WNT5A Promotes Tumor Invasion And Metastasis By Enhancing The HDAC7-SNAIL Signaling Pathway In Esophageal Squamous Cell Carcinoma

Background:Esophageal carcinoma is most frequently diagnosed cancer of the upper digestive tract,including esophageal squamous cell cancer(ESCC)and adenocarcinoma,and the incidence of ESCC is high in China.In recent years,with the application of targeted therapy and immunotherapy,the diagnosis and treatment of ESCC have made some progress.However,existing drugs are rarely used in ESCC and have side effects.Thus,it is necessary to further explore the regulatory mechanism of ESCC and find new targets.Discovered in 1990,WNT5 A is a typical non-canonical signaling pathway ligand of the WNT family,which plays an important role in tumors.Its targeted drugs have been developed and entered clinical trials.Studies have shown that WNT5 A has carcinogenic effects on lung carcinoma,gastric carcinoma,and breast carcinoma,but its role and molecular mechanism in ESCC remain unclear.Therefore,this research is going to reveal the WNT5 A expression in ESCC and its relationship with the survival prognosis of patients using ESCC patient’s tissues.In the meantime,we will reveal the role of WNT5 A in the invasion and metastasis of ESCC and its molecular mechanism in vivo and in vitro.Thus,we will supply new insights and theoretical basis for exploring the pathogenesis and targeted therapy of ESCC.Objectives:1.To clarify the expression of WNT5 A in ESCC tissues and its relationship with the survival prognosis of ESCC patients.2.To clarify the role of WNT5 A in the process of epithelial mesenchymal transformation(EMT),invasion,and migration of ESCC cells.3.To clarify the role of histone deacetylase 7(HDAC7)in the process of EMT,invasion,and migration of ESCC cells.4.To clarify the mediating role of HDAC7 in WNT5 A promoting ESCC progression.5.To investigate the expression levels of HDAC7 and EMT-related transcription factor SNAIL in ESCC tissues and their impacts on survival prognosis,and comprehensively analyze the significance of WNT5 A,HDAC7,and SNAIL expression on survival prognosis of ESCC patients.Methods:1.Immunohistochemistry(IHC)was applied to determine the expression levels of WNT5 A in 145 pairs of ESCC tissues and corresponding adjacent tissues.On this basis,the relationships between the expression level of WNT5 A in ESCC tissues and the clinical characteristics or prognosis of patients were analyzed.2.ESCC cell lines with WNT5 A overexpressing were constructed and the abilities of migration,invasion,and metastasis in these cells were determined via scratch assay,transwell assays,and nude mouse lung metastasis assay.Furthermore,Western blot was applied to detect the expression levels of EMT-related proteins.3.The expression levels of SNAIL in ESCC cancer tissues and adjacent tissues were analyzed as described in Method 1,and then the relationships between the expression level of SNAIL in ESCC and the clinical characteristics or survival prognosis of patients were analyzed.After that,the effects of WNT5 A and SNAIL expression levels on the survival prognosis of ESCC patients were comprehensively evaluated.4.Western blot was applied to determine the effect of WNT5 A overexpression on the expression levels of HDAC family in ESCC cells,and HDAC7 was screened out as the downstream molecule of WNT5 A.Lentivirus infection was used to construct ESCC cell lines with overexpression or knockdown of HDAC7,and the role of HDAC7 in ESCC cell invasion,metastasis,and EMT was determined according to Method 2 in this part.5.Based on the ESCC cell line with overexpression of WNT5 A,the expression of HDAC7 was down-regulated by lentivirus infection or inhibitors,and the mediating role of HDAC7 in WNT5 A promoting the progression of ESCC was determined by wound healing assay,transwell assays,and Western blot assay.6.The expression levels of HDAC7 in ESCC cancer tissues and adjacent tissues were analyzed as described in Method 1,and the relationships between the expression level of HDAC7 in ESCC tissues and the clinical characteristics or survival prognosis of patients were analyzed on this basis.At last,the effects of WNT5 A,HDAC7,and SNAIL expression levels on the survival prognosis of ESCC patients were comprehensively evaluated.Results:1.IHC analysis showed that the WNT5 A expression in ESCC specimens was notably higher than that in the paired specimens;WNT5A expression level was positively correlated to TNM stage and N stage of ESCC patients.Survival analysis suggested that high WNT5 A expression was linked to poor prognosis in ESCC patients.COX analysis indicated that WNT5 A was an independent prognostic factor for ESCC patients.2.The migratory,invasive,and metastatic potentials were enhanced by WNT5 A in ESCC cells via wound healing assay,transwell assays,and nude mouse lung metastasis assay.Western blot results showed that WNT5 A up-regulated the expression of SNAIL,a transcription factor promoting EMT,and down-regulated the expression of E-cadherin,a characteristic epithelial marker.3.IHC analysis showed that SNAIL expression level in ESCC tissues was notably higher than that in corresponding adjacent tissues.SNAIL expression level was positively correlated to TNM stage and N stage of ESCC patients.Survival analysis suggested that high SNAIL expression was associated with poor survival prognosis in ESCC patients.COX analysis indicated that WNT5 A was an independent prognostic factor for ESCC patients.Further analysis showed that there was a correlation between WNT5 A and HDAC7 expression in ESCC patients.ESCC patients with high co-expression of WNT5 A and SNAIL suffered poorest prognosis,while ESCC patients with low co-expressions of both were correlated to the best prognosis.4.Western blot analysis showed that overexpression of WNT5 A in ESCC cells could specifically up-regulate HDAC7 expression.The migratory,invasive,and metastatic potentials were enhanced by HDAC7 in ESCC cells via scratch assay,transwell assays,and nude mouse lung metastasis assay.Western blot results showed that HDAC7up-regulated SNAIL expression and down-regulated E-cadherin expression,thus promoting EMT in ESCC cells.These results are highly consistent with the role of WNT5 A in ESCC as described in Result 2.5.Wound healing assay,transwell assays,and Western blot assay confirmed that HDAC7 knockdown partially inhibited the effects of WNT5 A on activating SNAIL/EMT and enhancing cell migration and invasion.HDAC7 inhibitors SAHA and TMP269 also dose-dependently inhibited the effects of WNT5 A described above.6.IHC analysis showed that the WNT5 A expression in ESCC specimens was notably higher than that in the paired specimens;and HDAC7 expression was positively correlated to TNM stage and N stage of ESCC patients.Survival analysis suggested that high HDAC7 expression was associated with poor survival outcome in ESCC patients.Further comprehensive analysis showed that the expression level of HDAC7 was correlated with WNT5 A or SNAIL in ESCC patients.And patients with low expressions of WNT5A/HDAC7 and HDAC7/SNAIL had the best survival prognosis.Conclusions:This study clarified that WNT5 A promoted EMT,invasion,and metastasis by upregulating HDAC7-SNAIL signaling pathway in ESCC cells in vivo and in vitro.Then,it was verified by clinical study that the expression levels of WNT5 A,HDAC7,and SNAIL were increased in ESCC tissues,and their high expression levels were correlated to the poor survival prognosis of ESCC patients.Comprehensive analysis showed that their expression levels were correlated to each other,and patients with high expressions of WNT5A/SNAIL had the best survival prognosis,while patients with low expressions of WNT5A/SNAIL,WNT5A/HDAC7,and HDAC7/SNAIL had the worst survival prognosis.These results revealed the mechanism of WNT5 A promoting the invasion and metastasis of ESCC by enhancing the HDAC7-SNAIL signaling pathway,offering new insights for understanding the pathogenesis of ESCC and setting the stage for the novel therapeutic strategy of targeting the related molecules in ESCC.