Regulation On UPS-mediated Degradation Of Pro-apoptotic Protein NOXA In Colorectal Cancer

BackgroundColorectal cancer(CRC)accounts for approximately 10%of all diagnosed cancers annually and is currently the third most deadly cancer worldwide,the morbidity and mortality of CRC are increasing every year,and the continuous emergence of drug resistance and heterogeneity remain major challenges for CRC treatment.NOXA,a BH3only proapoptotic protein involved in regulating cell death decisions,is highly expressed but short-lived in colorectal cancer.Ubiquitin-E3 enzyme CRL5(Cullin5-RING Ligase)mediated ubiquitination and UPS(ubiquitin-proteasome system)-mediated degradation of NOXA is crucial to prevent its overaccumulation and maintain an appropriate action time.However,how this process is manipulated by CRC cells commonly exposed to oxidative stress remains unknown.The peroxiredoxin PRDX1,a conceivable antioxidant overexpressed in CRC tissues,has been shown to inhibit apoptosis and TRAF6 ubiquitinligase activity.However,whether the effect of PRDX1 on apoptosis is related to the key pro-apoptotic protein NOXA,and whether PRDX1 is involved in regulating the UPSmediated degradation of NOXA and its specific mechanism remain to be explored.ObjectivePrevious studies have suggested that NOXA has a short protein life in cells,but the regulatory mechanism of UPS-mediated NOXA degradation and its specific relation with cancer development are still unclear.In this study,colorectal cancer with high expression of NOXA was taken as the main research object to analyze the regulation and mechanism of PRDX1 on pro-apoptotic protein NOXA.The purpose of this study was to elucidate the regulatory mechanism of NOXA degradation and its effect on the development and drug resistance of colorectal cancer,to clarify the relationship between PRDX1 and protein ubiquitin/neddylation modification,and to explore the significance of PRDX1CUL5-NOXA as a therapeutic target or individual treatment differentiation marker for colorectal cancer.MethodsThe transcription and expression of NOXA and PRDX in colorectal cancer were detected by open database analysis and immunohistochemical experiment,and the relationship between NOXA protein half-life and cell proliferation in different cell lines was determined by cycloheximide(CHX)interfering protein synthesis method.Colorectal cancer cell lines with overexpression of PRDX1 and silencing of PRDX1 were constructed by gene cloning,plasmid construction and lentivirus infection,and the regulatory effect of PRDX1 on apoptosis-related cell proliferation was analyzed by cell proliferation,clone formation and flow cytometry assays.Gene transcription and protein expression were detected by RT-PCR and Western blotting assays,the effects of PRDX1 on NOXA and NOXA-induced apoptosis-related proteins were analyzed.Protein coimmunoprecipitation and Western blotting assay were applied to detect the effect and mechanism of PRDX1 on NOXA degradation rate and upstream ubiquitin/neddylation modification pathways.Non-reducing Western blotting was used to analyze the structural state of PRDX1 regulating NOXA degradation.A gene-damage inducer Etoposide was used to treat different cell models to verify the mechanism of PRDX1 to enhance cell resistance by inhibiting apoptosis through NOXA degradation.ResultsIn this study,we found that NOXA is a highly expressed but short-lived protein in CRC,and there is a positive between NOXA degradation rate and cell proliferation.PRDX1 inhibits CRC cell apoptosis by downregulating NOXA,and mechanistically,PRDX1 promotes NOXA ubiquitination and degradation,which completely depend on CUL5 neddylation.Further studies have demonstrated that PRDX1 oligomers bind with both the Nedd8-conjugating enzyme UBE2F and CUL5 and that this tricomplex is critical for CUL5 neddylation,since silencing PRDX1 or inhibiting PRDX1 oligomerization greatly dampens CUL5 neddylation and NOXA degradation.An increase in reactive oxygen species(ROS)is not only a hallmark of cancer cells but also the leading driving force for PRDX1 oligomerization.As shown in our study,although ROS play a role in upregulating NOXA mRNA transcription,ROS scavenging in CRC cells by N-acetyl-Lcysteine(NAC)can significantly reduce CUL5 neddylation and extend the NOXA protein half-life.Therefore,in CRC,PRDX1 plays a key role in maintaining intracellular homeostasis under conditions of high metabolic activity by reinforcing UBE2F-CUL5mediated degradation of NOXA,which is also evidenced in the resistance of CRC cells to etoposide treatment.Based on these findings,targeting PRDX1 could be an effective strategy to overcome the resistance of CRC to DNA damage-inducing chemotherapeutics.ConclusionThe UPS-mediated degradation of NOXA is regulated by PRDX1-CUL5-neddylation:PRDX1 oligomers induced by ROS can form a complex with CUL5 and the NEDD8conjugating enzyme UBE2F,which possibly facilitates the transfer of NEDD8 from UBE2F to CUL5,specifically enhancing CUL5 neddylation to active the ubiquitin-E3 ligase CRL5,promoting NOXA ubiquitination and subsequently UPS-mediated degradation.Collectively,these results support the tumor-promoting effect of ROS and PRDX1,clarify the mechanism of PRDX1 regulating NOXA ubiquitination and degradation,and reveal one of the pathways by which CRC cells maintain homeostasis under metabolic stress.