CDH1 Promotes Metastasis Of Hypopharyngeal Carcinoma By Regulating MMP-9 Expression And The Mechanism

Background and objectiveHypopharyngeal squamous cell carcinoma(HSCC)is a malignant tumor with the worst prognosis among head and neck tumors,which seriously threatens human life and health.The overall 5-year survival rate is 25%-40%.Despite the continuous improvement in the diagnosis and treatment of hypopharyngeal cancer,including surgical resection of the tumor,radiotherapy,chemotherapy and targeted therapy,the mortality of patients with hypopharyngeal cancer is still high.Invasion and metastasis are the main causes of failure of hypopharyngeal cancer treatment and death of patients.Therefore,it is of great significance to understand the molecular mechanisms of hypopharyngeal cancer invasion and metastasis fully for early diagnosis of hypopharyngeal cancer metastasis and improvement of survival rate.The change of intercellular adhesion is a key link in the process of tumor metastasis.Low epithelial cadherin(CDH1/E-cadherin)level is closely related to tumor metastasis and poor prognosis of patients.In addition,in malignant tumors,certain factors may overexpress MMP-9 and lead to matrix degradation,which is conducive to tumor invasion and metastasis.Studies have shown that decreased expression of CDH1 in head and neck squamous cell carcinoma and non-small cell lung cancer can transcriptionally activate epidermal growth factor receptor(EGFR)and ultimately promote cell proliferation.EGFR is a type of receptor tyrosine kinase(RTK)that phosphorylates signal transducer and activator transcription 3(STAT3).Once STAT3 is activated,it regulates the transcription of a number of target genes(such as MMP-9)that affect tumor cell proliferation,migration,and invasion.However,the precise molecular mechanism by which CDH1 down-regulation affects MMP-9 expression in hypopharyngeal cancer is unclear now.In conclusion,hypopharyngeal cancer metastasis is closely related to prognosis.Once metastasis occurs,5-year survival rate is lower and prognosis is worse.Therefore,it is of great clinical significance to study the mechanism of metastasis of hypopharyngeal cancer.Loss of CDH1 function can affect the regulation of extracellular matrix(ECM),which may be related to phosphorylation of STAT3.As an enzyme regulating ECM,MMP-9 plays a key role in tumor invasion,metastasis and angiogenesis.At present,the mechanism of CDH1/STAT3/MMP-9 signaling pathway in hypopharyngeal cancer is unclear.Therefore,it is necessary to study this molecular mechanism in depth.Research methods and grouping1.The paired tumor and adjacent normal tissue samples were obtained from 109 patients who were proceeded with radical surgery without adjuvant pre-operative treatment and were pathologically and/or cytologically diagnosed with HSCC in Shandong Provincial ENT Hospital Affiliated to Shandong University between June 2010 and December 2014 with a minimum 3 years follow-up.The enrolled 109 patients consisted of 31 postoperative lung metastases cases.Samples of matched tumor and adjacent normal tissue were obtained.The main research contents included patients' age,gender,smoking and alcohol history,duration of disease course,clinical manifestations,tumor site,pathological grade,tumor stage,whether there was postoperative distant metastasis,and follow-up of patients' survival time,cause of death and other survival conditions.2.Immunohistochemistry:The protein levels of CDH1 and MMP-9 were detected by rabbit monoclonal antibodies CDH1 and MMP-9.3.Cell line is human hypopharyngeal carcinoma FaDu cell line.4.The culture of FaDu cells includes FaDu cell resuscitation,subculture,inoculation,and cryopreservation.5.The western blot assay included extraction of total cell proteins,determination of protein concentration by Coomassie bright blue method and western blot method.6.FaDu cells siRNA transfection:FaDu cells were silenced until the coverage of FaDu cells reached 60-80%.The siRNA control group was targeted at the sequence of 5'-UUCUCCGAACGUGUCACGUTT-3',the siRNA group of STAT3 was targeted at the sequence of 5'-GCAAGAUUCAGACCCUCAATT-3',and CDH1 siRNA#1 and#2 duplexes targeted the sequences 5'-CAGACAAAGACCAGGACTA-3' and 5'-GCACGUACACAGCCCUAAU-3',respectively.The siRNA group of MMP-9 targeted 5'-CAUCACCUAUUGGAUCCAA-3'.7.Sulforhodamine B(SRB)method:FaDu cells were incubated with siRNA for 24 hours and then re-seeded in 96-well plates for 48 hours.Dyeing,rinsing,and measuring absorbance values.8.Cell migration assay:After 48 hours of siRNA transfection of FaDu cells,the cells were cultured in transwell chambers for another 24 h,followed by fixation,staining,cleaning and counting.9.Cell invasion assay:After transfecting with different combinations of CDH1,STAT3,and MMP-9 siRNAs for 48 h,FaDu cells were re-cultured in transwell chambers coated with Matrigel for another 36 h,followed by fixation,staining,cleaning and counting.10.CCK-8 assay:FaDu cells were transfected with siRNA for 48 h and then the cells were re-seeded and incubated in 96-well plates for 48 h,add 10 ?l of CCK-8 reagent solution to each well.After 2 hours of incubation,absorbance values were measured.Study contents and results1.Correlation between CDH1 expression and hypopharyngeal carcinoma metastasis and clinical prognosis1.1 CDH1 is associated with lung metastasis of hypopharyngeal squamous cell carcinoma(HSCC)In order to explore the role of CDH1 in the development of HSCC,we measured the expression level of CDH1 in cancer and adjacent normal tissues of 109 patients by immunohistochemical(IHC)staining,31 of which had postoperative lung metastasis of hypopharyngeal cancer.The negative expression of CDH1 in cancer tissues(16/109)was higher than that in adjacent normal tissues(5/109)(P=0.012,Mann-Whitney U-test).CDH1 negative expression was significantly increased in HSCC tissues with lung metastasis(11/31)(P<0.01,?2 test)compared with that in HSCC tissues without lung metastasis(5/78).The differences between negative expression and moderate or strong expression of CDH1 were significant(P<0.05,X2 test).Spearman's rank correlation coefficient was used to test the correlation between CDH1 expression and HSCC lung metastasis,and it was found that there was a significantly negative correlation between them(r=-0.43 1,P<0.01).1.2 CDH1 expression is correlated with clinical prognosis of HSCCUnivariate and multivariate analysis revealed that CDH1 down-regulation was associated with shorter OS and DFS(P<0.05,log-rank analysis),and the lymph node metastase were also connected with poor OS(P<0.05,log-rank analysis).CDH1 negative expression was significantly associated with HSCC patients relapse(P=0.001,?2 test).The correlation between CDH1 expression and the grade of the tumor was statistically significant(P=0.025,?2 test).1.3 Expression of CDH1 and MMP-9 in HSCCWe used immunohistochemistry staining to compare the expression of CDH1 and MMP-9 in a range of adjacent non-tumor tissue and HSCC tissue.Besides prominent localization to the membrane of tumor cells,CDH]also exhibited approximate 60%cytoplasmic expression.However,there was only basically membrane expression of CDH1 in the adjacent non-tumor cells.CDH1 expression was significantly different between the normal tissues(104/109)and the tumor groups(93/109)(P=0.012,Mann-Whitney U test).Intriguingly,we found that CDH1 expression was reduced,while MMP-9 expression was correspondingly increased in the same HSCC sample.2.Effect of loss of CDH1 regulates MMP-9 expression and the mechanism2.1 Loss of CDH1 induces MMP-9 up-regulation in FaDu cellsWe suppressed CDH1 expression in FaDu cells with siRNA treatment and detected the level of MMP-9 by western blot assay.The FaDu cells with decreased CDH1 expression showed elevated expression of MMP-9 than the control cells.2.2 Loss of CDH1 up-regulates MMP-9 expression via STAT3 phosphorylationOur data revealed that depletion of CDH1 elicited a significant increase in p-STAT3.And suppression of STAT3 elicited down-regulation of MMP-9.We inhibited STAT3 and CDH1 expression by siRNA transfection and discovered that blocking STAT3 attenuated the induction of MMP-9 that was caused by CDH1 down-regulation.In parallel,we suppressed STAT3 activation with AG490 treatment in CDH1-siRNA transfected FaDu cells,and found that pharmacological inhibition of STAT3 also alleviated the induction of MMP-9 triggered by CDH1 loss.2.3 Down-regulated CDH1 expression promoted FaDu cell proliferation through STAT3 and MMP-9The growth and proliferation of FaDu cells treated with CDH1 siRNA were enhanced while an obvious decline was observed in the ones that transfected with STAT3 or MMP-9 siRNAs.Moreover,additional STAT3 or MMP-9 knockdown apparently attenuated the enhanced cell grow and proliferation induced by CDH1 silence.2.4 STAT3 and MMP-9 are responsible for the enhanced metastasis induced by CDH1 reduction in FaDu cellsCompared with the control group,the migration and invasion capacity of CDH1-silenced FaDu cells were significantly enhanced.On the contrary,the migration and invasion ability of FaDu cells treated with STAT3 or MMP-9 siRNA decreased significantly.Furthermore,strengthened migration and invasion competences were observed in the cells interfered with CDH1 alone compared with the cells co-transfected CDH1 siRNA either with STAT3 or MMP-9 siRNA.ConclusionsCDH1 is a powerful indicator for HSCC lung metastasis and prognosis,further mechanical investigation reveals that the loss of CDH1 elevated MMP-9 expression via activation of STAT3 and promotes proliferation,invasion as well as metastasis of FaDu cells,which may contribute to the malignant development and lung metastases of HSCC.Taking together,our study may provide novel therapeutic targets for metastatic HSCC,and thereby develop potential avenues for further clinical prognosis and treatment.