Determination Of Molecular Mechanisms Involving CUL3-KEAP1-NRF2 Axis Regulated Oxidation Stress In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is one of the common malignant tumor in upper digestive system,which is characterized by high malignancy and poor prognosis,with 5-year overall survival less than 25%.Esophageal carcinogenesis is related with many factors,including smoking,alcohol consumption,poor diet habit,intake of nitrosamines and many other reasons.These divers pathogenic factors lead to a complicated gene background for ESCC,with alterations in proliferation,differentiation,cell cycle regulation,oxidative stress and epigenetics.Thus,no precision therapeutic strategy is suitable for ESCC.Oxidative stress is a critical biological process,which facilitates carcinogenesis by promoting tumor cell proliferation and genomic instability,but cell death will occur under overoxidation conditions.Oxidative stress related CUL3-KEAP1-NFE2L2 axis and TP53 are significantly mutated genes in ESCC,indicating the important role for them in ESCC.However,targetable downstream molecules for these two pathways and possible co-regulation in oxidative stress between them are not clear.In this study,we found that CUL3-KEAP1-NFE2L2 axis is significantly mutated both in ESCC and other squamous cell carcinomas including head and neck squamous cell carcinoma and lung squamous cell carcinoma,resulting in NRF2 functionally activated.We used a small-molecule inhibitor KI696 to interfere with the binding between KEAP1 and NRF2,this treatment introduced NRF2 hyper-activation and development of antioxidant ability in immortalized D3 rat esophagus epithelial cell and in NRF2 wild-type ESCC cell lines.Depending on the data of RNA-sequencing,we found that activation of NRF2 significantly up-regulated gene expressions of pentose phosphate pathway and glutathione metabolism,which are related with production of antioxidant substance NADPH and GSH,respectively.Using RRx001 and HG106 to inhibit G6PD and SLC7A11,which are corresponding critical regulating factors of pentose phosphate pathway and glutathione metabolism,significantly suppressed the proliferation of ESCC cells,while cell lines with NRF2 nutation were more sensitive to these drugs.Moreover,in organoid culturing experiments,we showed a combined therapeutic effect of these inhibitors in line with radiotherapy.To elucidate the effects of mutant p53 in ESCC oxidative stress regulation and the relationship with NRF2,we transduced D3 cell with p53R246W,a hot spot mutation of p53.The D3-p53R246W cell showed remarkable proliferating phenotype and elevation in anti-oxidation potency,but impaired cell cycle regulation effect.For the detection of relationships between p53 and NRF2,neither western blots nor bioinformatic analysis revealed distinct co-regulation.Mechanistically,we showed that the EGFR-ERK signaling is obviously activated in D3-p53R246W cell,with the ERK kinase mediated ETS1 phosphorylation and activation,which finally promoted expression of SLC7A11 and higher anti-oxidation potency.In addition,we showed that G6PD and SLC7A11 were highly expressed in the N-nitroso-N-methylbenzylamine induce rat ESCC model,carcinogenesis of ESCC would be evidently suppressed by artesunate,an oxidation inducer.In summary,NRF2 activation and mutant p53 both promote antioxidation effect through metabolic pathways,indicating a therapeutic strategy by inducing over oxidation in ESCC.Equally,intervening and destroying the redox balance could also suppress ESCC carcinogenesis.