| BackgroundCholangiocarcinoma constitutes one of the most malignant carcinoma of liver tumors,with an increasing incident and high motality.Surgical resection,chemotherapy and radiotherapy are the main treatment therapeutic for cholangiocarcinoma.However,the median survival of CCA remains less than two years and the prognosis is poor.At present,there is only one FDA approved targeted therapy,Pemigatinib is available for patients with advanced Cholangiocarcinoma.New therapeutic targets are urgent.Maternal embryo leucine zipper kinase(MELK)plays a critical role in various malignant tumors,including CCA.Previous studies have identified that MELK is high expressed in human CCA tissue,but the biological function and mechanism has not been well characterized.In addition,we found that small molecular inhibition of MELK,OTSSP167,suppressed the proliferation of CCA in vito and in vivo.These results suggest that targeting MELK could help to prevent CCA,thus,further studies to investigate whether MELK inhibition attenuate CCA are warranted.Methods1.MELK expression in TCGA database and CAA patient tissues1.1 To evaluate whether MELK expression is associated with CCA,we analyzed the publicly available TCGA database.We found that human MELK mRNA expression was significantly up-regulated in human CCA tissue compared to normal liver;1.2 We further examined MELK expression by immunostaining in human CCA tissues.We found that MELK protein expression was markedly higher in tumor tissues(n=56)compared to para-tumor tissues(n=16)tissue(P<0.01).The relationships between the expression of MELK and clinicopathological parameters were also analyzed.2.The biological function of MELK in Cholangiocarcinoma2.1 To assess MELK expression in human CCA celllines,HuCCT-1,HuH28,HCCC9810 and TFK-1,we performed qPCR and Western blot to dectect MELK mRNA and protein expression.We found significantly higher MELK mRNA and protein expression in HuCCT-1 and TFK-1;2.2 In addition,we found that knockdown of MELK expression with small interfering RNA(siRNA)(siMELK)in HuCCT-1 or TFK-1 significantly attenuated the proliferation,invasivness,metastasis and induced apoptosis of CCA.We further confirmed that MELK short hairpin RNA(shRNA)knockdown attenuated the progression of CCA in vitro.3.The mechanism of MELK in Cholangiocarcinoma3.1 To further investigate the potencial signaling pathway of MELK in CCA,we analyzed the National Cancer for Biotechnology Information Gene Expression Omnibus(GEO)database,and we found that MELK is correlated with FOXM1;3.2 we performed qPCR and Western blot to detect FOXM1 changes in CCA celllines following MELK siRNA knockdown;3.3 Co-Immunoprecipitation(CO-IP)validates the interaction between MELK and FOXM1.4.Small molecular inhibition of MELK,OTSSP167,attenuates the proliferation of Cholangiocarcinoma4.1 MELK is reported as a selective and potent oral inhibitor of MELK and has been in clinical evaluation for leukemia and breast cancer.We utilized OTSSP167 to investigate whether MELK antagonism has anti-tumor activity.T he semi inhibitory concentration of Cholangiocarcinoma cell line was detected by IC50 curve experiment;4.2 We found that OTSSP167 treatment inhibited cell proliferation in a dose dependent manner;4.3 We further tested the effects of the MELK inhibitor OTSSP167 on subcutaneous nude mouse models.Furthermore,we observed that OTSSP167 had not effects on total bilirubin(TBil),Cholyglycine(CG)and aspartate transaminase(AST).we found that at the dose of 5mg/kg OTSSP167 dramatically inhibited tumor proliferation compared to the sham group.OTSSP167 acts as a tumor suppressor in subcutaneous nude mouse model of Cholangiocarcinoma.Results1.MELK is up-regulated in CCA tissues compared to non-tumor tissues(p<0.01),and is correlated to tumor differeciation and serum AFP level;2.MELK promotes the proliferation,invasion,metastasis of Cholangiocarcinoma and induces apoptosis in vitro;3.MELK promotes CCA progression via modulating FOXM1 signaling pathway;4.MELK antagonism plays an anti-tumor role in vitro and in vivo.ConclusionIt can be concluded that MELK is a key molecule for CCA.MELK is highly expressed in CCA tissues and is correlated to tumor differeciation and serum AFP level.Down-regulated MELK in CCA celllines further attenuate the proliferation,invasivness,metastasis and induced apoptosis of CCA by modulating FOXM1 signaling pathway.MELK-targeted treatment with a MELK inhibitor resulted in a potent anti-tumor effect.Therefore,MELK is an attractive novel therapeutic target for CCA. |
