OTUD5 Regulates The Ferroptosis And Stemness Of Hepatoma Cells By SLC7A11

ObjectiveThere is a special cell population in cancer,namely tumor stem cells(CSCs),which have the potential of self-renewal and unlimited proliferation.They mainly lead to the heterogeneity and resistance to conventional radiotherapy and chemotherapy in malignant tumors.Reactive oxygen species(ROS)and iron metabolism can not only regulate the function of CSCs,but also induce cell death.Ferroptosis is a new type of cell death with iron-dependent and induced by ROS which catalyzes the lipid peroxidation of polyunsaturated fatty acids(PUFAs)on cell membrane.Ferroptosis plays a significant inhibitory role in a variety of tumors,especially targeting ferroptosis to kill CSCs has a broad application prospect.However,the sensitivity of CSCs to ferroptosis remains to be explored.Therefore,we carry out this project to explore the sensitivity and response mechanism of cells with different stemness to ferroptosis,and investigate the effects of the upstream regulator of this factor on cell ferroptosis and stemness,so as to provide new ideas for targeting ferroptosis and effectively killing CSCs and establish effective strategies,which is expected to eradicate tumors and prevent recurrence in combination with traditional treatment.MethodsFirst,we cultured two kinds of cells with different SP proportion,tumor sphere forming cells and all trans retinoic acid(ATRA)-induced differentiated cells,which were treated with ferroptosis inducer(Erastin,IKE,RSL3)and/or inhibitor ferrostatin-1(Fer-1).Further,CCK-8 reagent was used to detect their survival,589/591 C11 dye combined with flow cytometry was used to analyze their lipid ROS level,and transmission electron microscope(TEM)was used to observe their mitochondrial morphology.It was determined that cells with higher stemness were more sensitive to ferroptosis.Subsequently,RT-q PCR and Western Blot were used to detect the m RNA and protein expression of key ferroptosis-related factors(SLC7A11,GPX4,ACSL4 and COX2)in differentiated cells.We found that the expression of cystine transporter SLC7A11 protein of system X_C~-was significantly up-regulated in differentiated cells,but the m RNA level of SLC7A11 did not change significantly.Then,ATRA induced cells were treated with proteasome inhibitor MG132,the results showed that SLC7A11 may be regulated by ubiquitin proteasome system(UPS)in differentiated cells.Therefore,we transiently transferred 89 deubiquitination plasmids into 293T cells to identify deubiquitinase(DUB)OTUD5 stabilizing SLC7A11 and then verified the expression of OTUD5 in differentiated cells by Western Blot.Further,their interaction between OTUD5 and SLC7A11 was proved by Co-Immunoprecipitation(Co-IP)and immunofluorescence(IF)experiments.The half-life degradation experiment,ubiquitin proteasome pathway experiment and de-ubiquitination experiment were used to proved that OTUD5 and its mutants(S177A and C224S)up-regulated the protein expression of SLC7A11 through deubiquitination.Then,the effect of OTUD5 on ferroptosis of hepatoma cells was explored by cell survival experiment and lipid ROS level detection using RSL3 treatment.In addition,the effects of SLC7A11,OTUD5 and its mutants on cell stemness were investigated by tumor sphere formation test and flow cytometry(the proportion of SP cells was detected with Hochest 33342 dye,the activity of acetaldehyde dehydrogenase(ALDH)was analyzed by ALDEFLUOR probe and CD338+cells were measured with fluorescent CD338 antibody).Then,the effects of SLC7A11 and OTUD5 on the protein expression level of core stemness-related factors(c-Myc,OCT-4,SOX2 and KLF4)were analyzed by Western Blot.It was found that the change of c-Myc was the most obvious.Further,the effects of OTUD5 on cells induced by targeted drug Sorafenib and c-Myc inhibitor EN4 were analyzed by cell proliferation test and tumor sphere formation experiment.Finally,the effect of Sorafenib and EN4 on tumor cells was determined by detecting the activity of ALDH and the level of lipid ROS.ResultsIn this study,compared with the cells with lower stemness,we found that the cells with higher stemness(tumor cells with higher SP proportion,tumor sphere forming cells and undifferentiated cells)had lower survival number,higher lipid ROS level,mitochondrial shrinkage and ridge thickening after treatment with ferroptosis inducers.Moreover,we found that the expression of SLC7A11 protein increased in differentiated cells and was regulated by UPS.The expression of the deubiquitinase OTUD5,which upregulates SLC7A11,was also elevated in differentiated cells.Further studies found that OTUD5 interacted with SLC7A11 and OTUD5could stabilize SLC7A11 by removing the ubiquitin chain connected to SLC7A11 protein,while the C224 mutation of OTUD5 could not regulate the deubiquitination of SLC7A11.Moreover,under the induction of RSL3,OTUD5 could increase the number of viable cells and reduce the level of lipid ROS in cells.Further studies showed that both SLC7A11 and OTUD5 could accelerate the formation of tumor spheres,increase the proportion of SP cells,enhance the ALDH activity of cells and increase the number of CD338+cells by up-regulating the expression of c-Myc.In addition,under the induction of Sorafenib and c-Myc inhibitor EN4,OTUD5 could promote cell proliferation and accelerate the formation of tumor spheres.Finally,compared with Sorafenib or EN4 alone,the combined induction of Sorafenib and EN4 greatly inhibited the activity of ALDH and increased the level of lipid ROS in hepatoma cells.Conclusions1.The tumor cells with higher stemness are more sensitive to ferroptosis,and OTUD5 promotes the resistance of differentiated cells to ferroptosis by deubiquitylating SLC7A11.2.SL7A11 and OTUD5 can enhance cell stemness by up-regulating c-Myc.The combination of Sorafenib and EN4 can inhibit hepatoma cells proliferation.