SUMO/De-SUMOylation Of Nrf2 In The Regulation Of Tumor Metabolism And Proliferation

BackgroundNuclear factor erythroid-2 related factor 2?Nrf2?that belongs to the Cap'n'collar?CNC?transcription factor family,is a pivotal activator of genes encoding cytoprotective and detoxifying enzymes that protect against oxidative and electrophilic stress,to maintain cellular redox homeostasis.Abundant expression of Nrf2 has been found in various cancers,facilitating malignant phenotypes of cancer including aggressive proliferation and resistance to anticancer therapy.These abnormal expression of Nrf2 enhances its anti-oxidative capacity and mediates metabolic reprogramming in tumor cells by regulating metabolic-related target genes.In recent years,it has been found that Nrf2 regulates the activation of pentose phosphate pathway at transcriptional as well as at post-transcriptional levels,and enhances the syntheses of serine and glutathione promoting tumor growth and proliferation.SUMO?Small ubiquitin related modifier?modification is a crucial post-translational modification of proteins that plays regulatory roles in biological functions of cellular proteins,which can affect the localization,activity or stability of the protein in cells.SUMOylation is dynamically reversible,as SUMO can be removed from the substrate protein by SUMO-specific protease SENPs?SUMO-specific proteases?.It has been reported that Nrf2 could be modified by SUMO1 and SUMO2/3.Modified by SUMO2/3,Nrf2 could be degraded by proteasome in the nucleus,while the SUMO1modification of Nrf2 could enhance the expression of downstream target genes during liver fibrosis.But the biological role of Nrf2 SUMOylation in cancers,is largely unknown and its modified sites have not yet been detected.ObjectiveSince Nrf2 is reported as a SUMOylated protein,the first questeion we addressed is which lysine is modified by SUMO1 in Nrf2.Secondly,we aim to know whether the SUMOylation at K110 is regualted by SENP1 in tumor cells.And thirdly,the function of SUMO1 modification of Nrf2 at K110 in HCC tumorigenesis and growth,and the mechanims underlying are determined.Methods1.Nrf2 could be modified by SUMO1,being confirmed by Ni²⁺-NTA enrichment and Western blot with co-transfection of exogenous Nrf2 and SUMO1 in HEK-293T cells.Predicted by the softwares,mutations of thepotential SUMOylation sites in Nrf2 were performed respectively.Ni²⁺-NTA enrichment and Western blot were used to detect the changes of SUMOylation bands of Nrf2 in order to determined the modified sites.2.Ni²⁺-NTA Enrichment and co-immunoprecipitation were carried out to verify Nrf2could be de-SUMOylated by SENP1.SUMOylation of Nrf2 was accumulated in SENP1 knockdown cell lines.The effects of SENP1 knockdown on Nrf expression,stability and activity were analyzed by Western blot,Realtime PCR and half-life assays.Colony formation assays were performed to determine if SENP1 knockdown would affect tumor cell proliferation and colony formation in vitro.3.Protein stability,cellular localization and transcriptional activity of wildtype and K110 mutant Nrf2,are determined by half-life assay,nucleocytoplasmic separation and immunofluorescence experiments,and luciferase reporter assay,individually.By using the lentivirus infection system,wildtype and K110 mutant Nrf2 overexpressed stable HCC cell lines were established.In addition,shRNA was used to knockdown the endogenous Nrf2 expression in the cells.Tumorigenesis and growth in vitro and in vivo of HCCs were analyzed by plate colony formation and nude mice xenografts.ROS level of the cells were analyzed by FACS.Expression of Nrf2 downstream target genes were identified by RNA-seq,Realtime PCR and Western blot.Results1.Nrf2 is conjugated by SUMO1 at K110.2.Nrf2 can be de-SUMOylated by SENP1.SENP1 knockdown can up-regulate the expression of Nrf2 and downstream target genes related with PPP,and the stability of Nrf2 is enhanced,leading to the tumorigenesis and growth of cancer cells.3.SUMOylation of Nrf2 at K110 could promote the clearance of intracellular ROS,by activating GPx2 at the transcriptional level.The reduced ROS level up-regulates the PHGDH expression at the translational level,which is the controlling enzyme for cellular de novo serine synthesis.This progression could provide one-carbon unit for de novo nucleotide synthesis,thereby promoting the development of tumors.The SUMO1modification of Nrf2 at K110 is induced by serine starvation,accelerating tumor growth.ConclusionThe results of this study collectively suggested that SUMOylation of Nrf2 at K110 plays an important role in tumorigenesis by regulating the ROS level and de novo serine synthesis in tumor cells,which may provide new ideas for the targeted therapy in HCCs.