Notch1 Serves As A Prognostic Factor And Regulates Metastasis Via Regulating EGFR Expression In Hypopharyngeal Squamous Cell Carcinoma

Part Ⅰ Notch1 serves as a prognostic factor for distant metastasis and outcome in hypopharyngeal squamous cell carcinomaObjectiveHypopharyngeal squamous cell carcinoma(HSCC)had the worst outcome among head and neck cancers.The highest rate of subsequent distant metastases was observed among patients with HSCC.Distant metastasis is the most important cause of death in these patients.At present,there are no molecular markers to predict distant metastasis.Notchl signaling pathway is involved in the carcinogenesis and progression of many kinds of tumors,and the deregulation of Notchl expression is significantly related to the prognosis of many tumors.However,very little was known with regard to the relationship between Notch1 expression and the clinicopathological characteristics of HSCC.The purpose of this study was to investigate the expression of Notchl in HSCC and its correlation with distant metastasis and prognosis of HSCC.MethodsThe clinical data of 71 patients with HSCC admitted to our department from June 2010 to September 2014 were retrospectively analyzed,including age,sex,smoking and alcohol history,family history,site of onset,T stage,N stage,M stage,clinical stage,survival time after operation,pathological type,differentiation degree,size of tumor,proportion of metastatic positive lymph nodes,etc.A total of Seventy-one pairs of hypopharyngeal cancer tissue samples and adjacent noncancerous formalin-fixed paraffin embedded tissue specimens were analyzed by immunohistochemistry.The expressions of Notchl and EGFR in hypopharyngeal carcinoma and adjacent noncancerous tissues were detected by immunohistochemistry.The correlation between Notchl expression level and clinical and pathological parameters such as age,sex,T/N/M stage and differentiation degree was statistically analyzed.The correlation between Notchl expression and clinical prognosis was also analyzed.ResultsWe found that the expression of Notchl and EGFR in HSCC tissues was upregulated compared with those in the adjacent noncancerous tissues.Further clinicopathological characteristics analysis revealed that the expression of Notchl was positively correlated with distant metastasis(P=0.003).The expression level of Notchl was also positively correlated with tumor differentiation(P =0.031),the expression level of Notchl in the middle and low differentiation group was higher than that in the high differentiation group.But there was no significant difference between Notchl expression and T/N stages.The overall 3-years and 5-years survival rates of all 71 HSCC patients were 54.93%and 35.43%,respectively.Kaplan-Meier survival analysis showed that the high expression of Notchl is an independent prognostic factor for a poor overall survival in patients with HSCC,we found that the patients with high-expression of Notchl showed markedly shorter overall survival than that of low-expression group and there was statistical significance(P = 0.015,χ2= 10.403).The 3-years overall survival rates of Notchl low-expression group and high-expression group were 78.26%and 43.75%,respectively.In Notchl high-expression group,84.38%of HSCC patients died.ConclusionsTaken together,the expression of Notchl and EGFR in hypopharyngeal carcinoma tissues is upregulated.Overexpression of Notchl is closely related to the risk of distant metastasis in patients with HSCC,we found a positively cross-linked role of Notchl signaling in the outcome of HSCC.Notchl may be used as a novel valuable prognostic marker and potential therapeutic target for the treatment of HSCC patients.Part Ⅱ Notchl regulates metastasis via regulating YBX1-EGFR pathway in hypopharyngeal squamous cell carcinomaObjectiveHSCC is the most malignant tumor in head and neck cancers,it is often found in the advanced stage of the tumor.Many HSCC patients show cervical lymph node metastasis or distant metastasis.Lymph node metastasis and distant metastasis are independent factors affecting the prognosis of HSCC.However,the mechanism of metastasis of HSCC remains unclear.Previous studies have shown that Notchl is involved in the metastasis of cancer cells through a variety of mechanisms.The purpose of this study was to investigate the effect of down-regulation of Notchl expression on the metastasis of FaDu cells,and to further study the potential mechanism of Notchl regulating the metastasis of FaDu cells.MethodsHuman HSCC cell line FaDu was used in our study.siRNA-Notchl(si-Notchl)treated FaDu cells as the experimental group,and siRNA-negative control(siRNA-NC)treated FaDu cells as the control group.To verify the efficiency of knockdown on Notchl expression,we examined the relative protein and mRNA levels of Notchl expression in siRNA-NC and si-Notchl FaDu cells by Western blot and RT-PCR analyses.After confirming that Notchl expression level was down-regulated,the invasion and migration ability of FaDu cells were detected by Transwell chamber test,and the changes of YBX1 and EGFR expression at the level of mRNA and protein were detected by Real time PCR and Western blot,and p-YBX1 expression at the level of protein were also detected.At the same time,Cell Counting Kit-8(CCK-8)assay was used to investigate the influence of Notchl expression on the growth of FaDu cells.To further examine whether the YBX1 was involved in the Notchl-induced EGFR regulation in FaDu cells,PCDNA 3.1-YBX1 overexpression plasmid and siRNA were co-transfected into FaDu cells.Western blot analysis was used to detect the changes of EGFR,YBX1 and p-YBXl expression levels after co-transfection of siNotchl and PCDNA 3.1-YBX1 plasmid.Transwell chamber was also used to detect the effect of YBX1 overexpression on invasiveness of FaDu cells after si-Notchl treatment.ResultsThe results of Real time-PCR and Western blot showed that the expression of Notchl in si-Notchl group FaDu cells was decreased compared with siRNA-NC group,suggesting effectively suppression of Notchl expression by si-Notchl(P<0.05).After down-regulation of Notchl expression,the migration and invasion ability of FaDu cells in si-Notchl group was significantly inhibited than that in siRNA-NC group(P<0.05).Real time-PCR and Western blot results showed that the expression of EGFR was decreeased at the level of mRNA and protein.Interestingly,we further found that Notchl knockdown also decreased the expression of YBX1,which is a upstream transcription factor of EGFR.CCK-8 assay results showed that siNotchl treatment for 24h and 48h did not reduce the proliferation of FaDu cells compared with control group(P=0.93 vs 0.22,respectively).Moreover,the upregulation of YBX1 reverses the suppression of Notchl on EGFR(P<0.05).Following the cells were co-transfected of siNotchl and PCDNA 3.1-YBX1 overexpression plasmids,we detected the expressions of EGFR,YBX1 and p-YBX1 were elevated than those in control group.Furthermore,forced overexpression of YBX1 induced the invasion of FaDu cells.ConclusionsNotchl can regulate the invasion and migration ability of FaDu cells,and its potential mechanism may be related to the regulation of EGFR expression mediated by Notchl through YBX1.Notchl may be an important factor involved in the metastasis of HSCC and provide a new target for clinical treatment.