Dub3 Deubiquitinates And Stabilizes NRF2 In Chemotherapy Resistance Of Colorectal Cancer

Colorectal cancer?CRC?is the third most commonly diagnosed cancer among both men and women worldwide,causing more than half a million deaths per year.Many researchers have attempted to unravel the complex role of NRF2 in CRC.The NRF2 transcription factor plays a dual role in colorectal cancer.5-Fluorouracil?5-FU?and camptothecin?CPT?are the most common chemotherapeutic agents used for the treatment of CRC.Unfortunately,multidrug resistance remains one of the main obstacles to efficient chemotherapy of CRC.Several studies have revealed the relation between NRF2 overexpression and increased resistance to 5-FU in CRC.Resistance to paclitaxel in mammary cancer was also found to be related to NRF2 overexpression.In this study,we focused on the molecular mechanism of resistance to camptothecin and paclitaxel in CRC cells.NRF2 is an important transcription factor regulating intracellular homeostasis.NRF2 regulates a battery of downstream genes by recognizing enhancer sequences called antioxidant response elements?AREs?in their promoters.These ARE-genes encode a network of cooperating enzymes that participate in various pathophysiological processes.Under cell homeostatic conditions,the NRF2 protein level and transcriptional activity are relatively low.It has been reported that there are three E3ubiquitin ligase complexes controling the ubiquitination and proteasomal degradation of NRF2.Although the E3 ubiquitin ligase has been very well studied,the DUBs of NRF2 remain largely unknown.Consequently,identifying the DUB?s?of NRF2 will make it easier and more effective to explore the regulatory network of NRF2-ARE signaling.DUB3,a member of the highly conserved human DUB/USP17 family,is induced in response to cytokine stimulation.Cys89 in DUB3 functions as the deubiquitinating catalytic active site,and its mutation to Ser(DUB3^C89S)blocks the deubiquitinating catalytic activity.Recently,accumulating evidence has highlighted the important function of DUB3 in multiple cellular processes and diseases.The high protein level of DUB3 in tumors regulates cell proliferation,metastasis,and epithelial–mesenchymal transition,indicating that DUB3 is a potential therapeutic target for treating cancer.In this study,we detected the expressions of DUB3 and NRF2 in 24 pairs of samples collected from colorectal cancer patients.The results suggested that DUB3 and NRF2 were positively correlated,and both of them had the tendency of high expression in cancer tissues.At the same time,we also validated this conclusion in seven colorectal cancer cell lines,and selected HCT116 and RKO cell lines for subsequent molecular experiments.We found that DUB3 upregulated NRF2 protein level and transcriptional activity,and activated the NRF2-ARE signaling pathway,depending on its ubiquitinase activity.At the same time,we excluded the effect of DUB3 on the transcription level of NRF2,and found that DUB3 did not affect the NRF2 mRNA level.Further,in order to verify the relationship between them,we proved that DUB3,NRF2 and KEAP1 may form a complex to regulate the dynamic equilibrium of NRF2.DUB3,relying on its deubiquitinase activity,protected NRF2 from proteasome degradation by removing the ubiquitination of the K48 junction of NRF2.Subsequently,we explored the effects of DUB3 stabilizing NRF2 on colorectal cancer cells.The results showed that DUB3caused resistant to paclitaxel and camptothecin in colorectal cancer cell lines by stabilizing NRF2.In this study,we reported for the first time that DUB3 was the deubiquitinase stabilizing NRF2 protein level.DUB3 decreased the NRF2 K48-ubiquitination level and promoted NRF2 stability by forming a complex with NRF2 and KEAP1.More importantly,we revealed the molecular mechanism by which DUB3 caused chemotherapy resistance by promoting NRF2 stability in colon cancer.Our study fills in the blank in the research of NRF2 deubiquitination system and provides novel insights into the protein homeostasis of NRF2 in tumorigenesis.