The Role And Potential Molecular Mechanism Of NRF2 In Esophageal Squamous Cell Carcinoma

Background and AimEsophageal carcinoma is the sixth most common malignant tumor in the world.It is mainly divided into two subtypes:adenocarcinoma and squamous cell carcinoma,of which 70%of esophageal squamous cell carcinoma occurs in China.Studies have shown that timely intervention for early esophageal cancer can improve the 5-year survival rate of patients to more than 90%.Therefore,it is very important to further clarify the pathogenesis of esophageal squamous cell carcinoma.Nuclear factor erythroid-derived 2 like 2(NRF2),a member of the basic leucine zipper(BZIP)family,is one of the most important antioxidant stress proteins in the body,which can protect the body cells from ROS damage caused by various internal and external stimuli.Physiologically,NRF2 is mainly localized in the cytoplasm,and binds to Kelch-like ech-associated protein 1(KEAP1)and Cullin3 to mediate the ubiquitination degradation of NRF2 and maintain its dynamic homeostasis.When the organism is subjected to various stimuli,the oxygen free radicals or electrophilic substances in cells can cause the change of cysteine residues of KEAP1 protein,promote the depolymerization of KEAP1-CUL3-NRF2 complex and the nuclear transport of NRF2,and then regulate the expression of various antioxidant stress-related genes(such as GST,SRXN1)to exert the anti-ROS function.NRF2signaling pathway plays an important role in chronic inflammatory diseases,diabetes,neurodegenerative diseases,aging and tumors,but it has dual functions of anti-tumor and pro-tumor in tumors.Bioinformatics analysis of gastroesophageal carcinoma in TCGA database showed that the mutation rate of the NRF2 gene in esophageal squamous cell carcinoma was significantly high.Further RNA sequencing and immuno-histochemistry showed that NRF2 expression was higher in infiltrated tumor cells despite its lower RNA and protein expression in the ESCC tissues than paracancer tissues.GO enrichment analysis demonstrated the expression of NRF2 was not only highly enriched in epithelial cell differentiation,metabolism and other functions,but also had a good correlation with a variety of esophageal stem cell markers.This study aims to further clarify the role of NRF2 in esophageal squamous cell carcinoma and explore its main molecular mechanism in the maintenance of cancer stem cells,which will provide us a new theoretical basis and research direction for the treatment of esophageal squamous cell carcinoma or other squamous cell carcinoma.Part one Gene status,expression and clinical significance of NRF2 in ESCC1 ObjectiveTo investigate the gene status,expression and clinical significance of NRF2 in ESCC.2 Methods2.1 The genomic sequencing data of 559 cases of gastric-esophageal carcinoma were downloaded from the Cancer and Tumor Genome Atlas Project(TCGA)database,and the differences of the mutated genes were analyzed by Venny and c Bio Portal software in esophageal squamous cell carcinoma,esophageal adenocarcinoma,and gastric adenocarcinoma.2.2 The expression of NRF2 related molecules in esophageal squamous cell carcinomaThe relative expression values of 120 pairs of Chinese esophageal squamous cell carcinoma RNA sequencing were used to analyze the expression of NRF2,KEAP1,CUL3,and STK11 in esophageal squamous cell carcinoma by paired-sample t-test.The protein expression and distribution of NRF2 in esophageal squamous cell carcinoma(ESCC)were detected by immunohistochemistry.2.3 The expression distribution of NRF2 in esophageal squamous cell carcinomaThe protein expression and distribution of NRF2 in esophageal squamous cell carcinoma(ESCC)were detected by immunohistochemistry.2.4 The clinical significance of NRF2 expressionThe protein expression of NRF2 was evaluated with a tissue microarray and the clinical significance of NRF2 expression was analyzed.3 Results3.1 The mutation rate of NRF2 in esophageal squamous cell carcinoma.The mutation rate of NRF2 in esophageal squamous cell carcinoma(19%)was significantly higher than that in gastric adenocarcinoma(2.5%)and esophageal adenocarcinoma(5%),and this difference also existed in lung squamous cell carcinoma(17.8%vs 2.8%).Mutation site analysis showed that most NRF2mutations were located in the N domain which participated in ubiquitination degradation of NRF2 mediated by the NRF2-KEAP1 complex.Other molecules in the NRF2 pathway,including KEAP1,CUL3,STK11,were mutated both in esophageal squamous cell carcinoma and adenocarcinoma.The mutation rate of CUL3 and STK11 in squamous cell carcinoma were significantly higher than that in adenocarcinoma,while there was no significant difference in KEAP1 in squamous cell carcinoma and adenocarcinoma.The total mutation rate of NRF2,Keap1,Cul3,STK11 was three times that of adenocarcinoma(33.33%vs.10.23%).NRF2,KEAP1,CUL3,STK11 mutations have certain incompatibilities.3.2 The expression of NRF2 related molecules in esophageal squamous cell carcinoma.RNA sequencing results showed that the expression of NRF2,CUL3,and STK11in cancer tissues was significantly lower than that in adjacent tissues,while the expression of KEAP1 was not significantly different.3.3 The expression distribution of NRF2 in esophageal squamous cell carcinoma and the clinical significance of NRF2 in ESCCImmunohistochemistry showed that NRF2 was mainly localized in the basal layer cells in normal esophageal epithelial cells,while in esophageal squamous cell carcinoma tissues,NRF2 was mainly highly expressed in infiltrated tumor cells and presented as nuclear localization.Higher expression of NRF2 was associated with TNM stage(p=0.046),while had no significant correlation with age,sex,tumor size and lymphic metastasis(p>0.05).Patients with higher NRF2 level is associated with poor overall survival of ESCC patients(p=0.022).4 ConclusionThe NRF2 signaling pathway is highly mutated in esophageal squamous cell carcinoma.Patients with higher NRF2 level is associated with poor overall survival of ESCC patients.Part two The function of NRF2 in esophageal squamous cell carcinoma1 ObjectiveNRF2 overexpression and knockdown stable cell lines were established to investigate the function of NRF2 in ESCC by in vivo and in vitro cell biology experiments.2 Methods2.1 Establishing stable cell lines with overexpression and knockdown of NRF2The expression of NRF2 in esophageal squamous cell carcinoma cell lines was detected by Western blot.Stable cell lines including NRF2 overexpression and knockdown were established with lentivirus and validated using Western-blot assay.2.2 Effect of NRF2 on proliferation in esophageal squamous cell carcinomaNRF2 overexpression and knockdown cells were seeded into 96-well plates,and the effect of NRF2 on the proliferation of esophageal squamous cell carcinoma cells was dynamically analyzed by Incucyte system.2.3 Effect of NRF2 on migration and invasion of esophageal squamous cell carcinomaTranswell was used to detect the effect of NRF2 on the migration and invasion ability of esophageal squamous cell carcinoma cells,and statistical analysis was performed.2.4 The effect of NRF2 on proliferation of ESCC was evaluated with tumor-bearing nude miceNRF2 knockdown cells and control cells were respectively inoculated into bilateral armpits of CD1 nude mice,and the effects of NRF2 knockdown on the growth of esophageal squamous cell carcinoma cells were measured and analyzed.3 Results3.1 Establishing stable cell lines with overexpression and knockdown of NRF2NRF2 expression in ESCC cell lines including KYSE-30,KYSE-140,KYSE-150,KYSE-270 and KYSE-410 was low.The expression of NRF2 was relatively high in KYSE70,KYSE-180 and KYSE-520 which have mutations of NRF2 pathway.Overexpression and knockdown of stable cell lines were successfully constructed,and protein level was validated by Western-blot(p<0.05).3.2 Effect of NRF2 on proliferation in esophageal squamous cell carcinoma cellsIncucyte analysis showed that overexpression of NRF2 promoted the growth of esophageal squamous cell carcinoma cells(p=0.024),while knock-down of NRF2inhibited cell growth(p<0.01).3.3 Effect of NRF2 on migration and invasion of esophageal squamous cell carcinomaThe Transwell migration and invasion assay showed that NRF2 overexpression had no significant effect on the migration and invasion ability of esophageal squamous cell carcinoma cells(p>0.05).3.4 The effect of NRF2 on proliferation of ESCC was evaluated with tumor-bearing nude miceThe tumor volume of NRF2 knockdown cells was significantly lower than that of control cells in nude mice(87mm~3 vs 1510mm~3)(p=0.0442).4 ConclusionThe protein expression of NRF2 was elevated in cells with mutations of NRF2pathway.NRF2 promoted the growth and proliferation of esophageal squamous cell carcinoma cells and enhanced the formation ability of tumor stem cell balls,but had no significant effect on cell migration and invasion ability.Part three Potential molecular mechanism of NRF2 in esophageal squamous cell carcinoma stemness1 ObjectiveExploring the potential molecular mechanism of NRF2 in the maintenance of ESCC stemness,and evaluating the clinical significance of NRF2 expression.2 Methods2.1 Go enrichment analysis of differentially expressed genes in NRF2 knockdown cellsR studio was used to standardize the RNA sequencing data,and GO enrichment analysis of differentially expressed genes were performed with Metascape software.2.2 Effect of NRF2 in cancer stemness of esophageal squamous cell carcinoma by the stem cell clone formation experimentNRF2 overexpressed and knocked down cells were seeded into 12-well plates and cultured in serum-free stem cell medium for 7 days to quantitatively analyze the changes in the formation ability of tumor stem cell spheres.2.3 The regulation of cancer stem cell related genes by NRF2 in ESCCThe expression and distribution of different tumor stem cell markers in esophageal squamous cell carcinoma were detected by immunohistochemistry.Potential binding sites of NRF2 in the LGR6 promoter region were predicted with Jaspar software and validated by Western blot assays.2.4 The expression and function enrichment analysis of NRF2 in ESCC tissuesGroups with high or low expression of NRF2 was divided using the median of RNA sequencing data,then function enrichment analysis was performed with GSEA software.3 Results3.1 Function enrichment analysis of differentially expressed genes in NRF2knockdown cellsThe differentially up-regulated NRF2 genes were mainly enriched in epidermis development,cell junction organization,transmembrane receptor protein tyrosine kinase signaling pathways.3.2 Effect of NRF2 in cancer stemness of esophageal squamous cell carcinoma by stem cell clone formation experimentStem cell clonal sphere formation experiments showed that the number and volume of stem cell clonal spheres in the NRF2 overexpression group increased(p=0.005),and inhibited in the NRF2 knockdown group(p=0.008).3.3 Regulation of NRF2 on Stem Cell Related Genes in ESCCImmunohistochemistry showed that NRF2 and stem cell indicator SOX2 were co-located in the basal cell layer in paracancerous tissues.In ESCC tumor tissues,NRF2 expression was weak in the region with high SOX2 expression.Both NRF2and stem cell indicator LGR6 were highly expressed in infiltrated tumor cells.Bioinformatics analysis showed that there were two potential binding sites of NRF2in the promoter region of LGR6.Western blot proved that NRF2 was able to increase the expression of LGR6 in ESCC cells.3.4 The expression and main function enrichment of NRF2 in ESCC tissuesThe m RNA enrichment analysis of 120 cases of esophageal squamous cell carcinoma was carried out according to the level of NRF2.The data showed that NRF2 was mainly concentrated in the pathway of epithelial differentiation,oxidative stress,and glyceraldehyde triphosphate metabolism.4 ConclusionNRF2 is involved in ESCC stem cell maintenance,it was able to bind the stem cell marker LGR6 promoter and promotes its transcription.NRF2 is able to bind the promoter of stem cell marker LGR6 and promote LGR6 transcription.