| Oral squamous cell carcinoma(OSCC)is the sixth most common malignant tumor in the world.It is characterized by differences in differentiation degree and tendency of lymph node metastasis.Intra-tumor heterogeneity(ITH)is caused by continuous mutation in the process of tumor progress.With the development of tumor,single cell and clone continue to compete for nutrition,oxygen and space in tumor microenvironment.In this selective environment,cloning evolves and acquires mutations that enable it to survive and proliferate,essentially becoming dominant subcloning,while others either perish or remain static.Based on the genomic study of total exon sequencing(WES),the whole exon sequence of OSCC was studied.According to the analysis of the sequencing data,the mutation of the genome,in particular the mutation of the somatic cell,and the tumor necrosis tree are more obvious.In order to better understand that the ITH of OSCC is carried out in different regions of a single tumor space,the uniqueness of different fragments and the change of common copy number are revealed after the analysis of these different tumor fragments.Previous studies have shown that individual tumor clones have a wide range of genetic and biological properties.Our results can further reveal the complexity and heterogeneity of OSCC.These findings may help guide the development of future clinical trials,overcome the spatiotemporal heterogeneity of tumors,and improve the clinical treatment status of patients with OSCC.OBJECTS:Oral squamous cell carcinoma(OSCC)is the most common type of oral cancer,but there are few studies on the driving mechanism of its occurrence,development and metastasis.In this study,we aimed to explore the driving effects of OSCC intra-tumoral heterogeneity and subclonal mutations on lymph node metastasis to identify novel potential therapeutic targets.METHODS:To address this problem,86 tumor regions of 18 patients with oral squamous cell carcinoma(OSCC)were subjected to full exome sequencing.The bases were read from the Illumina machine containing the sequencing adapter and the low quality fragments with more than five unknown bases were removed to obtain clean data.Clean data were mapped to the human reference genome(Grch38)and annotated with variation using ANNOVAR.The changes of somatic copy number were analyzed with GATK4Alpha,normalized with the proportional coverage of the tumor and the proportion of the normal tissue,and then the tangent normalized.Finally,the cyclic binary segmentation method is used to segment the copy ratio.On this basis,the tumor-driven genes were selected,and the tumor evolution tree of OSCC was constructed.RESULTS:1.Heterogeneity in oral squamous cell carcinoma.Tumor heterogeneity exists not only in different patients but also within the same patient.Oral squamous cell carcinoma in the same sample also showed different subtypes.In primary tumors,chromosomal instability is mainly manifested as triploid or even polyploid,with less haploid.Metastatic enlarged lymph nodes are mainly haploid.Results showed that heterogeneous tumor cells were present in both OSCC primary foci and metastatic lymph nodes,including in the patient’s blood.these heterogeneous tumor cells play an important role in tumorigenesis,metastasis,distant colonization,and maintenance of the tumor microenvironment.2.Whole exome sequencing(WES).Tthe genomic DNA of 18 primary OSCC patients was used for WES detection.A total of 86 tumor regions were sequenced with an average coverage depth of 100×.A total of 1,720 non-sinking cell mutations(affecting 1,287 genes)and 433 silent mutations were identified,with a validation rate of 90%.At the same time,the potential driver mutations were identified based on the available OSCC sequencing data.According to previous findings in other tumor types,driving mutations are relatively early in tumor development.Then,the putative driver mutations were divided into oncogene mutations and tumor suppressor gene mutations.Half of the subclonal mutations are located on the branches of the tumor evolution tree,and target genes including TP53、MROH8、MUC4、KMT2D、DMD and NOTCH can influence tumor evolution and metastasis.These findings suggest that extra care should be taken when considering the suppression of these mutants in the OSCC.3.Copy number variation(CNA).We examined the cloning status of somatic mutations in a single region.Calculation drivers include many oncogenes,including TP53、MROH8、MUC4、KMT2D、DMD and NOTCH1,they highlight the important role of this subclonal mutation in OSCC formation and metastasis.chromosome 7p11.2(including KMT2D)amplification was observed in the T1 and T3 regions,for example,but not in the T2 and T4 regions.Similarly,deletion of chromosome 9p21.3(containing MROH8 and AADACL3)is prevalent in some cases,but heterogeneity aberrations also occur in other samples.The only driving CNA found to be ubiquitous is the copy number gain of 11q13,which includes many oncogenes,such as NOTCH1、MUC4 and KLHL4,highlighting the importance of this distortion as a founding genomic injury in OSCC development.These results suggest that similar to somatic mutations,CNAs also show significant spatial ITH,consistent with observations from several other cancers.Somatic mutation rates(an average=238 mutations per case)were higher than in each tumor region(an average=187 mutations per region).For example,KMT2D mutations were only detected in 20.9%of tumor regions(consistent with previous results)and 1.33%of cases.In addition,the proportion of subclonal mutations detected in each tumor region is significantly lower than in each case.These results suggest that subclonal mutations also show ITH,similar to somatic mutations.This feature is consistent in several other cancers.Therefore,sequencing data analysis of individual biopsies may underestimate the incidence of mutations,especially those acquired at the late stage of onset.4.ITH and subclonal mutations affect tumor evolution.To investigate ITH and OSCC genomic evolution,a phylogenetic tree was constructed based on the identification of somatic mutations(silencing and non-sinking)in each tumor region.The trunk,"shared "and" private "branches of each tree represent mutations in all tumor regions,some tumor regions(but not all tumor regions),and only one tumor region,respectively.Phylogenetic trees vary greatly under different conditions.All OSCC patients showed evidence of spatial ITH.Somatic variation of spatial heterogeneity averaged 60.68%(range 29.7-97.2%).Potential driver mutations were identified based on large-scale OSCC sequencing data.Overall,the enrichment of trunk-driven mutations was significantly higher than that of passenger mutations(77.8%versus 63.8%;P=0.023).This result suggests that driving mutations are relatively early events during tumor evolution,consistent with previous findings of other tumor types.then,the putative driver mutation is isolated as a driver mutation that occurs in oncogenes or tumor suppressor genes.Nnotably,half of the driver mutations localized to the branch(50.0%)were located in oncogenes,including TP53、MROH8、MUC4、KMT2D、DMD and NOTCH.Potentially operable mutations,such as those targeting PRUNE2 and CASP8,are often carcinogenic branching events.For example,18 of the 5 cases had KMT2D mutations that were located at the proximal end of the branch in all mutation cases.5.Role of subclonal mutations in tumor metastasis.By mutation profiling,we can know the number of various types of mutations(C>T/G>A)in each tumor sample and whether the sample prefers some type of mutation.By analyzing the characteristics of somatic mutation,the characteristics of somatic point mutation in different cancer species can be studied.Silencing mutations accounted for the largest proportion of several subclonal mutations with high mutation frequency.There may be synergistic or mutually exclusive relationships between high frequency mutant genes in 86 samples.Synergistic effects suggest that both genes may have to mutate simultaneously to produce oncogenic effects.The mutex effect suggests that the two genes may be carcinogenic if mutated alone.TP53 synergistic and mutually exclusive genes,for example,can serve as targets for further research OSCC progress and drug resistance.Most subclonal mutation enrichment pathways in tumors are associated with tumor progression and metastasis,such as Hippo signaling,p53 signaling,and apoptosis pathways.These results suggest that subclonal mutations promote oral squamous cell carcinoma metastasis by driving tumor-associated pathways.6.Temporal dissection of mutational spectra and signatures.To determine the temporal dynamics of OSCC mutations,the tree trunk and branch mutations were analyzed using deconstruction plots and signatures.Calculated the contribution of a single mutation signal to each tumor and identified several features in the examined tumor.The inferred signatures included signature 1(age-related),signature 2 and 13(APOBEC-related),signature 6 and 15(associated with DNA mismatch repair),signature 3(associated with DNA double-strand break repair),and signature 7(associated with uv exposure in squamous cell carcinoma).In some cases,increased contributions to features associated with DNA mismatch repair(including features 6 and 20)and microsatellite unstable tumors(including feature 21)were observed in branching mutations(OSCC95309 and OSCC94377).Data suggest that various mutation processes may play an important role in subclonal diversity in OSCC progression.CONCLUSIONS:Intratumoral heterogeneity and subclonal mutations in OSCC occur mainly in tumor suppressor genes,which are more frequently driven,including TP53,MROH8,MUC4,KMT2D,DMD and NOTCH,related the signal pathways associated with tumorigenesis and metastasis signaling pathways.Our study of ITH and subclonal mutations provides an important molecular basis for further understanding of OSCC occurrence and metastasis and provides potential targets for the treatment of this disease.More than 90%of oral cancers are squamous cell carcinomas.Oral squamous cell carcinoma(OSCC)is a very aggressive neoplasm.OSCC progresses through the four stages of cancer and metastasises to distant sites,including the buccal mucosa,the gingival,floor of mouth,palate and anterior tongue.Despite advances in cancer diagnosis and treatment,the overall 5-year survival rate for OSCC remains the lowest among all malignancies.The epithelial-mesenchymal transition(EMT)is a process wherein an epithelial cell loses its morphology and attains the morphological characteristics of a mesenchymal cell,which is important for the cancer metastasis.In case of OSCC,the invasive front has been reported to express EMT markers,suggestive of the occurrence of this process during cancer progress.Hence,EMT has a prognostic significance in OSCC.The induction of EMT in OSCC has been attributed to multiple oncogenic pathways,including the phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(AKT)signalling pathway,Transforming Growth Factor-β(TGF-β)/Smad pathway,Wnt pathway and Notch pathway.Our previous research revealed the overexpression of EphA2 and ephrinA1 in OSCC and salivary adenoid cystic carcinoma.EphrinA3 plays an important role in the growth and development of the embryonic nervous system,but its role in the occurrence and progression of tumors is unclear.OBJECTS:This study aims to improve survival rate in OSCC patients and provide new therapeutic targets by investigating the role of eprinA3 signaling mediated Epithelial-Mesenchymal Transition(EMT)in regulating the mechanism of oral squamous cell carcinoma(OSCC)metastasis.METHODS:We collected primary tumor samples from 53 patients with oral squamous cell carcinoma(OSCC)from Oral Maxillofacial-Head and Neck Tumor Surgery in Wuhan University Oral Hospital between 2013 and 2015,and simultaneously selected normal mucosal tissue beside their cancer as a negative control.We prepared tissues into microarrays for eprinA3 and E-Cadherin staining evaluation and observed correlation between them.Two cell lines,CAL-27 and SCC25,were subsequently used for experiments in vitro to explore the association of eprinA3 with Epithelial-Mesenchymal Transition(EMT)and the main signaling pathways.Finally,nude mice with tumor-bearing experiments were performed in vivo to verify the regulatory effect of eprinA3 on oral squamous cell carcinoma(OSCC)metastasis.RESULTS:1.The expression of EprinA3 and E-cadherin was negatively correlated in oral squamous cell carcinoma.We measured the average optical density values of eprinA3 and E-Cadherin staining in tissue chips and found that eprinA3 was not only negatively correlated with E-Cadherin expression,but also negatively correlated with pathological grade,which meant decreased eprinA3 expression in high-grade tumors.This difference was statistically significant between grade Ⅰ and Ⅱ and between gradeⅠ and Ⅲ(p<0.01),but not between grade Ⅱ and Ⅲ(p=0.357).2.Protein mass spectrometry results showed that EphrinA3 may be associated with tumor progression.We extracted total proteins from d CAL-27 cells which transfected with eprinA3-RNAi and vector and quantified differentially expressed proteins using proteomics.Differentially expressed proteins were named using a Homo sapiens database,and database searches were performed using MASCOT 2.6.Using isobaric markers for relative and absolute quantification of differentially expressed proteins.A total of 24 differentially expressed proteins met the screening criteria,of which 17 were up-regulated and 7 were down-regulated.Using enrichment analysis of go annotations,it was found that the biological behavior involved in these differential proteins was partially associated with tumor progression.3.EphrinA3 regulates epithelial-mesenchymal transition(EMT)-related biological behavior in oral cancer cells.Within 24h,CAL-27 and SCC25 cells in the eprinA3-RNAi group(representing SCC25 and CAL-27 cell lines constructed after virus transfer that knocked out the EFNA3 gene)had a stronger scratch healing capacity than the eprinA3-mimics group(representing the SCC25 and CAL-27 cell lines of the overexpressing protein eprinA3).At 12 hours,Cal-27 and SCC25 cells in the eprinA3 knockout group were more invasive than those in the eprinA3 overexpression group.when treated with PTX(paclitaxel),the apoptosis rate in the eprinA3-RNAi group was lower than that in the negative control group(with the SCC25 and CAL-27 cell lines transfected with an empty vector virus as the blank control group).the apoptosis rate in the eprinA3-mimics group was higher than in the negative control group.In the eprinA3 knockout group,CAL-27 and SCC25 cells were more proliferative than the eprinA3 overexpression group.4.EprinA3 signaling regulates epithelial-mesenchymal transition(EMT)via the PI3K/AKT pathway.The decrease of EphrinA3 expression can lead to the decrease of E-Cadherin expression,the increase of N-Cadherin expression,and the increase of p-Akt(Ser473)expression,indicating that the PI3K/Akt pathway is activated.E-cadherin overexpression,N-cadherin expression decreased,and p-Akt expression decreased after transfection of EphrinA3-mimics,indicating that the PI3K/Akt pathway was inhibited.5.Inhibition of eprinA3 expression can promote tumor proliferation in vivo.The tumor volume of eprinA3-RNAi group was larger than that of eprinA3-mimics group,and the difference was statistically significant(P<0.05).We also used Western Blot to analyze the total protein and found the same trend as in vitro testing.Therefore,knockdown of eprinA3 can promote tumor proliferation,and overexpression of eprinA3 can inhibit tumor proliferation in vivo.6.MiR-210-3p targeting gene EFNA3 regulates tumor progression.we used three databases to predict the microRNAs of four targeting genes,EFNA3,and validated the high expression of miR-210-3p in SCC-25 and CAL-27 cell lines.To further identify the target group of miR-210-3p,we performed double luciferase test to confirm our prediction.After transfection of miR-210-3p in SCC-25 and Cal-27 cells,the expression of EprinA3 decreased with the increase of miR-210-3p through real-time PCR.And the decrease of miR-210-3p expression also leads to the increase of EprinA3 expression.Therefore,miR-210-3p can regulate the EMT process of oral cancer cells by targeting gene EFNA3 through PI3K/AKT signaling pathway.CONCLUSIONIn oral squamous cell carcinoma(OSCC),the miR-210-3p/ephrinA3/PI3k/Atk signaling axis plays an important role.Down-regulation of eprinA3 expression through this signaling axis may inhibit the progression of epithelial-mesenchymal transition(EMT)in OSCC and the development and metastasis of oral cancer.Therefore,eprinA3 can be a new target for the treatment of oral cancer. |
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