The Research Of The Expression Patterns And Biological Functions In Hypopharyngeal Squamous Cell Carcinoma

Part 1:Overexpression of S100A4 Predicts Migration,Invasion,and Poor Prognosis of Hypopharyngeal Squamous Cell CarcinomaBackground and objectiveHypopharyngeal squamous cell carcinoma(HSCC)is among the most lethal tumors encountered in the head and neck.The presence of metastasis in the cervical lymph nodes is considered the most important prognostic factor for HSCC:ipsilateral cervical nodal metastasis in 60%-80%of patients and contralateral occult nodal metastasis in up to 40%of cases.In addition,cervical lymph node metastasis is recognized as an important predictive indicator of early regional recurrence and distant metastasis,which also negatively impact the prognosis for HSCC patients.Despite several lines of evidence identifying multiple metastasis-related signaling pathways or mediators in HSCC,the mechanism underlying aggressiveness of HSCC remains elusive,particularly for HSCC.Therefore,it is essential to identify therapeutic targets with improved efficacy of inhibiting tumor invasiveness and metastasis in HSCC.S100 A4 belongs to S100 calcium binding protein family,and the link between S100A4 and tumor dissemination has been causally implicated using experimental animal models.It has been well established that S100A4 is able to exert its metastasis-promoting effects through multiple signaling pathways,such as epithelial-mesenchymal transition(EMT)-pathway,Wnt/β-catenin pathway,RAGE/NF-κB pathway,ERBB2 signaling pathways.Moreover,S100A4 can enhance pro-metastatic activities by modifying the tumor microenvironment through promoting angiogenesis,regulating matrix metalloproteinases(MMPs)activity,mediating interactions between tumor and stroma cells,and stimulating immune cell recruitment.Taken together,S100A4 widely participates in the biological processes that contribute to cancer metastasis through the modulation of both primary tumor and pre-metastatic niche.As a member of the S100 calcium binding protein family,the roles of S100A4 in promoting metastatic phenotype have been reported in a variety of human malignancies.Moreover,the effect of S100A4 on prognosis has been observed in many types of solid tumors including colorectal cancer,breast cancer,and bladder cancer.Accumulating evidence has shown that S100A4 is a promising biomarker for cancer diagnosis and metastasis,and a potential therapeutic target for cancer treatment,including buccal and tongue cancer.However,the expression status and biological function of S100A4 in HSCC remain to be verified.Therefore,this study aims to 1)explore the expression levels of S100A4 in HSCC tumors versus adjacent normal tissues,and its association with clinicopathological parameters and clinical prognosis of HSCC patients and 2)confirm the role of S100A4 in the metastatic process of HSCC FaDu cell line in vitro.MethodsImmunohistochemistry(IHC)was used to detect S100A4 expression in HSCC tumors and adjacent normal tissues.Nuclear and cytoplasmic S100A4-staining in HSCC tumor cells was scored as separate variables.The staining intensity and percentage of S100A4-positve cells were assessed and scored semi-quantitatively.Differences between HSCC tumors and adjacent normal tissues were determined by the Wilcoxon signed ranks test.Chi-square test or Fihser exact test was conducted to explore the correlation of S100A expression and clinicopathological parameters.Kaplan-Meier analysis was used to analyze the association between S100A 4 expression and prognosis.Univariate and multivaruate analysis was performed utilizing the Cox proportional hazards regression model.In vitro loss-of-function experiments were performed in HSCC FaDu cells.CCK-8 proliferation assay,scratch migration assay,Transwell migration and invasion assay,apoptosis and cell cycle assay were performed to explore the effect of S100A4 knockdown on the phenotype of FaDu cells.Differences between the experiment group and control group were determined by the Mann-Whitney U test.P<0.05 was considered to be statistically significant.Results1.The expression of S100A4 was significantly elevated in HSCC tumors compared with their corresponding normal tissues(P<0.0001).When we scored the intensity and frequency of S100A4 expression in the tumor/normal tissues,we counted the S100A4 signal within the carcinoma cells in tumor tissues or the epithelial cells in the normal tissues,but not in the stromal cells.The IHC results showed that Fifty-seven tumor samples exhibited strong expression of S100A4 in HSCC tumors.Amongst these slides,all the samples showed cytoplasmic expression and 20(28%of all the samples)had nuclear expression.2.The results of chi-square test or Fisher exact test showed that high cytoplasmic expression of S100A4 was positively correlated with cervical lymph node metastasis and poor pathological differentiation(P = 0.007 and 0.028,respectively).In addition,high expression of nuclear S100A4 was positively correlated with advanced clinical stage,cervical lymph node metastasis,poor pathological differentiation(P = 0.046,0.017,and 0.042,respectively).3.Kaplan-Meier analysis showed that the overall survival in high expression of cytoplasmic S100A4 was lower than low expression group;however,the difference was not statistically significant(P = 0.071).Whereas there was no significant difference in overall survival between high and low cytoplasmic expression groups,we found that the overall postoperative survival time of HSCC patients with low nuclear S100A4 expression was significantly longer than those with high nuclear S100A4 level(P<0.001).The Cox proportional hazard model suggested that nuclear S100A4 expression was an independent predictor of HSCC prognosis.4.To further explore the prognostic value of nuclear S100A4 expression in HSCC patients,the enrolled HSCC patients were stratified into two groups based on their clinical stages,i.e.early(clinical stages Ⅰ and Ⅱ)and advanced(clinical stages III and IV).In advanced patients,overall survival of cases with high expression of nuclear S100A4 was significantly poorer than those with low nuclear S100A4 level(P= 0.001).However,no obvious impact of nuclear S100A4 expression was identified on postoperative survival time in early-stage patients(P = 0.101).5.In vitro experiments showed that S100A4 expression was significantly inhibited in FaDu cells with the transfection siRNA targeting S100A4 in comparison with the control one(P = 0.0101).cell motility decreased significantly after S100A4 knockdown(P<0.0001).Transwell assay for migration also showed that the cell numbers traversing the membrane were also significantly reduced upon S100A4 depletion(P = 0.0032).Transwell assay for invasion revealed that invasive abilities of FaDu cells were also significantly impaired in depletion of S100A4(P = 0.0171).6.Cell-cycle analysis revealed that compared with the control,transfection with S100A4 siRNA caused increased accumulation of FaDu cells at G0/G1 phase(P<0.01).However,neither the proliferation nor apoptosis in FaDu cells was obviously affected in absence of S100A4(P = 0.0987 and 0.5948,respectively).ConclusionThe expression of S100A4 is upregulated in HSCC tumors and this upregulation is positively correlated with cervical lymph node metastasis of this malignancy.The metastasis-promoting role of S100A4 is further validated in the HSCC FaDu cell line,indicating that S100A4 is a potential therapeutic target for HSCC.Furthermore,this study suggests that nuclear S100A4 expression could be considered as a prognostic biomarker for HSCC.Our study suggests that S100A4 might be used as a prognostic biomarker and potential treatment target for HSCC in the future.Part 2:Aberrant Expression of PAFAH1B3 Associates with Poor Prognosis and Affects Proliferation and Aggressiveness in Hypopharyngeal Squamous Cell CarcinomaBackground and objectiveRegional lymph node metastasis is recognized as an important predictive indicator of early regional recurrence and and poor prognosis for HSCC patient.it has become clear that many of the signaling pathways that were affected by genetic mutations and the tumor microenvironment had a profound effect on tumor metabolism,making this topic one of the most intense areas of research in cancer biology.The altered metabolism allowed tumor cells to sustain higher proliferative rate and resist some cell death signals.The metabolic alterations created a phenotype that was essential for tumor cell growth and survival,altering the flux along key metabolic pathways.Some of the mechanisms used by tumors to bring about thsese changes included the altered expression,mutation,and post-transcriptional inactivation of an enzyme of the substitution of a different enzyme isoform.Therefore,the development of treatment that target tumor metabolism is receiving renewed attention,with several potential drugs targeting metabolic pathways currently in clinical trials.Platelet-activating factor acetylhydrolase 1B3(PAFAH1B3)is the one of the catalytic subunits of PAFAH.PAFAH1B3 is reported to be among the 50 most commonly upregulated metabolic enzymes across>1000 primary human tumors across 19 types of cancers,and is dysregulated broadly across many types of cancers.PAFAH 1B3 can maintain tumor cell aggressiveness via regulating tumor-suppressing lipids.In addition,PAFAH 1B3 participates in multiple signaling pathways,including PAF-signaling pathways,Wnt pathways,Reelin pathways.Furthermore,PAFAH1B3 is identified as a target for combination therapy with TKIs in BCR-ABL1+ BCP-ALL.Despite the critical role that PAFAH 1B3 plays in tumor progression and malignancy,it remains obscure what the clinical value of PAFAH 1B3 is in HSCC and whether it makes a difference to the biological phenotype of hypopharyngeal cancer cells.Here,we first explored the expression patterns of PAFAH 1B3 in HSCC tumor tissues and adjacent non-tumor samples,and the correlation of PAFAH1B3 expression with overall survival and clinicopathological parameters based on the IHC results.Next,in vitro loss-of-function assays were performed to explore impacts of PAFAH 1B3 on biological phenotype of the human HSCC FaDu cell line.MethodsImmunohistochemistry(IHC)was used to detect PAFAH IB 3 expression in HSCC tumor tissues(n=83)and adjacent normal tissues(n=44).The staining intensity and percentage of PAFAH1B3-positve cells were assessed and scored semi-quantitatively.Differences between HSCC tumor tissues and adjacent normal tissues(n=44 pairs)were determined by the Wilcoxon signed ranks test.Chi-square test or Fishser exact test was conducted to explore the correlation of PAFAH1B3 expression and clinicopathological parameters.Kaplan-Meier analysis and Cox model analysis were used to analyze the association between PAFAH 1B3 expression and prognosis.Loss-of-function assays were conducted to explore the biological function of PAFAH1B3 in HSCC FaDu cells.PAFAH1B3 expression was significantly knocked down in FaDu cells transfected with PAFAH1B3-specific siRNA1 and siRNA2 compared with the negative control(NC)siRNA.CCK-8 proliferation assay,wound healing assay,Transwell migration and invasion assay,apoptosis and cell cycle assay were performed to explore the effect of PAFAH1B3 knockdown on the phenotype of FaDu cells.Differences between the experiment group and control group were determined by the student’s t test.P<0.05 was considered to be statistically significant.Results1.PAFAH1B3 was mainly observed in the cytoplasm of hypopharyngeal squamous cell carcinoma cells.PAFAH1B3 was overly expressed in 62.7%(52/83)of the HSCC tumor specimens which was markedly higher than adjacent non-tumor samples(22.7%,10/44)(P<0.0001).Moreover,we statistically compared the PAFAH1B3 staining in the 44 paired tumor and non-tumor specimens,confirming that PAFAH1B3 was significantly overexpressed in HSCC tumor tissues(P<0.0001).2.The results of chi-square test or Fisher exact test showed that high expression of PAFAH1B3 was significantly correlated with advanced clinical stage and cervical lymph node metastasis(P=0.032 and 0.010,respectively).Moreover,the overall survival time of HSCC patients with low PAFAH1B3 expression was significantly longer than those with high PAFAH1B3 level(P=0.022).However,the Cox multivariate analysis showed that only clinical stage was an independent prognostic factor(HR,1.73;95%CI,0.86-3.47;P = 0.123).3.Loss-of-function assays were conducted to explore the biological function of PAFAH1B3 in HSCC FaDu cells.PAFAH1B3 expression was significantly knocked down in FaDu cells transfected with PAFAH1B3-specific siRNA1 and siRNA2 compared with the negative control(NC)siRNA.4.Cell proliferation was significantly suppressed in FaDu cells treated with PAFAH1B3 siRNAs in comparison with the NC group(siRNA1 vs NC:P=0.0001;siRNA2 vs NC:P=0.0046).PAFAH1B3 silencing induced more early-phase cell apoptosis than NC group(siRNAl vs NC:P=0.0002;siRNA2 vs NC:P=0.0061).Furthermore,the expression of cleavage of poly-ADP-ribose polymerase(c-PARP),a marker of apoptosis,was also increased in FaDu cells transfected with PAFAH1B3 siRNAs.In cell cycle analysis,the proportion of cells in G1-phase was significantly increased(siRNAl vs NC:P=0.0106;siRNA2 vs NC:P=0.0192),and the percentage of cells in S-phase was markedly reduced(siRNA1 vs NC:P=0.0196;siRNA2 vs NC:P=0.0376)upon PAFAH1B3 knockdown.5.The wound healing assay showed that percentage of wound closure in PAFAH1B3 siRNAs transfected FaDu cells was significantly lower than NC(siRNAl vs NC:P=0.0012;siRNA2 vs NC:P=0.0039).Transwell assay for migration also showed that the number of cells traversing the membrane was significantly decreased after PAFAH1B3 depletion(siRNA1 vs NC:P=0.0005;siRNA2 vs NC:P=0.0017).Transwell invasion assays showed that the cell number on the bottom of the membranes in PAFAH1B3 siRNAs groups were also significantly reduced(siRNAl vs NC:P=0.0002;siRNA2 vs NC:P=0.0013).ConclusionOverall,our study suggests that PAFAH1B3 is overly expressed in HSCC tumor tissues and its overexpression is correlated with poor prognosis and cervical lymph node metastasis and advanced clinical stage.Furthermore,loss-of-function experiments demonstrate that PAFAH1B3 knockdown suppresses cell proliferation and aggressiveness in FaDu cells.Therefore,inhibiting PAFAH1B3 might serve as a novel therapeutic strategy for patients with HSCC.