FGFR/VEGFR Dual Inhibitor: A Potential Targeted Therapy In Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma(HCC)ranks in the forefront of global morbidity and mortality.Only a small number of patients with early diagnosis of liver cancer can receive surgical treatment.For advanced liver cancer,sorafenib is the first to be approved for systemic treatment,and the median overall survival(m OS)was improved by nearly 3 months.The second-line treatment drugs,including regorafenib,ramucirumab,and cabozantinib have failed to show significant efficacy,or the efficacy is not as good as sorafenib in the clinical study of first-line treatment,and the first-line treatment drug is limited to sorafenib for a long time.This situation was not changed until the treatment models of lenvatinib,atezolizumab combined with bevacizumab were approved.While the prognosis of patients with HCC is still poor,which may be caused by the heterogeneity of liver cancer,there is still a need to improve the treatment of patients.Advanced HCC is hypervascular.The VEGF family is the most important angiogenic factor,and VEGFR1-3 is its important receptor.After combining these two components,they can promote angiogenesis and vascular permeability,supply tumor nutrition and oxygen,promote tumor metastasis and lead to immunosuppression through activating Ras-RAF-MAPK,PI3K-AKT,PLCγand STATs signalling pathways.The main mechanism of sorafenib used to treat HCC is mainly inhibiting the VEGF/VEGFR signalling pathway,but its median time to progress(m TTP)was only 5.5 months.Studies have found that FGFs and FGFRs levels increase after anti-VEGF treatment.Alternative activation of the FGF/FGFR signalling pathway accounts for resistance to anti-VEGF/VEGFR therapy.The activation of FGF/FGFR signalling pathway can promote the proliferation,survival,migration,and invasion of cancer cells through triggering Ras-RAF-MAPK,PI3K-AKT,PLCγ,and STATs.In addition,FGF is an effective angiogenesis factor in hepatocellular carcinoma.The abnormal overexpression and activation of FGF/FGFR pathway also promote the proliferation,differentiation,and metastasis of HCC,and is associated with poor prognosis.In other words,the FGF/FGFR pathway may be a target for HCC treatment.However,the current drugs used to inhibit the FGF/FGFR pathway in the treatment of liver cancer have not yet achieved a good clinical efficacy,which may be due to a large number of crosstalk and redundancy in the signalling pathway of liver cancer.Studies have shown that the FGF/FGFR and VEGF/VEGFR pathways can synergistically promote angiogenesis and immune tolerance,and promote tumor cells.Therefore,the combination therapy designed to block FGF/FGFR and VEGF/VEGFR signalling cascades may be an alternative therapy for patients with HCC.Therefore,the FGF/FGFR and VEGF/VEGFR signalling pathways have roles in tumors,tumor blood vessels,and tumor microenvironment.This study explored the relationship between FGFR1-4 and VEGFR2 expression and the prognosis of HCC,and evaluated the effect of blocking FGFR/VEGFR in the treatment of HCC.This article studied the anti-tumor activity and mechanism of a novel small molecule compound,FGFR/VEGFR dual inhibitor WXFL11100033,hoping for providing a new treatment strategy for the targeted therapy of HCC.Materials and Methods:The information of FGFR1-4 m RNA and clinical data of patients with HCC were retrieved through the TCGA database,and the RNA-seq data and clinical data were merged using R language.ROC curve was used to determine the best cut-off value of FGFR1-4 m RNA expression related to OS.The optimal cut-off point was used as the standard to divide the included patients into FGFR1-4 m RNA high or low expression groups.The clinical data of patients were listed according to the grouping,and the differences in clinical characteristics were counted.According to the patients’survival time and survival status,the Kaplan-Meier method was used to draw the survival curve.Simultaneously,the log-rank test was used to calculate the statistical difference,and the cox analysis to calculate the HR and its 95%CI.ROC curve was used to get the best cut-off value of FGFR1-4 and VEGFR2 protein expression related to prognosis.The expression of FGFR1-4 protein in different HCC cell lines was detected by western-blot,and CCK8 was used to detect the inhibitory effect of WXFL11100033 on HCC cell lines.The plate cloning experiment was used to detect the proliferation inhibitory effect of WXFL11100033 on HUH-7 and Bel-7074 cell lines.The pro-apoptotic effects of WXFL11100033 on HUH-7 and Bel-7074 were detected by flow cytometry(FCM)after annexin-V/PI double staining,and western-blot was applied to detect the expression of apoptosis-related proteins.What’s more,the changes in the cell cycle were analyzed by FCM.The effect of WXFLl11100033 on the migration of HCC cell lines was observed by scratch test,and the effect of WXFLl11100033 on the invasion of HCC cell lines was detected by transwell test.Western-blot was used to detect the mechanism of WXFL11100033on tumor cells.CCK8 detected the effect of WXFL11100033 on the proliferation activity of HUVEC,the scratch test detected the impact on HUVEC migration,the transwell test detected the impact on HUVEC invasion,and the western-blot detected the mechanism of action.HUH-7 and Bel-7074 subcutaneous tumor models were established,and the treatment started when the tumor grew to about 100mm~3.During the treatment,the mice’s general condition was observed,and their body weight was recorded every three days.At the end of the treatment,the mice,their tumors,and their major organs were weighed.FCM was used to the immune microenvironment in tumor tissues,and the orbital blood was taken for biochemical detection.Ki-67,CD31,and Tunel were detected after paraffin embedding of the tumor,and the organs were stained by H&E.Results:A total of 370 HCC patients with FGFR1-4 m RNA data were retrieved from the TCGA database.Among them,6 patients had no follow-up information,5 patients had unknown G stage,2 patients had unknown T stage,and 24 patients had unknown TNM stage.33 patients without complete information were excluded,and the remaining 337 patients were used for analysis.The information included FGFR1-4 m RNA expression,age,gender,tumor grade,TNM stage,OS,and survival status.The results showed the m RNA expression levels of FGFR3 and FGFR4 in HCC were significantly higher than those of FGFR1 and FGFR2.The cut-off values of FGFR1-4 m RNA were 0.29,0.35,15.19,and 30.84,respectively.We found that the high m RNA expressions of FGFR3 and FGFR4 were associated with poor differentiation of tumor.High expressions of FGFR4 m RNA were associated with short OS.Immunohistochemical staining of FGFR1-4 and VEGFR2 was performed in 90patients with HCC.According to the relationship between the protein expressions of FGFR1-4 and VEGFR2 and disease-free survival(DFS)status,the cut-off values were 9,1.75,9,6.75,and 5,respectively;cox univariate analysis showed that high protein expressions of FGFR2 and VEGFR2 were associated with DFS shortening in HCC patients,while cox multivariate analysis suggested that the high expression of FGFR2 protein shortened DFS.According to the relationship between the protein expressions of FGFR1-4 and VEGFR2 and OS status,the cut-off values were 7,1.75,7,5,and 5,respectively;the results suggested that the high protein expressions of FGFR1,FGFR2,FGFR4,and VEGFR2 were associated with short OS in cox univariate analysis.However,only high expressions of FGFR2 and FGFR4 protein shortened OS in cox multivariate analysis.WXFK11100033,a dual FGFR/VEGFR inhibitor,was synthesized by Wu Xi App Tec.Its molecular weight was 493.5g/mol.The values of IC50 for FGFR2,FGFR4,and VEGFR2 were 2n M,39n M,and 2n M,respectively.The maximum tolerated dose(MTD)in mice was greater than 100mg/Kg for 7 days of continuous dosing.We first tested the effect of WXFL11100033 on HCC cell lines in vitro.Western-blot analysis showed that HCC cell lines express FGFR1-4 protein,among which HUH-7 and Bel-7074 were representatives,with high expressions of FGFR1-4 and relatively low expressions of FGFR1-4,respectively.CCK8 experiment showed that WXFL11100033 had an inhibitory effect on HCC cell lines.The IC50 of WXFL11100033 fluctuated from 2.36μM to 20.86μM at 72 hours.The IC50 of HUH-7 was 6.15μM,and that of Bel-7074 was 12.13μM.HUH-7 and Bel-7074cell lines were selected for subsequent experiments.WXFL11100033 can inhibit the proliferation and promote the apoptosis of the two cell lines.The inhibition of proliferation of HUH-7 was better than that of Bel-7074,and the promotion of apoptosis of Bel-7074 was better than that of HUH-7.After treatment with WXFL11100033,apoptotic proteins’expression was consistent with the phenotypic changes in these two cell lines.After treated by WXFL11100033,the cell cycle of HUH-7 and Bel-7074 cells was arrested in G0/G1 phase.At the same time,we observed that WXFL11100033 could inhibit the migration and invasion of HUH-7and Bel-7074 cell lines.Western-blot also showed that the inhibitory effect of WXFL11100033 on tumor cells was achieved by inhibiting Ras-RAF-MAPK,PI3K-AKT,PLCγ,and STATs pathways.WXFL11100033 inhibited p-FGFR2/4,p-m TOR,p-ERK,and p-STAT3 in both cells,but the total protein content of these proteins did not change.As an FGFR/VEGF dual inhibitor,WXFL11100033 can not only directly act on tumor cells but also HUVEC.CCK8 experiment found that after treatment by WXFL11100033 for 48 hours,the IC50 for inhibiting the proliferation of HUVEC stimulated by VEGF165 and b FGF was 0.91μM and 0.24μM,respectively.In the HUVEC special medium containing various growth factors,the IC50 was 2.74μM.At the same time,WXFL11100033 also can inhibit migration,invasion,and tube formation of HUVEC.To further verify the effect of WXFL11100033,we conducted in vivo experiments,which proved that the tumor inhibition rate of WXFL11100033 on HUH-7 tumor model was 95%,and 83%of inhibition rate was observerd in Bel-7074 tumor model.In vivo,WXFL11100033 can achieve the purpose of inhibiting tumor growth by improving the tumor microenvironment,inhibiting tumor proliferation,inhibiting tumor angiogenesis,promoting apoptosis and necrosis.FCM was performed on the tumor tissue to detect immune cells.We found that the proportion of MDSCs(CD45+CD11b+Gr-1+)in the tumor tissue was much lower in the WXFL11100033 group in the HUH-7 subcutaneous tumor model.At the same time,the proportion of DC cells(CD45+CD11b+CD11C+)was significantly higher,and the proportion of M2 type macrophages(CD45+CD11b+CD206+)was significantly lower in the WXFL11100033 group in the Bel-7074 subcutaneous tumor model.WXFL11100033 can inhibit angiogenesis in both tumor models.The inhibitory effects of WXFL11100033 on the proliferation of the two tumor models are not the same.In the HUH-7 model,the proportion of Ki-67 in the WXFL11100033 group decreased by nearly 70%compared with the control group,which only slightly decreased in the Bel-7074 model.The proportion of apoptosis and necrosis in the Bel-7074 model was significantly higher than that of the control group.We also conducted a toxicological evaluation of WXFL11100033,and we did not observe side effects,including lack of energy,skin ulcers,peeling,bleeding,vomiting,and diarrhea.The the mice’weight in the WXFL11100033 treatment group had a downward trend,but the difference had no statistical significance.The treatment of WXFL11100033 did not obviously change the organ/body weight coefficient of major organs.The biochemical blood test found that the AST of the WXFL11100033 treatment group was higher,indicating that liver function was damaged,but in a controllable range.The main organs of Bel-7074 subcutaneous tumor model mice were stained with H&E,and no noticeable pathological changes of heart,liver,spleen,lung,and kidney between groups were found.Conclusion:High expressions of FGFR3 and FGFR4 m RNA were correlated with poor differentiation of hepatocellular carcinoma.High expressions of FGFR4 m RNA indicated poor prognosis.High protein expression of FGFR2 was associated with shorter DFS,and high protein expressions of FGFR2 and FGFR4 were associated with shorter OS.WXFL11100033 has a direct inhibitory effect on HCC cell lines and HUVEC.WXFL11100033 can regulate immunity and improve the tumor microenvironment.WXFL11100033 has significant anti-tumor activity on subcutaneous transplantation tumor models of HCC.WXFL11100033 is well tolerated.It is feasible to simultaneously inhibit the FGF/FGFR and VEGF/VEGFR signalling pathways in HCC.The high expression of FGFR1-4 protein can predict the therapeutic effect.The FGFR/VEGFR dual inhibitor WXFL11100033 is a very promising targeted drug.