Transcription Factor RUNX1 Activates OPN To Promote The Progression And Drug Resistance Of Head And Neck Malignancies

Part I Identification and bioinformatics analysis of differentially expressed genes in head and neck malignanciesBackground: Head and neck squamous cell carcinoma(HNSCC)is the sixth leading cause of cancer related deaths worldwide,and affects approximately 550,000 people annually.Due to the critical location in the upper aerodigestive tract,these types of cancers and their treatments significantly affect the quality of life of patients.The 5 year overall survival(OS)remains approximately 40%-60% in the past several decades.Obstacles to the treatment of this cancer include progressive disease progression and drug resistance,which are caused by the inherent heterogeneity of cancer cells.Therefore,the identification of candidate genes in progression and drug resistance of HNSCC has attracted extensive attention.Objectives: Identification and bioinformatics analysis of differentially expressed genes in HNSCC.Methods: In this study,the microarray datasets were downloaded from Gene Expression Omnibus(GEO)database.The differentially expressed genes(DEGs)were identified and functional enrichment analyses were performed.Cytoscape software was used to construct the protein-protein interaction(PPI)network,and hub genes were selected for functional enrichment analysis again.The expression of hub genes in HNSCC was verified in The Cancer Genome Atlas(TCGA).Results: The overlap among the two GEO databases(GSE58911,GSE107591)contained 316 differentially expressed genes.The results of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses of DEGs indicated that the main biological processes(BP)included extracellular matrix organization,cell adhesion,collagen catabolic process,collagen fibril organization,and epidermis development.The primary cellular component(CC)included extracellular space,extracellular exosome,extracellular region,extracellular matrix(ECM),and proteinaceous ECM.For the primary molecular function(MF)included extracellular matrix structural constituent,heparin binding,collagen binding,metalloendopeptidase activity,and serine type endopeptidase activity.Furthermore,KEGG pathway analysis indicated that the DEGs were primarily involved in ECM receptor interaction,amebiasis,focal adhesion and drug metabolism cytochrome P450,respectively.The protein-protein interaction(PPI)network of DEGs was constructed and the most significant module contained 13 genes,which were identified as hub genes.Among these hub genes,SPP1 showed a higher node degree,and was significantly overexpressed in HNSCC in the different data sets.Conclusions: The DEGs and hub genes identified in the study contribute to our understanding of the molecular mechanisms underlying continuous development and drug resistance of HNSCC,and provide candidate targets for diagnosis and treatment of HNSCC.Part II Roles of OPN(SPP1)influencing malignant progression and mediating CTX resistance via KRAS/MEK pathway in HNSCCBackground: Acquired resistance,largely attributed to genetic alterations,in the process of cancer treatment.Cetuximab(CTX)is the first immunotherapy drug approved for HNSCC.CTX overcomes the immunosuppressive environment of HNSCC and increases the clinical response to cancer treatment.However,for patients treated with CTX monotherapy,the clinical response shows that HNSCC patients are intrinsically resistant in the process of treatment.This highlights the clinical need to identify both the molecular determinants of sensitivity to CTX and the mechanisms of resistance to CTX in HNSCC.SPP1 also known as osteopontin(OPN),is a multifunctional secreted acidic glycoprotein encoded by the SPP1 gene,and is involved in various physiological and pathophysiological processes,including immune responses,and cancer progression.However,little is known about the regulation of the aberrant expression of SPP1 in HNSCC and whether it is involved in tumor resistance remain to be further elucidated.Objectives: To investigate the expression and role of SPP1 gene in HNSCC,and to explore the mechanism of CTX resistance.Methods: In this study,the expression of SPP1 in HNSCC tissues and cells was detected by real-time quantitative PCR and Western blot,and stable SPP1 overexpression and down-regulation were established in cell lines.CCK-8,Transwell assay and flow cytometry were used to study the roles of SPP1 in malignant phenotype and CTX resistance.The xenotransplantation models of nude mice were used to verify the role of SPP1 and inhibitors of MEK pathways.Results: The expression of SPP1 was up-regulated in HNSCC tissues and in Fa Du and SCC-9 cells.Overexpression of SPP1 promoted proliferation,migration and invasion,and inhibited apoptosis,whereas silencing of SPP1 showed opposite results to those of SPP1 overexpression.Mechanistic experiments showed that activation of the KRAS/MEK pathway contributed to the SPP1-induced malignant progression of HNSCC and resistance to CTX.Furthermore,SPP1 knockdown or an MEK inhibitor overcame this CTX resistance pattern.Conclusions: Our findings indicate that SPP1 promotes malignant progression,and confers resistance to CTX via KRAS/MEK pathway in HNSCC,which may be a new potential therapeutic target of HNSCC.Part III Roles of transcription factor RUNX1 activating OPN to promote malignant progression of HNSCCBackground: Metastatic progression remains a major burden for HNSCC and is associated with eventual resistance to prevailing therapies.Significantly,complex molecular transcriptional programs and downstream signaling pathways have been implicated in the development of HNSCC from premalignancy and progression to invasion,metastasis,and treatment resistance.Runt-related transcription factor 1(RUNX1),also known as acute myeloid leukemia 1 protein(AML1),is a member of RUNX family of transcription factors,and is an essential master transcription factor implicated in basic cellular and developmental processes,stem cell biology and tumorigenesis.Although much is known about the roles of RUNX1 in different cancers,the mechanisms in the regulation of genes(such as OPN)that are intimately associated with tumor progression,invasion and metastasis remain unclear.Objectives: To investigate the role of RUNX1 in malignant progression and drug resistance of HNSCC,and the role of RUNX1 in OPN transcriptional regulation.Methods: In this study,RUNX1 expression levels in HNSCC cells and tissues were detected by quantitative real-time PCR(q PCR),Western blotting and immunohistochemistry(IHC).In vitro and in vivo assays were performed to investigate the function of RUNX1 in the metastatic phenotype and the tumorigenic capability of HNSCC cells.Luciferase reporter and chromatin immunoprecipitation(Ch IP)-q PCR assays were performed to determine the underlying mechanism of RUNX1-mediated HNSCC aggressiveness.Results: RUNX1 was increased with disease progression in patients with HNSCC.Furthermore,we found that silencing of RUNX1 significantly decelerated the malignant progression of HNSCC cells and reduced Osteopontin(OPN)expression in vitro,and weakened the tumorigenicity of HNSCC cells in vivo.Mechanistically,we demonstrated that RUNX1 played an important role in activating MAPK signaling by directly binding to the promoter of OPN.Conclusions: Our findings indicate that RUNX1 plays an important role in tumor cell migration and invasion by regulating OPN transcription in HNSCC.Our results provide new insight into the mechanisms underlying the facilitate metastasis ability of RUNX1 and reveal the therapeutic potential of targeting RUNX1 in HNSCC.