Prognostic Significances Of PD-L1 Expression And Its Tumor-intrinsic Functions In Hypopharyngeal Squamous Cell Carcinoma

Part Ⅰ The clinical and prognostic significances of PD-L1 expression in hypopharyngeal squamous cell carcinomaObjectiveHypopharyngeal squamous cell carcinoma(HSCC)is a kind of squamous cell carcinoma of head and neck(squamous cell carcinoma)with high degree of malignancy.Despite the tremendous progresses that have been made in the comprehensive treatments,However,the prognosis for patients with advanced HSCC remains poor,with a 5-year survival rate of less than 40%.Distal metastasis is a key factor in HSCC patients,especially the distant metastasis which patients developed after treatments,which makes it a disastrous disease.So it is urgent to investigate the new mechanisms by which HSCC develops and find novel biomarkers for predicting the prognosis.Programmed death-ligand 1 is a coinhibitory molecule which belongs to the B7 family.The interaction between PD-L1 and its receptor,PD-1,plays a pivotal role in driving immune suppression and contributes to tumor immune evasion in the context of tumor progression.PD-L1 is rarely expressed on normal tissues but up-regulated expression of PD-L1 is detected on many human cancers.The levels of PD-L1 expression on tumors are significantly associated with aggressive biological behaviors and poor prognosis.However,the contributions of PD-L1 to tumor pathogenicity,especially in HSCC,are incompletely understood.MethodsClinicopathological features of ninety-five patients with hypopharyngeal squamous cell carcinoma in our department during January 2005 and November 2014 were collected,including the gender,age,T stage,N stage,clinical stage,differentiation,distant metastasis and patient prognosis.PD-L1 expression in HSCC tumor tissues and paired adjacent hypopharyngeal mucosal tissues was detected using immunohistochemistry assay.The association between the expression of PD-L1 in tumor tissues and the clinicopathological features of patients was analyzed by chi-square test or Fisher’s exact probability method.The correlation between PD-L1 expression and prognosis was analyzed by Kaplan-Meier method,and the survival curve was drawn.The log-rank test was used for comparative analysis of survival indicators.The influence of each variable on survival was examined by the Cox-multivariate regression analysis.ResultsPD-L1 expression was detected in HSCC samples but we found no positive expression in matched normal hypopharyngeal mucosal tissues.PD-L1 expression was significantly correlated with distal metastasis,N stage and clinical stage(χ2=20.850,14.317,7.878,all P values were below 0.05).Survival analysis showed that compared with patients with negative expression of PD-L1,patients with positive expression of PD-L1 had a poor prognosis(χ2=37.01 and P value below 0.001).Multivariate Cox regression analysis showed that positive expression of PD-L1 was an independent risk factor associated with the prognosis of HSCC patients(P=0.002,HR=2.703,95%CI:1.455-5.022).ConclusionThe expression of PD-L1 was closely correlated with adverse clinicopathological features and worse prognosis of HSCC patients.PD-L1 expression could act as a biomarker for predicting the progression and prognosis of patient with HSCC.Part Ⅱ The tumor-intrinsic functions of PD-L1 in the regulation of invasion and metastasis of FaDu cellsObjectiveMost studies associated with PD-L1 signals in cancers focused on its immunomodulatory effects on T cells.Nevertheless,recent work showed that tumor-expressed PD-L1 prevented tumor apoptosis,regulated cell growth and autophagy,and regulated tumor glucose metabolism in sarcomas.Thus,PD-L1 has important tumor-intrinsic functions which are still needed to be further elucidated.Epithelial-mesenchymal transition(EMT),a process in which epithelial cells interact with surrounding mesenchymal cells and obtain mesenchymal phenotypes,is important for invasion and metastasis of carcinoma.Several authors have demonstrated that EMT had connections with PD-L1 expression in cancers.However,to our acknowledge,the analysis regarding the relationship between PD-L1 expression and EMT in HSCC has not yet been reported.MethodsIn vitro assays including cell viability assays,migration assays,invasion assays as well as western blot assays were performed to illuminate the biological functions and underlying molecular mechanisms of PD-L1 in HSCC development.The expression of PD-L1 was down-or up-regulated by siRNA or overexpression plasmid.The CCK-8 assays were conducted to assess the ability of proliferation of FaDu cells.The invasion and migration of FaDu cells were measured by Transwell assay.Western blot(WB)was used to determine the expression levels of related EMT-related proteins(including E-cadherin,Vimentin and N-cadherin)after the changes of PD-L1 expression in FaDu cells.In order to evaluate the effect of PD-L1 expression on the AKT-mTOR signaling pathway,the expression of p-AKT and p-mTOR was detected by western blot.The AKT inhibitor was adopted to treat FaDu cells with PD-L1 overexpression and the changes of the expression of EMT-related proteins was assessed by western blot.ResultsFunctional experiments showed that when the expression of PD-L1 was down-regulated,the ability of proliferation,migration and invasion was inhibited significantly.Conversely,the corresponding abilities were markedly promoted when the expression of PD-L1 was up-regulated.Mechanistically,investigations demonstrated that PD-L1 could promote the epithelial-mesenchymal transition of FaDu cells.Meanwhile,PD-L1 knockdown inhibited,while PD-L1 overexpression activated the Akt-mTOR signaling pathway in FaDu cells.The EMT induced by PD-L1 overexpression could be reversed by the Akt inhibitor.ConclusionPD-L1 can promote the proliferation,migration and invasion of FaDu cells and consequently enhance the aggressiveness.Moreover,PD-L1 induces EMT through AKT-mTOR signaling pathway.These suggest that PD-L1 has important tumor-intrinsic functions independent of its immunopathogenic effects.