Synergistic Inhibitory Effect And Molecular Mechanism Of Combined Targeting PLK1 And Aurora-A On Esophageal Squamous Cell Carcinoma/Inhibitory Effect Of ICG-001 On The Proliferation Of Esophageal Squamous Cell Carcinoma And Underlying Mechanism

Esophageal squamous cell carcinoma(ESCC)is one of the most common malignant tumors in China,with poor prognosis and high mortality.For many years,the five-year survival rate for advanced patients has hovered around 15%to 25%.At present,there is a lack of effective drugs for the treatment of ESCC,so it is urgent to develop new effective drugs and therapeutic strategies.Our previous research results suggest that Polo-likekinasel(PLK1)is an important functional gene and a potential therapeutic target in ESCC.Since the efficacy of targeting PLK1 alone in the treatment of malignant tumors have not achieved satisfactory in most clinical trials,we explored PLK1-inhibition-based combination therapy strategies.In this study,we screened a compound library containing 346 small molecular inhibitors and found that the combination of small molecular inhibitor BI 6727 of PLK1 and inhibitor TC-S 7010 of protein kinase Aurora-A had a significant synergistic inhibitory effect on the proliferation of ESCC cells.Based on the analysis of the dataset GSE23400 in the public gene expression database GEO,it was found that the mRNA expression levels of PLK1 and Aurora-A in ESCC tissues were significantly higher than those in adjacent normal tissues(n=53,P<0.0001).The mRNA expression levels of PLK1 and Aurora-A were positively correlated in ESCC(n=53,P<0.001,r=0.5052).Subsequently,we investigated the effects of the combination of the two drugs on the malignant phenotype of ESCC cells,and found that the combination of BI 6727 and TC-S 7010 synergistically inhibited the proliferative activity and colony formation ability of ESCC cell lines KYSE450 and KYSE510.In addition,combination treatment synergistically promoted G2/M cycle arrest and induced cell apoptosis in these two cell lines(both P values were less than 0.05).Similarly,BI 6727 in combination with MLN 8237,another inhibitor of Aurora-A,also showed a synergistic inhibitory effect on proliferation and survival of KYSE450 and KYSE510 cells.The results at the molecular level showed that compared with monotherapy,combination therapy significantly enhanced the expression of cell cycle(CyclinB1 and p-HistoneH3)and apoptosis-related molecular markers(Cleaved-Caspase3 and Cleaved-PARP).Additionally,the expression levels of other molecules related to cell proliferation and survival,including YAP,c-MYC,Bcl-2,Bcl-xL,were also markerdly reduced after combination therapy.Furthermore,we constructed a nude mouse transplanted tumor model with KYSE450 cells for tumor inhibition experiments,and the results showed that the inhibition rate of BI 6727 combined with TC-S 7010 on the growth of transplanted tumor was 82.7%.Compared with the monotherapy group,the combination therapy showed synergistic anti-tumor effect(Deviation value was 0.25).In summary,the combination of small molecular inhibitor BI 6727 of PLK1 and inhibitor TC-S 7010 or MLN 8237 of Aurora-A synergistically inhibited the malignant proliferation and survival of ESCC cells,suggesting that the co-targeting of PLK1 and Aurora-A may be a promising therapeutic strategy for ESCC.Previous studies have shown that abnormal activation of Wnt/β-catenin signaling pathway is closely related to the occurrence,development and prognosis of ESCC,suggesting that it may be a potential target for ESCC treatment.ICG-001 is a small molecule inhibitor of Wnt/β-catenin signaling pathway.The results of in vitro and in vivo studies have shown that ICG-001 can significantly inhibit the proliferation of various malignant tumor cells,but the effect of ICG-001 on ESCC cells has not been reported.In this study,the ESCC cell lines KYSE410 and KYSE450 were treated with small molecule inhibitor ICG-001 of 50 and 100 μmol/L,and the cells treated with solvent DMSO were used as control to detect the cell proliferation viability,colony formation ability,cell cycle distribution and apoptosis.The results showed that ICG-001 treatment significantly inhibited the proliferation and colony formation ability of ESCC cell lines KYSE410 and KYSE450,and significantly induced G0/G1 phase arrest(P<0.01).Correspondingly,Western blot results showed that the expression of cyclin-dependent kinase CDK4 and CDK6 was obviously decreased,while the negative regulator of cell cycle p27 Kip1 was markedly up-regulated upon ICG-001 treatment.The expression of downstream target molecules SKP2 and Survivin of β-catenin/TCF was significantly decreased,and the expression of transcription factor TCF4 which could bind to β-catenin was also significantly decreased(P<0.01).At the same time,qRT-PCR analysis showed that the mRNA expression of SKP2,Survivin and TCF4 was significantly decreased,while the mRNA expression of p27 Kipl was significantly up-regulated in ICG-001-treated ESCC cells(all P values were less than 0.01).Collectively,the results of this study showed that ICG-001 significantly inhibited the proliferation viability and colony formation ability of ESCC cell line KYSE410 and KYSE450,and induced G0/G1 phase arrest,which may be attributed to the suppression ofβ-catenin/TCF4 transcriptional activity and the expression of its target molecules Survivin、SKP2 and TCF4,and the up-regulation of p27 Kip1.Our findings suggest that ICG-001 may be a potential therapeutic drug for the treatment of ESCC,and it may improve the clinical efficacy alone or in combination with other therapeutic methods.