated their prognostic significance with univariate analysis.The overall survival time was calculated from the operation to the date of death or censored at the date of the last follow-up examination. The follow up was realized mostly by phone. Clinical data, including age, gender and other clinicopathologic features were abstracted from the patients’ medical records. The clinical follow-up was at least3months after surgery, with the median follow-up time24.6months (from3to98months). Samples of all the199patients were made tissue microarray(TMA) with1mm diameter cylinder. The TMAs were made using buffered formalin-fixed and paraffin-embedded tissue sections from all the229patients. Before immunohistochemistry detection, hematoxylin and eosin staining were performed to confirm the histological characterization of all samples.ResultsAll the patients were divided into positive and negative group according to the results of IHC. Biomarkers which had a positive expression more than20%were VEGF, EGFR, FGFR1, FGFR2, FGFR4, FGF19, ErbB3, NGF, TrkA and CREB. With univariate analysis, we found that only VEGF, EGFR, FGFR4and NGF/TrkA double positivity had prognostic value.ConclusionsAs candidate biomarkers, FGFR1, FGFR2, FGFR4, FGF19, ErbB3, NGF, TrkA and CREB had positive expression rate more than20%. VEGF, EGFR, FGFR4and NGF/TrkA double positivity were identified to be correlated with poor prognosis in CCA. Second part FGF19-FGFR4signaling pathway correlates to progression and prognosis of CCAObjectiveFGFR4has been proved to be involved in many kinds of cancers. However, the correlation between FGFR4expression and CCA has not been well elucidated. This study aimed to investigate the FGFR4expression and prognostic significance in BTCs and FGFR4role in BTCs progression. Methods Tissue microassay of83intrahepatic cholangiocarcinomas (IHCCs),75perihilar cholangiocarcinomas (PHCCs), and41distal cholangiocarcinomas (DCCs) was used to measure the expression of FGFR family by immunohistochemistry. Associations of FGFR4with clinicopathologic parameters, other biomarkers and survival rate were subsequently evaluated. Additionally, a series of functional assays were performed by knocking down and overexpressing FGFR4in three BTC cell lines, including proliferation and invasion assay.ResultsThe positive rate of FGFR4was61.4%(51/83) in IHCCs,36.7%(35/75) in PHCCs,56.1%(23/41) in DCCs, and56.7%(17/30) in GBCs. FGFR4expression was significantly related to poor prognosis of IHCC (P=0.002), PHCC(P=0.019) and GBC(P=0.004) with univariate analysis. FGFR4was also identified as an independent prognostic factor in IHCC (P=0.045), PHCC (P=0.049) and GBC (P=0.018) with multivariate analysis. With experiments in vitro, we demonstrated that FGFR4activation by FGF19could induce proliferation and invasion of BTC cell lines.ConclusionsFGFR4expression in biliary tract cancers was extremely high and could be identified as a significant independent prognostic marker of IHCC, PHCC and GBC. FGFR4played a pivotal role in proliferation and invasion of BTC cell lines, indicating that FGFR4may act as a potential therapeutic target. Third partClinical significance of NGF-TrkA signaling pathway on CCA progression and prognosisBackgroundNerve growth factor(NGF) and its receptor TrkA have been identified to be correlated to tumorgenesis and prognosis in several kinds of cancers. However, the importance of NGF-TrkA signaling pathway incholangiocarcinoma (CCA) is poorly elucidated. We aimed at revealing the importance of NGF-TrkA signaling pathway in CCA progression and prognosis.MethodsNGF and TrkA expression in83samples of IHCC,75samples of PHCC and41samples of DCC was assessed by immunohistochemistry (IHC). Correlations between NGF-TrkA expression and clinicopathological features were analyzed by Chi-Square test. Moreover, we evaluated the association of NGF-TrkA with overall survival with univariate and multivariate analysis. With experiments in vitro, we assessed the crucial role of NGF-TrkA in proliferation and invasion of IHCC cells with recombinant NGF-p stimulation.ResultsIn IHCC, we found that NGF and TrkA expression was significantly related with differentiation (P=0.024) and intraneural invasion (P=0.003), respectively. Additionally, NGF-TrkA double positivity was identified as an independent prognostic factor in IHCC (P=0.003). Moreover, we demonstrated that NGF-TrkA signaling pathway can promote IHCC proliferation and invasion. ConclusionsNGF-TrkA double positivity 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.