Sprouty4 Expression Correlates With Favorable Prognosis In Perihilar Cholangiocarcinoma By Blocking The FGFR-ERK Signaling Pathway And Arresting The Cell Cycle

BackgroundCholangiocarcinoma(CCA)is a type of malignancy arising from the biliary tree.Cholangiocarcinoma accounts for 3%of all gastrointestinal tumors.Globally,hepatobiliary malignancies account for 13%of cancer-related deaths;10%-20%of these are attributable to CCA.The mean age at diagnosis of CCA is 50 years.The global incidence of iCCA varies widely.Over the past 3 decades,the overall incidence of CCA appears to have increased,especially in East and Southeast Asia.Based on the anatomical location of the tumor,CCA can be further classified into subtypes including intrahepatic(ICC),perihilar(PHCC),and distal(DCC)cholangiocarcinoma,with distinct risk factors,molecular pathogenesis,biological features,clinical characteristics and treatment strategies.Intrahepatic cholangiocarcinomas(ICC)are located within the hepatic parenchyma.The second order bile ducts serve as the point of separation between Intrahepatic cholangiocarcinomas(ICC)and perihilar cholangiocarcinomas(PHCC)or distal cholangiocarcinomas(DCC)—the cystic duct is the anatomic boundary between these latter 2 subtypes.It is a great challenge to diagnose CCA because of its anatomic location,and silent clinical character.Patients with CCA usually suffer from late diagnosis and poor outcomes.PHCC is the most common type of CCA,accounting for more than 50%of cases.Radical surgery is a curative option for all CCA subtypes but is extremely difficult for PHCC because of the anatomical complexity of the perihilar region.The prognosis of PHCC is still very dismal(<30%in most studies),although surgical techniques and adjuvant therapy have been dramatically improved.Technological revolution,such as second-generation sequencing,provides more insights into the molecular characteristics and therapeutic strategies for tumor treatment.This is especially important to biliary cancer,including CCA,because more than 65%of patients with biliary cancer are diagnosed with unresectable disease.Emerging evidence from comprehensive genetic analyses reveal several actionable mutations in CCA,such as fibroblast growth factor receptor(FGFR)fusion rearrangements and isocitrate dehydrogenase(IDH)-1 and IDH2 mutations.However,studies on the molecular patterns and features of PHCC are lagging behind those for ICC,despite PHCC having the highest prevalence.No study has regarded PHCC as a distinct cancer type in comprehensive genetic analysis thus far,although PHCC and DCC have been identified as different extrahepatic CCA since 2007 by the 7th American Joint Committee on Cancer/Union for International Cancer Control(AJCC/UICC)system.In all subtypes of CCA,Kirsten ras sarcoma viral oncogene homolog(KRAS)mutations and FGFR2 fusions are well-identified somatic genetic alterations.KRAS mutations are associated with poor overall survival,and several independent lines of evidence have demonstrated the role of FGFR2 fusion in CCA tumorigenesis and progression.FGFR2 is a receptor tyrosine kinase involved in cellular processes such as proliferation mainly by activating downstream pathways,including Ras/Raf/MEK/MAPK and PI3K/AKT signaling.RAS is a member of the FGFR2 signaling pathway,and its common downstream signaling pathway is the MEK/MAPK pathway.Both KRAS mutations and FGFR2 fusions constitutively stimulate the MEK/MAPK pathway,and this ectopic activation finally leads to excessive proliferation in tumor cells.ERK,one of the most famous MAPKs,is a main effector downstream of both KRAS and FGFR2.It is well accepted that RAS activation can initiate compensatory feedback mechanisms that attenuate signaling output.The Sprouty(SPRY)family,comprising SPRY 1-4,is the most important negative regulator of the Ras/Raf/MEK/MAPK signaling pathway.SPRY can inhibit ERK phosphorylation by modulating the FGFR/Ras/Raf/MEK/MAPK pathway at different levels.Dysfunction in the SPRY family has been reported to be correlated with progression in several types of cancers,including gastric cancer,breast cancer,liver cancer and prostate cancer.However,emerging evidence indicates that the expression of SPRY members is tumor-specific and that not all SPRY members are involved in the progression and prognosis of different cancer types.For example,SPRY 2,but not SPRY 4,is an independent prognostic biomarker for human epithelial ovarian cancer.Moreover,loss of SPRY 4 but not of other SPRY members,amplifies RAS signaling in acute myeloid leukemia.In our previous study,we demonstrated that different SPRY members had different expression patterns in ICC and that SPRY 2 suppressed the progression of ICC by antagonizing FGFR2 signaling.Nevertheless,the expression,function and clinical significance of the SPRY family in PHCC are still unknown.In our study,we investigated the expression of SPRY family members in ICC,PHCC and DCC to identify the key effector responsible for Ras/Raf/MEK/MAPK signal feedback in PHCC cells and evaluated its clinical significance by analyzing the correlations between clinicopathological factors and survival time.Through in vitro and in vivo experiments,we further evaluated the oncogenic function of SPRY 4 in PHCC progression and explore the underlying mechanism of SPRY 4-suppressed progression of PHCC.Part ? Expression and clinical significance of SPRY family in cholangiocarcinomaObjectives:CCA is an aggressive malignancy with a dismal prognosis.According to the anatomical position,the cholangiocarcinoma is divided into intrahepatic cholangiocarcinoma,perihilar cholangiocarcinoma and distal cholangiocarcinoma.Perihilar cholangiocarcinoma(PHCC)is the most common subtype of cholangiocarcinoma(CCA).We previously investigated the expression pattern of SPRY in intrahepatic cholangiocarcinoma(ICC),but the expression and clinical significance of SPRY family members in PHCC are still unknown.We investigate the expression of SPRY family in cholangiocarcinoma and its correlation with clinicalpathologic features and prognosis of cholangiocarcinoma patients.Methods:The primary cohort of our study comprised 424 patients who underwent surgery for PHCC at Qilu Hospital of Shandong University from 2007 to 2018.A validation cohort consisting of 142 patients was further selected.We collected CCA tissues and paired adjacent normal tissues,the expression of SPRY family members(SPRY 1-4)was detected in different subtypes of CCA and corresponding adjacent tissues by qRT-PCR.SPRY family expression in 142 cases of PHCC was detected by immunohistochemistry.Survival curves were plotted using Kaplan-Meier method and survival rate was analyzed by log-rank test.The clinical significance was evaluated using univariate and multivariate analyses.Findings:The mRNA levels of SPRY 2 in ICC tumor tissues were significantly lower than those in paired adjacent tissues.SPRY 2 expression was not remarkably different between PHCC tumor and paired adjacent tissues,while the expression of SPRY 1,SPRY 3 and SPRY 4 was notably lower in PHCC tumor tissues than in adjacent tissues.Moreover,we detected the expression of SPRY 1-4 in 142 cases of PHCC with IHC and divided these cases into subgroups according to the H-scores of SPRY 1-4 expression.All SPRY members were expressed in the cytoplasm.SPRY 1 and 4 had relatively higher expression than SPRY 2 and 3 in PHCC.Among the four SPRY members,SPRY 4 was the only marker of favorable prognosis in PHCC(P=0.006).High expression of SPRY 4 indicated a longer survival time.In addition to SPRY4 expression,patient age>65(P=0.013),positive lymph node metastasis(P=0.025),positive metastasis(P<0.001),and advanced TNM stage(P<0.001)were all significantly associated with low survival rates in our study.High expression of SPRY4 was significantly associated with small tumor size(P=0.007),negative lymphatic invasion(P=0.031)and early TNM stage(P=0.002).Conclusion:SPRY 4 was the only SPRY family member associated with PHCC prognosis,and high expression of SPRY 4 was an independent biomarker of favorable prognosis in PHCC.Part ? SPRY4 correlates with favorable prognosis in perihilar cholangiocarcinoma by blocking the FGFR-ERK signaling pathway and arresting the cell cycleObjectives:In the previous section,we found high expression of SPRY 4 was an independent biomarker of favorable prognosis in PHCC,the mechanism was not yet clear.The SPRY family was identified as a negative regulator of ERK activation,and we speculated that SPRY4 may affect cellular function by inhibiting ERK activation and thus the prognosis of perihilar cholangiocarcinoma patients.In this section,we aim to investigate the correlations between SPRY4 and FGFR family.To investigate the role of SPRY4 in PHCC cell lines and mechanisms by which SPRY4 correlates with favorable prognosis in perihilar cholangiocarcinoma.Method:The expression of FGFR1-4 was detected with IHC and divided into low and high expression.We further detected the correlations between SPRY4 and FGFR family.The functions of SPRY4 in the FGFR-induced proliferation and migration of PHCC cells were investigated through in vitro and in vivo experiments.The expression of SPRY4 in 8 pairs of fresh PHCC tissues and corresponding tumor adjacent tissues was detected by western blotting.SPRY4 expression was detected in ICC cell lines RBE and HCCC-9810 and PHCC cell lines FRH0201 and QBC939.After silencing SPRY4 with shRNAs in QBC939 and overexpressing SPRY4 in FRH0201 with lentiviruse carrying LV5-SPRY4,we evaluated the proliferation and migration of QBC939 cells.Stable QBC939 cells with SPRY4 knockdown and stable FRH0201 cells with SPRY4 overexpressing were established and subcutaneously transplanted into nude mice for successful xenograft formation.We further investigated the effects and mechanisms of SPRY4 on FGFR-induced ERK phosphorylation and cell cycle distribution in the presence of FGFR and ERK inhibitors.Findings:High expression of FGFR2 and FGFR4 indicated poor prognosis of PHCC.In PHCC,SPRY4 expression was extensively associated with FGFR2,and its expression can be induced by ectopic FGFR2 activation.SPRY4 expression was detectable in all these cell lines and was most prominent in QBC939 cells.Both CCK-8 and colony formation assays showed that SPRY4 knockdown substantially promoted the proliferation of QBC939 cells,and SPRY4 overexpression facilitated the progression of FRH0201 cells.Similar results were obtained in transwell and wound healing assays,suggesting that SPRY4 also played an essential role in the migration of QBC939 cells.Through in vitro and in vivo experiments,we demonstrated that SPRY4 suppressed FGFR-induced proliferation and migration by inhibiting ERK phosphorylation.Moreover,SPRY4 knockdown was shown to decrease the percentage of cells in the G1 phase and promote the percentage of cells in the S and G2/M phases by increasing cyclin D1 expression,which also required FGFR-induced ERK phosphorylation.Conclusion:SPRY4 expression can be induced by ectopic FGFR2 activation in PHCC.SPRY4 arrested the cell cycle at G1 phase and suppressed FGFR-induced proliferation and migration by inhibiting ERK phosphorylation,indicating that SPRY4 may be a potential therapeutic target in PHCC.