CREB Promotes Laryngeal Cancer Proliferation And Migration Via MYCT1/NAT10 Axis

Introducion: Laryngeal cancer is the most common head and neck cancer and its incidence has been recently increasing.Laryngeal cancer has higher incidence rates in men compared with women.Both environmental and genetic factors are risk factors for laryngeal cancer.Surgery treatment combining radiotherapy and chemotherapy is the current treatment option and has been shown to improve prognosis and survival of patients with laryngeal cancer.However,recurrent laryngeal cancer is a clinically common problem faced by physicians.Hence,it's necessary to elucidate the molecular mechanism of laryngeal cancer development to find new treatment strategies.Transcription factors regulate gene expression via binding to promoter of their target genes.Transcription factors have been indicated to play vital role in pathogenesis and development of cancer.CREB belongs to the leucine zipper class of transcription factors and participates in memory formation and development of cancer.Several studies have reported that CREB is overexpressed in lots of cancer such as lung cancer,gastric cancer,liver cancer and breast cancer.CREB also regulates cancer various biological processes including proliferation,apoptosis and autophagy via its targets.For an example,CREB could up-regulate YAP expression and promote tumorigenesis in liver cancer.In melanoma,CREB inhibits AP-2alpha expression to regulate malignant phenotype of melanoma.Meanwhile,CREB is also be regulated by other genes.For an instance,mi R-9 could inhibit CREB expression and moderate glioma phenotype.These results imply that CREB participates in various cancers depend on different pathways.Our bioinformatics analysis result revealed a cyclic AMP response element(CRE)in MYCT1 promoter,suggesting that MYCT1 is a potential target of CREB.To further understand the downstream factor regulated by MYCT1,we performed a protein microarray in stable MYCT1-expressed Hep2 cells.As a result,we found that NAT10(N-acetyltransferase 10)is one of downregulated proteins in the cells,displaying that NAT10 was a downstream factor of MYCT1.NAT10 has been reported to be associated with cancer in several studies.For an example,NAT10 overexpression was found in liver cancer and is associated with shorter survival time of patients.NAT10 decreases E-cadherin expression and induce epithelial mesenchymal transition leading to hepatocellular carcinoma metastasis.Thus,we hypothesize that CREB can play a role in laryngeal cancer by regulating MYCT1 and NAT10.Up to now,role and molecular mechanism of CREB in laryngeal cancer have not been reported.In this study,we would like to detect CREB expression in laryngeal cancer,analyze the relationship between CREB expression and clinical features of the patients,confirm regulatory relationships among three proteins and explore effects of three genes on laryngeal cancer proliferation and migration to elucidate function and molecular mechanism of CREB in laryngeal cancer.These will provide novel clues for further investigation of CREB-related network and its clinical significance in laryngeal cancer.Materials and methods: 1.Materials Thirty-five cases of laryngeal cancer and paired normal tissues were obtained from Department of Otolaryngology,463 Hospital of PLA.Human laryngeal cancer cells Hep2 was purchased from Key GEN biotech company(Nanjing,China).pc DNA3.1-CREB,pc DNA3.1-NAT10,p GL3-P795(-795/+12)and p GL3-P760(-760/+12)vectors were bought from Gen Script company(Nanjing,China).2.CREB expression detection Western blot was used to detect CREB protein level in laryngeal cancer and paired normal tissues.Relationship between CREB and clinicopathological features of the patients was analyzed by one-way ANOVA.3.Regulation of CREB on MYCT1 transcription The bioinformatics analysis found that there is a CREB binding site in promoter region of MYCT1.Luciferase reporter assay was performed to detect effect of CREB on MYCT1 promoter activity.Real-Time PCR and Western blot were applied to monitor effect of CREB on MYCT1 m RNA and protein levels in CREB-transfected Hep2 cells,respectively.Chromatin immunoprecipitation was used to detect binding of CREB to MYCT1 promoter region.4.Effects of CREB and MYCT1 on NAT10 expression Real-Time PCR and Western blot were used to monitor effects of CREB and MYCT1 on NAT10 m RNA and protein levels in CREB-,MYCT1-transfected,CREB and MYCT1-cotransfected Hep2 cells,respectively.5.Effects of CREB/MYCT1/NAT10 axis on laryngeal cancer proliferation and migration CCK8 and Transwell assay were performed to examine effects of CREB/MYCT1/NAT10 axis on larygeal cancer proliferation and migration in CREB-,MYCT1-,NAT10-transfected,CREB-and MYCT1-,MYCT1 and NAT10-cotransfected Hep2 cells,respectively.Results: 1.CREB is overexpressed in laryngeal cancer Western blot result revealed that CREB was up-regulated in 28 of 35(80%)cases of laryngeal cancer tissues.Moreover,average protein CREB level was significantly higher in laryngeal cancer tissue than in normal tissue.Statistics analysis result showed that CREB over-expression was associated with tumor stage,lymphatic metastasis and differentiation,but not with age and sex.2.CREB directly inhibits of MYCT1 transcription Bioinformatics analysis result revealed a cyclic AMP response element(CRE)in the MYCT1 gene promoter.Luciferase reporter gene assay result displayed that CREB significantly reduced MYCT1 promoter activity.Real-Time PCR and Western blot results indicated that CREB significantly reduced MYCT1 expression both at m RNA and protein levels.Ch IP results demonstrated that CREB directly bound CRE of MYCT1 promoter.3.CREB promotes NAT10 expression via inhibiting MYCT1 expression Protein microarray detection result showed that NAT10 was significantly downregulated in stable MYCT1-transfected Hep2 cells compared to controls,suggesting that it is a potential downstream protein of MYCT1 in larygeal cancer.Real-time PCR and Western blot results indicated that CREB and MYCT1 significantly enhanced and inhibited NAT10 expression both at m RNA and protein levels,respectively.Meantime,MYCT1 significantly rescued the effect of CREB on NAT10 expression.These results suggest that there is a CREB/MYCT1/NAT10 axis in laryngeal cancer.4.CREB regulates laryngeal cancer cells proliferation and migration through MYCT1/NAT10 axis CCK8 and Transwell detection results displayed that CREB and MYCT1 significantly promoted and suppressed Hep2 cell proliferation and migration,respectively.MYCT1 significantly rescued the effects of CREB on Hep2 cell proliferation and migration.Meanwhile,MYCT1 and NAT10 significantly inhibited and enhanced Hep2 cell proliferation and migration,respectively.NAT10 significantly rescued the effects of MYCT1 on Hep2 cell proliferation and migration.These results imply that CREB regulates laryngeal cancer cells proliferation and migration via MYCT1/NAT10 axis.Conclusions: 1.CREB is upregulated in laryngeal cancer tissues and is associated with tumor stage,lymphatic metastasis and differentiation.2.In laryngeal cancer Hep2 cells,CREB is a transcription suppressor of MYCT1.3.In laryngeal cancer Hep2 cells,CREB upregulates NAT10 expression via MYCT1.4.CREB promotes laryngeal cancer cells proliferation and migration through MYCT1/NAT10 axis.