Clinical And Experimental Study Of Fibroblast Growth Factor Receptor 4 In Colorectal Cancer

BackgroundColorectal cancer(CRC)is the second most common cause of cancer death in the Western world,and the incidence of colorectal cancer in the Asia-Pacific region is increasing.Surgical resection of primary tumors and adjuvant chemotherapy improved patient survival,but nearly half of the patients eventually died of local recurrence and metastasis.At present,targeted therapy has become an important treatment for various malignant tumors(including CRC).Bevacizumab(targeted to VEGF)and cetuximab(targeted to EGFR)are two drugs approved for use in the treatment of progressive and/or metastatic colorectal cancer.However,molecular targeted therapy also has the problem of multi-targeted combination therapy.Therefore,we need to find new and effective targets for the treatment of colorectal cancer.The fibroblast growth factor receptor(FGFRs)is a polygene family,an immunoglobulin gene superfamily member,which is distributed in a variety of cells on the membrane protein.FGFRs are members of FGFR1,FGFR2,FGFR3 and FGFR4.FGFR4 is a class of transmembrane tyrosine kinase receptors with autophosphorylation activity that plays a very important role in embryonic development,tissue repair and angiogenesis.However,there are relatively few studies on FGFR4 in malignant tumors.FGFR4 is overexpressed in common malignancies such as gastric cancer,pancreatic cancer,breast cancer,and renal cell cancer.Our previous findings showed that knockdown of FGFR4 expression inhibited the proliferation of gastric cancer cells and promoted apoptosis.The apoptosis rates were obviously increased in GC cells treated with the single and combination of 5-Fu and PD173074(an inhibitor of FGFR).Blu9931 is the first potent and selective inhibitor of FGFR4,and it was reported that it inhibited the proliferation of hepatocellular carcinoma cells.However,it has not been reported in colorectal cancer.Epithelial-mesenchymal transition(EMT)is considered as a key event in tumor metastasis and plays an important role in the progression of colorectal cancer.During EMT,cells lose the epithelial phenotype(such as E-Cadherin)and obtain a mesenchymal phenotype(such as Vimentin).In hepatoma cells,FGF19 promoted EMT progression by promoting FGFR4 expression.However,the role of FGFR4 in EMT in colorectal cancer is not well understood.To explore the mechanism of FGFR4 in the tumorigenesis,development and EMT progression of colorectal cancer,we examined the expression of FGFR4 in colorectal cancer tissues and adjacent normal colorectal tissues by immunohistochemistry(IHC)staining.The possible correlation between FGFR4 protein expression and clinicopathological features and prognosis of CRC patients was evaluated.FGFR4was silenced in colorectal cancer cell line SW620 by constructing a small hairpin RNA(shRNA)lentivirus.A series of in vitro functional assays were performed to investigate the effect of FGFR4 silencing on colorectal cancer.To explore the effects of single agent treatments and combination of Blu9931 and 5-Fluorouracil(5-Fu)on the biological characteristics of colorectal cancer cells and its mechanism.Find a new target for the treatment of colorectal cancer.Part One The role of fibroblast growth factor 4 in the growth and metastasis of colorectal cancerBackground and projectiveColorectal cancer(CRC)is one of the most common gastrointestinal cancers in the world and is the second most common cause of cancer death in the Western world.The incidence of colorectal cancer is on the increase in China.Effective resection of primary tumor and adjuvant chemotherapy can improve the survival rate of patients.And tumor recurrence and metastasis seriously affect patient survival.At present,targeted therapy has become an important treatment for various malignancies,including colorectal cancer.The emergence of targeted drugs has opened a new avenue for the treatment of colorectal cance.FGFRs is a multi-gene family that belongs to the immunoglobulin gene superfamily and is a membrane protein distributed on many kinds of cells.Members of the FGFR family are FGFR1,FGFR2,FGFR3 and FGFR4.FGFR4,a type of transmembrane tyrosine kinase receptor with autophosphorylation activity,plays a very important role in embryonic development,tissue repair and angiogenesis.Studies have shown that FGFR4 is highly expressed in a variety of malignant tumors,and our previous experiments show that silencing of FGFR4 can inhibit gastric cancer cell growth and promote apoptosis.However,there is relatively little research on FGFR4 in colorectal.This study aimed to detect the expression of FGFR4 in human colorectal cancer by immunohistochemistry and its clinical significance.A series of in vitro functional assays were performed to investigate the effect of FGFR4 silencing on colorectal cancer cells.To investigate whether the effect of FGFR4 on EMT progress through the Wnt/?-catenin pathway.MethodIn this study,we purchased human colorectal cancer tissue microarray,including100 colorectal cancer tissues and 60 adjacent tissues.Immunohistochemistry was used to detect the expression of FGFR4 in colorectal cancer and normal tissues.Then,correlation analysis and survival analysis were performed.Small hairpin RNA(shRNA)was used to silence the expression of FGFR4,which were detected by immunofluorescence and western blot.Then a series of in vitro functional experiments were performed including proliferation assay,invasion assay,scratch assay,apoptosis detection and cell-cycle evaliation.Western blot was used to detect the expression of apoptosis-related,cell cycle-related and epithelial-mesenchymal transition-related proteins.The effect of FGFR4 on EMT progression was explored by inhibiting the Wnt/?-catenin signaling pathway using the?-catenin inhibitor XAV-939.Results:FGFR4 is overexpressed in CRC tissues compared to normal tissues.Patients with positive FGFR4 expression had a lower 5-year survival than patients with negative FGFR4 expression(64%vs 74%).FGFR4 silencing reduced the proliferation of colorectal cancer cells,inhibited cell invasion,arrested cells in S phase,and promoted apoptosis.FGFR4 silencing reversed EMT progression and FGFR4 silencing further reversed EMT progression in the presence of XAV939(a?-catenin inhibitor).Conclusion:Our data suggest that FGFR4 may be associated with the prognosis of patients with colorectal cancer.In vitro functional tests show that FGFR4 is an effective therapeutic target for colorectal cancer.FGFR4 may affect EMT process through Wnt/?-catenin pathway.Chapter Two Combination of FGFR4 inhibitor Blu9931 and 5-?uorouracil effects on the biological characteristics of colorectal cancer cellsBackground and projectiveCurrently the treatment of colorectal cancer is improving.In addition to surgery,radiotherapy,chemotherapy three kinds of the main treatment,the molecular targeted therapy also plays an important role.Targeted drugs open up a new avenue for the treatment of colorectal cancer.Some of the results of this study suggest that FGFR4may be a new therapeutic target.Blu9931 is the first potent and selective inhibitor of FGFR4,and it has been reported that it can inhibit the proliferation of hepatocellular carcinoma cells.However,the role of Blu9931 in colorectal cancer cells is unknown.The purpose of this part of the study is to investigate the effects of single agent treatments and combination of Blu9931 and 5-Fluorouracil(5-Fu)on the biological characteristics of colorectal cancer cells and its mechanism.Find a new target for the treatment of colorectal cancer.Method:Detection of FGFR4 expression in five common colorectal cancer cell lines using PCR and Western blot.Two FGFR4-overexpressing colorectal cancer cell lines were selected for subsequent experiments.HCT116 and SW620 cells was treated by the single and combination of 5-Fu and Blu9931.Then,a series of function assays were performed,including viability assay,proliferation assay,apoptosis detection and cell-cycle evaluation by flow cytometry.Western blot detected apoptosis and cell cycle related proteins through different treatments.Western blot was used to detect the expression of EMT-related proteins under different treatment conditions.Result:FGFR4 protein expression was stronger in HCT116 and SW620,while in LS47,DLD1 and SW116 were weaker.Reverse transcription PCR results showed that FGFR4 mRNA expression was significantly higher in HCT116 and SW620 than in the other three colorectal cancer cell lines.The quantitative analysis results by real-time PCR confirmed that FGFR4 mRNA expression in HCT116 and SW620were much higher than the other colorectal cancer lines(P<0.05).The concentration of 5-Fu and Blu9931 increased,the cell viability of HCT116 and SW620 cells progressively decreased.Proliferation experiments showed that the absorbance of the single and combination of 5-Fu and Blu9931 was significantly lower than that of the control group(P<0.05).Compared with the single of 5-Fu and Blu9931,the proliferation ability significantly decreased in HCT116 and SW620 cells treated with the combination of 5-Fu and Blu9931(P<0.05).In both HCT116 cells and SW620cells,the apoptotic rate of each treatment group increased,the difference was statistically significant(one-way ANOVA;P<0.05).But in HCT116 cells,the apoptosis rate of 5-Fu plus Blu9931 was higher than that in the single of 5-Fu and Blu9931(one-way ANOVA;P<0.05).However,there was no statistically significant difference among 5-Fu,Blu9931 and 5-Fu plus Blu9931 in SW620 cells.(one-way ANOVA;P<0.05).Compared to the control group,cleaved caspase-3 expression in HCT116 and SW620 cells was obviously increased in the single and combination of5-Fu and Blu9931.When compared with the control group,expression of STAT3 was markedly increased in the single agent 5-Fu group.The HCT116 cells treated with the single and combined of Blu9931 and 5-Fu in S stage evidently increased while those in G0/G1 stage remarkably decreased as respect to mock group(one-way ANOVA;P<0.05).But in sw620 cells,the G2/M stage cells in the three treatment groups were much less than those in mock group.The SW620 cells treated with the single Blu9931 in S stage significantly increased while 5-Fu group and 5-Fu plus Blu9931group remarkably increased in G0/G1 stage.When compared to the control group,P27~kip1ip1 expression was obviously increased in the single and combination of 5-Fu and Blu9931 while cyclinD1 expression was prominently weakened.In HCT116 cells,expression of E-cadherin was increased in the single and combination of 5-Fu and Blu9931 compared with control group while vimentin expression was weakened.We can see the same result in SW620 cells.Conclusion:Inhibiting the activity of FGFR4 may be one of main mechanisms of Blu9931 to inhibit the proliferation of colorectal cancer cells,increase the apoptosis rate,prevent the cell cycle and inhibit EMT.The combination of 5-Fu and Blu9931 has a synergistic effect in reducing colorectal cancer cell proliferation and preventing cell cycle.The results of this study further demonstrate that the first specific inhibitor of FGFR4,Blu9931,can be a novel target drug for the treatment of colorectal cancer.