Dasabuvir Suppresses Esophageal Squamous Cell Carcinoma Growth As A Novel ROCK1 Inhibitor

Esophageal cancer is a common gastrointestinal tumor originating from the esophageal epithelium.According to the World Health Organization,the prevalence and mortality of esophageal cancer in China ranks fifth in the world,the incidence and mortality of esophageal cancer accounts for about 55% of the world,and ninety percent of these cases are esophageal squamous cell carcinoma.Esophageal squamous cell carcinoma(ESCC)is characterized by high recurrence rate and low 5-year survival rate.At present,we mainly use surgical treatment,chemotherapy,radiotherapy and other comprehensive treatment methods,but the treatment effect is poor.Due to the lack of effective recurrence prevention methods after initial treatment.Therefore,there is a urgent need for effective chemopreventive drugs that can extend the survival rate of patients.Through screening of FDA-approved drugs,dasabuvir was found to suppress ESCC proliferation.Dasabuvir is a drug used in combination with other antivirals for hepatitis C treatment.This study found that dasabuvir is a novel inhibitor of Rho-associated protein kinase 1(ROCK1).Dasabuvir inhibited the growth and clone formation of the KYSE150 and KYSE450 ESCC cell lines in a time-and concentration-dependent manner,inducing cell stagnation at the G0/G1 cell cycle phase.The antitumor activity was validated in vivo using a patient-derived xenograft tumor model in mice.Dasabuvir can significantly inhibit the growth of esophageal squamous cell carcinoma in vivo.Through phosphoproteomics and proteomics analysis,combined with computer molecular simulation and pull-down assay,we found that dasabuvir acted on ROCK1,thereby inhibiting ERK1 and ERK2 activation by ROCK1 and down-regulating CDK4 and cyclin D1 expression.These results suggest that,as a ROCK1 inhibitor,dasabuvir inhibits the growth of esophageal squamous cell carcinoma in vivo and in vitro,and acts on the cell cycle through the ROCK1/ERK signaling pathway,thereby inhibiting the progression of esophageal squamous cell carcinoma.Methods:1.To explore the inhibitory effect of dasabuvir on ESCC in vitro Cytotoxicity assay was used to screen the inhibitory effect of FDA-approved drugs on ESCC cells and to verify the toxic effect of dasabuvir on ESCC cells.The inhibitory effect of dasabuvir on the proliferation of ESCC cell lines was detected by cell proliferation assay.Anchorage-independent cell growth assay was used to detect the inhibitory effect of dasabuvir on the anchorage-independent growth ability of ESCC cell lines.Anchorage-dependent cell growth assay was used to detect the inhibitory effect of dasabuvir on the anchorage-dependent growth ability of ESCC cell lines.2.To explore the inhibitory mechanism of dasabuvir against esophageal squamous cell carcinoma Phosphoproteomics was used to study the changes of corresponding phosphorylation sites in ESCC cells induced by dasabuvir;western blotting was used to verify the down-regulated phosphorylation sites in phosphoproteomics analysis;i GPS1.0 was used to predict the kinases which were dasabuvir acted;Swiss Target Prediction was used to predict the targets which were dasabuvir acted;the TCGA database was used to investigate the association between the kinase and downstream targets of dasabuvir in ESCC.3.To explore whether ROCK1 is the direct target of dasabuvir The molecular simulation docking experiment was used to analyze whether dasabuvir could combine with ROCK1;in vivo and in vitro pull down assays were used to analyze the combination of dasabuvir and ROCK1;in vitro kinase assay was used to verify the activation effect of ROCK1 on ERK1 and ERK2 and the inhibition of ERK1 and ERK2 phosphorylation by dasabuvir on ROCK1.4.To explore how dasabuvir affects the proliferation of ESCC Immunofluorescence assay was used to detect the inhibitory effect of dasabuvir on p-ERK1/2 and its effect on the interaction between ROCK1 and p-ERK1/2;GSEA enrichment analysis was used to further analyse phosphoproteomics and proteomics,so as to study how dasabuvir affects the signal pathways.5.To explore the effect of ROCK1 on the proliferation of ESCC cells and the effect of ROCK1 on the inhibitory effect of dasabuvir on ESCC ROCK1 knockdown cells were constructed by transfection with sh ROCK1 plasmid,then ROCK1 knockdown cells were used to detect the effect of ROCK1 knockdown on the proliferation and anchorage-dependent growth ability of ESCC cell lines and the effect of dasabuvir on the inhibition of ROCK1 knockdown cells.6.To explore the inhibitory effect of dasabuvir on ESCC in vivo PDX models were established to detect the inhibitory effect of dasabuvir on ESCC in vivo;immunohistochemical staining was used to investigate the changes of Ki67 and p-ERK1/2 in ESCC tissues after dasabuvir treatment.Results:1.Dasabuvir inhibits ESCC cell proliferation in vitro Dasabuvir has significant cytotoxic effect on ESCC cell lines,and has a time and dose dependent inhibitory effect on proliferation of ESCC cell lines;anchorage-independent cell growth assay shows dasabuvir can inhibit anchorage-independent cell growth in a dose dependent manner;anchorage-dependent cell growth assay shows dasabuvir can inhibit anchorage-dependent cell growth in a dose dependent manner.2.Dasabuvir plays a role in the ROCK1/ERK signaling pathway Through enrichment and analysis of phosphoproteomics,we found that dasabuvir downregulated p-MAPK1;western blotting results confirmed the down-regulation of p-ERK1/2 altered by dasabuvir;we found that dasabuvir down-regulated the kinase activity of ROCK1 by enrichment of upstream kinases at phosphorylation sites altered by dasabuvir;Swiss Target Prediction predicted dasabuvir was most likely to act on ROCK1;the TCGA database showed a significant association between ROCK1 and MAPK1 in ESCC.3.Dasabuvir binds and inhibits ROCK1 The molecular simulation docking experiment showed that dasabuvir could bind directly to ROCK1;in vitro and in vivo pull-down assays showed that dasabuvir binded to ROCK1 in vitro and in vivo;in vitro kinase assay showed that ROCK1 activated ERK1 and ERK2,and dasabuvir inhibited the phosphorylation of ERK1 and ERK2 by inhibiting ROCK1.4.Dasabuvir induces G0-G1 cell cycle arrest of KYSE150 and KYSE450 cells through the ROCK1/ERK signal pathway Immunofluorescence showed that dasabuvir inhibited p-ERK1/2 and the interaction between ROCK1 and p-ERK1/2;GSEA enrichment of phosphoproteomics and proteomics showed that dasabuvir significantly affected the cell cycle pathway;cell cycle experiments showed that dasabuvir blocked the cell cycle in the G0/G1 phase.5.Dasabuvir exerts cancer cell inhibition through ROCK1ROCK1 knockdown significantly inhibited the proliferation and anchor-dependent growth of ESCC cell lines,and weakened the inhibitory effect of dasabuvir on ESCC cells.6.Dasabuvir reduces ESCC PDX xenograft growth in vivo Animal experiments showed that dasabuvir had a significant inhibitory effect on ESCC in vivo.Immunohistochemical staining results showed that Ki67 and p-ERK1/2were significantly down-regulated in ESCC after dasabuvir treatment.Conclusion:1.This study provides the first evidence that dasabuvir is a novel inhibitor of ROCK1.2.Dasabuvir inhibits ESCC in vivo and in vitro.3.Dasabuvir inhibits the kinase activity of ROCK1 by binding to ROCK1.4.Dasabuvir acts on the cell cycle through the ROCK1/ERK signaling pathway and blocks the ESCC cells in the G0/G1 phase,thus blocking the progression of ESCC.