Expression Of Nuclear Transcription Factors E2Fs In Skin Cutaneous Melanoma And The Mechanisms Regulating The Growth Of Skin Cutaneous Melanoma Cells

BackgroundSkin cutaneous melanoma is one of the most common malignant solid tumors.Skin Cutaneous melanoma(SKCM)is a highly aggressive malignant neoplasm that develops from uncontrolled proliferation and transformation of melanocytes.Current treatment strategies used for the management of melanoma patients include observation alone,surgery,chemotherapy,radiation therapy,biologic therapy,and immunotherapy.Despite enormous international efforts and great achievement in multidisciplinary approaches,SKCM remains a major clinical challenge worldwide and a serious public health problem,laying heavy burdens on society.One of the leading causes of poor tumor prognosis is the lack of early diagnostic method and effective prognostic indicator for guiding the clinical diagnosis,prognosis,and therapy.Therefore,it is imperative to explore the molecular mechanisms underlying the progression in SKCM.Early detection(screening)of melanoma remains crucial to improve prognosis and survival rates of the patients.Besides,as new methods and treatment approaches for precise medicine are available,there is an urgent need for utilizing more precise and effective biomarkers to detect melanoma before distant metastasis formation and to assess the risk of relapse.E2Fs is a collection of genes that encode higher eukaryotic transcription factors.Beyond simply engaging in the regulation of cell cycle,the roles of E2 Fs have also been found to be involved in other numerous physiological processes,including cell proliferation,apoptosis,DNA damage repair,senescence and autophagy,which are known to be crucial in tumor cell motility and tumor progression.Although these experimental results indicated that E2 Fs may function as reliable biomarkers for SKCM,the various expression patterns,multiple biological functions,specific prognostic values and molecular pathogenesis of the majority of E2 Fs members remain evasive.E2 F transcription factor 1 is essential as a transcription factor in tumor cell motility and tumor progression.the specificity of E2 F transcription factor 1expression in different tumor tissues suggests that the mechanism of E2 F transcription factor 1action may differ in different tumors.Previous studies suggest that E2 F transcription factor 1is a key factor in melanoma development,but its different expression patterns,multiple biological functions,specific prognostic value and molecular pathogenesis remain to be further investigated.Materials and MethodsTranscriptional expression levels of E2 Fs were analyzed through ONCOMINE database and Tumor Immune Estimation Resource(TIMER)site.Survival analysis plots were drawn with Gene Expression Profiling Interactive Analysis(GEPIA)and TIMER databases.Furthermore,the correlations between E2 Fs and cancer immune infiltrates were also investigated by TIMER.The sequence alteration data for E2 Fs was evaluated using The Cancer Genome Atlas(TCGA)and c-Bio Portal.Protein-protein interaction network analyses for differentially expressed E2 Fs in SKCM were detected via STRING and Gene MANIA.Gene functional enrichment analyses were performed in Metascape and Database for Annotation,Visualization and Integrated Discovery(DAVID).Immunohistochemistry was performed on paraffin sections of cutaneous melanomas to detect the expression of E2 Fs protein.The countenance of E2 F transcription factor 1 in the SKCM tissues was examined by means of the data acquired from the Gene Expression Omnibus(GEO)and the Cancer Genome Atlas(TCGA)databases.The expression of E2F1 was also analyzed in human biopsies using immunohistology,and its expression was detected in SKCM cell lines using q PCR and WB technology.The prognostic value of E2 F transcription factor 1 in SKCM was investigated using Kaplan-Meier survival and Cox analysis.The Tumor Immunization Estimation Resource(TIMER)was used to analyze the invasion of immune cells and associated markers of immune cells.The prognostic value of E2 F transcription factor 1 DNA methylation levels for each Cp G was analyzed by means of the Meth Surv.In addition,cell function experiments were performed to validate the biological behavior of E2 F transcription factor 1 in cutaneous melanoma.The proliferation ability of A375 and SK-MEL-28 cells was detected by CCK8 assay and Edu assay after treatment with different concentrations of HLM006474 inhibitor,cell migration ability by scratch assay,apoptosis by PI flow cytometric staining,and intracellular autophagosomes by electron microscopy.Subcutaneous tumor-forming animal experiments in nude mice to assess the effect of in vivo use of HLM006474 small molecules interfering with E2 F activity on melanoma proliferation.ResultsWe observed a statistically meaningful increased messenger RNA(m RNA)expression in E2 F transcription factor 1/3/5/7 compared with matched normal tissues.A significant correlation was observed between the expression of E2F7/8 and the pathological stage of SKCM patients.In addition,survival analysis revealed that SKCM patients with low transcriptional levels of E2F1/2/3/6 were associated with a significantly better prognosis.Moreover,immune infiltrations analysis indicated that transcriptional levels plus somatic copy number alterations(SCNA)of the E2 F family were significantly correlated with several immune cell recruitments,including CD4+ T cells,CD8+ T cells,B cells,dendritic cells,macrophages and neutrophils.The function of differentially expressed E2 Fs and their neighboring genes were mainly linked to P53 signaling pathway,oocyte meiosis and cell cycle.The transcription levels of E2 F transcription factor 1 were significantly elevated in SKCM and human melanoma cell line compared with normal tissues.The consequences were confirmed in the Human Protein Atlas(HPA)database.Individuals with elevated E2 F transcription factor 1 expression had a worse prognosis(HR=1.43,p=0.01).Using the expression level of E2 F transcription factor 1,a reliable distinction between tumor and normal tissues can be made(Area Under the Curve =0.949).Moreover,immune infiltrations analysis showed that the m RNA expression levels and somatic copy number alterations(SCNA)in E2 F transcription factor 1 were drastically correlated with recruitment of several immune cells,including CD4+ T cells,Th2 cells and dendritic cells.Gain and loss-of-function assays showed that deletion of E2 F transcription factor 1 inhibited the growth of SK-MEL-28 cells,while high expression of E2 F transcription factor 1 led to the opposite result in A375 cells.HLM006474 treatment significantly inhibited A375 and SK-MEL-28 migration.Our study also revealed that inhibition of E2F1 expression with HLM006474 induced autophagy in melanoma cells by blocking the AKT-m TOR pathway.ConclusionOur study showed that overexpression of E2 F transcription factor 1 was significantly associated with poor prognosis and malignant phenotype of melanoma cells in SKCM patients.These results suggest that the use of small molecules to interfere with the activity of E2 F may have clinical applications in cancer therapy.Our results may provide novel strategies for the selection of prognostic biomarkers and immunotherapeutic targets in patients with SKCM.These findings may be beneficial to get a better understanding of molecular underpinning in SKCM,which may accelerate progress towards the achievement of more appropriate prognostic appliance and new immunotherapeutic for SKCM.