AKT1 Participates In Ferroptosis Vulnerability By Driving Autophagic Degradation Of FTH1 In Cisplatin Resistant Ovarian Cancer

AKT1 participates in ferroptosis vulnerability by driving autophagic degradation of FTH1 in cisplatin resistant ovarian cancerOvarian cancer is a platinum-sensitive tumor with a high response rate to first-line conventional treatment.However,75% of advanced patients still undergo metastasis and/or recurrence after being treated with cisplatin(DDP)for a period of time,and DDP resistance ultimately affects the long-term survival rate of ovarian cancer patients.The mechanism of DDP resistance in ovarian cancer is complex,and research suggests that it may be related to increased drug efflux,epigenetic changes,enhanced DNA repair,and activation of autophagy.Although the growth-promoting and deathpromoting effects of autophagy in the process of tumor formation are still controversial,a large number of studies support that under the microenvironmental pressure caused by chemotherapeutic drugs,tumor cells will stimulate autophagy as a protective mechanism to fight against the cytotoxicity of chemotherapeutic drugs ultimately leads to the emergence of chemotherapy resistance.Drug-resistant ovarian cancer cells may be more sensitive to ferroptosis activators.Ferroptosis is a new type of iron-dependent programmed cell death.Recent studies have pointed out that ferroptosis may be autophagy-dependent.This is because the response to the ferroptosis activator leads to the accumulation of autophagosomes,and the components of the autophagy mechanism contribute to the occurrence of ferroptosis.A cystine glutamate antiporter,called x CT(SLC7A11),contributes to de novo synthesize antioxidant glutathione(GSH).The function of GSH in catalyzing the detoxification of phospholipid hydroperoxides is essential for cell survival.GSH is also the substrate of phospholipid hydroxyperoxidase glutathione peroxidase(GPX4).Therefore,cysteine deprivation and GPX4 inhibition can effectively induce ferroptosis.GPX4 inhibitor RSL3,as well as Erastin,the direct inhibitor of SLC7A11,are widely used to induce ferroptosis.Our study aimed to explore the susceptibility of DDP resistant ovarian cancer cells to ferroptosis.Using two ovarian cancer parents and DDP resistant cell lines,we found that DDP resistant ovarian cancer cells were more sensitive to the ferroptosis inducer Erastin.We further explored the molecular mechanism and found that the high autophagy level of DDP resistant cells led to an increase in the level of free iron ions.Importantly,animal experiments show that the combination of Erastin and DDP has a good therapeutic effect on DDP resistant ovarian cancer cells.Thus,the present research may implicate targeting to ferroptosis as a promising therapeutic candidate to overcome DDP chemoresistance in ovarian cancer.Methods:1.Increased sensitivity of cisplatin-resistant ovarian cancer cells to Ferroptosis(1)Verification of cisplatin-resistant ovarian cancer cell lineSKOV3 and A2780 cisplatin-resistant ovarian cancer cell lines(Res)and parent cell lines(Par)were constructed by the research group in the early stage.Before the experiment,they were treated with 0,2.5,5,10 and 20μM cisplatin,respectively.The viability of parent cells and drug-resistant cells was detected by CCK8 method.(2)Increased sensitivity of cisplatin-resistant ovarian cancer cells to FerroptosisThe ovarian cancer parents and drug-resistant cell lines were treated with 20μM Erastin for 24 h,and the cell viability was evaluated by CCK8 method.The levels of MDA,GSH and SOD were detected by ELISA,and the levels of free iron were detected by iron ion colorimetry.ROS level was detected by fluorescent probe staining.The cells were pretreated with ferristatin-1 and then killed by Erastin.The cell viability was evaluated by CCK8 method to determine the increased sensitivity of cisplatin-resistant ovarian cancer cells to Ferroptosis.2.Targeted Ferroptosis pathway reverses cisplatin resistance in ovarian cancer(1)Erastin enhances the killing effect of cisplatin on drug-resistant ovarian cancer cellsSKOV3-Res and A2780-Res cells were treated with cisplatin alone or cisplatin combined with Erastin.CCK8 method was used to determine the cell viability,and calcein/PI was used to stain the living/dead cells to determine the apoptosis rate.(2)Erastin enhances the therapeutic effect of cisplatin on ovarian cancer drug-resistant model in miceAdd 2×106 SKOV3-Res cell suspensions were intraperitoneally injected into C57BL/6 female mice to prepare the peritoneal metastasis model of ovarian cancer.After one week,the mice received three different treatments: control(DMSO,0.05%),DDP(4mg/kg)or DDP(4mg/kg)+ Erastain(1mg/kg).Mice were injected intraperitoneally once every 3 days for 4 weeks.The survival time of mice,ascites volume,number of abdominal wall metastatic tumors were counted,and the invasion of abdominal wall by metastatic tumor was detected by HE staining.3.Cisplatin-resistant ovarian cancer cells are susceptible to Ferroptosis through FTH1 autophagy degradation(1)Erastin sensitivity of cisplatin-resistant ovarian cancer does not depend on Ferroptosis defense systemThe levels of GPX4,NRF2 and SLC7A11 proteins in the parent and DDP-resistant SKOV3 and A2780 cells were detected by Western Blot with or without Erastin treatment.NRF2 target genes HO1 and NQO1 were detected by RT-PCR.(2)FTH1 decreased in cisplatin-resistant ovarian cancer cellsThe ferritin FTL1 and FTH1 in the parent and DDP-resistant SKOV3 and A2780 cells were detected by Western Blot with or without Erastin treatment.The m RNA expression of ferritin FTL1 and FTH1 was detected by RT-PCR.(3)FTH1 degradation increased in cisplatin-resistant ovarian cancer cellsThe cells were treated with 10μ/m L of cycloheximide(CHX)to block the synthesis of new proteins to observe the protein stability of FTH1 in parent cells and cisplatin-resistant cells.After the FTH1 plasmid was transfected into SKOV3-Res and A2780-Res cells,the response of cells to Erastin-induced Ferroptosis was detected by live/dead staining and CCK8 method.(4)Increased autophagy level of cisplatin-resistant ovarian cancer cellsThe autophagic bodies of cisplatin-resistant ovarian cancer cells and their parents were observed by transmission electron microscopy,and the expression of autophagic marker LC3 b in cisplatin-resistant ovarian cancer cells and their parents was detected by immunofluorescence staining.After treatment with autophagy inhibitor 3-MA,the expression of FTH1 protein was detected by Western Blot,and the sensitivity of cisplatin-resistant ovarian cancer cells to Erastin was evaluated by CCK8 method.4.AKT1 deletion mediates FTH1 autophagy degradation in cisplatinresistant ovarian cancer cells(1)Screening of cisplatin-resistant genes in ovarian cancer based on GEO databaseBy comparing the sequencing results of 17 patients with complete response(CR)to cisplatin and 10 patients with incomplete response(IR)in the GEO database GSE23603,we analyzed the differential genes of cisplatin resistance,and KEGG analyzed the molecular pathway enriched by the differential genes.(2)Association between AKT1 deletion and prognosis of ovarian cancerThe relationship between AKT1 expression level and prognosis of ovarian cancer was analyzed by TCGA database.AKT1 was divided into high expression group and low expression group by the median expression level.Nomogram model was used to evaluate the risk of AKT1 loss in ovarian cancer patients.(3)AKT1 deletion up-regulates autophagy level of cisplatin-resistant ovarian cancer cellsTransduce exogenous AKT1 in SKOV3-Res and A2780-Res cells,and detect the expression of AKT1,LC3 b,p-ULK1 and FTH1 by Western blot.CCK8 method was used to detect the Ferroptosis induced by Erastin after the recovery of AKT1 expression in cisplatin-resistant ovarian cancer cells.Results:1.Increased sensitivity to Ferroptosis in cisplatin-resistant ovarian cancer cells(1)Verification of cisplatin-resistant ovarian cancer cell lineCompared with parent cells,SKOV3-Res cells have stronger resistance to cisplatin(IC50/SKOV3-Par=10.717,IC50/SKOV3-Res=23.830).Compared with parent cells,A2780-Res cells have stronger resistance to cisplatin(IC50/A2780-Par=9.528,IC50/A2780-Res=25.591).The above results showed that SKOV3 and A2780 cisplatinresistant ovarian cancer cell lines were successfully constructed.(2)Increased sensitivity of cisplatin-resistant ovarian cancer cells to Ferroptosis(1)The ovarian cancer parents and cisplatin-resistant cell lines were treated with20μM Erastin for 24 h,and the cell viability was evaluated by CCK8 method.Compared with the parent cells,the viability of SKOV3-Res cells and A2780-Res cells were significantly reduced.Compared with the parent cells,the levels of SOD and GSH in SKOV3-Res and A2780 cells were significantly decreased and the level of MDA was increased after the treatment of Erastin.Cell iron accumulation is one of the typical signs of Ferroptosis.After Erastan treatment,the level of ferrous ion in SKOV3-Res and A2780 cells was significantly higher than that of their parent cells.The increase of lipid ROS is an important feature of cell Ferroptosis.The ROS level in SKOV3-Res and A2780-Res cells treated with Erastin was significantly higher than that of their parent cells.(2)After pretreatment with 10μM Fer-1 for 2 hours,the killing effect of Erastin was evaluated.It was found that inhibiting the Ferroptosis pathway saved the killing effect of Erastin on the parent and cisplatin-resistant ovarian cancer cells.These results suggest that cisplatin-resistant ovarian cancer cells have high Ferroptosis sensitivity compared with their parent cells.2.Targeted Ferroptosis pathway reverses cisplatin resistance in ovarian cancer(1)Erastin enhances the killing effect of cisplatin on drug-resistant ovarian cancer cellsCompared with cisplatin alone,cisplatin combined with Erastin treatment significantly reduced the viability of drug-resistant ovarian cancer cells and significantly increased the apoptosis rate.It is suggested that Erastin enhances the killing effect of cisplatin on drug-resistant ovarian cancer cells in vitro.(2)Erastin enhances the therapeutic effect of cisplatin on ovarian cancer drug-resistance model in miceCompared with the mice in the DDP treatment group alone,the mice in the DDP+Erastin combined treatment group had fewer abdominal wall metastatic tumors,and the mice in the DDP+Erastin combined treatment group had lower invasion of abdominal wall,smaller ascites volume,and longer median survival time.The above results show that the combination of Erastin and cisplatin has a stronger killing effect on cisplatin-resistant ovarian cancer in vitro and in vivo.3.Cisplatin-resistant ovarian cancer cells are susceptible to Ferroptosis through FTH1 autophagy degradation(1)Erastin sensitivity of cisplatin-resistant ovarian cancer does not depend on Ferroptosis defense system(1)The expression of key molecules of Ferroptosis defense system in SKOV3-Res cells was significantly higher than that in SKOV3-Par cells without the killing of Erastin.Erastin significantly induced the up-regulation of NRF2 and SLC7A11,and did not change the expression difference of SKOV3-Res cells and SKOV3-Par cells.However,Erastin has a strong inhibitory effect on GPX4 expression in SKOV3-Res cells.After Erastin treatment,the expression of GPX4 in SKOV3-res cells was significantly lower than that of its parent cells.(2)The expression of GPX4,NRF2 and SLC7A11 in A2780-Res cells was significantly higher than that in A2780-Par cells without Erastin killing.Erastin treatment resulted in significant down-regulation of GPX4 in A2780-Res cells.RT-PCR results showed that the NRF2 target genes HO1 and NQO1 increased in cisplatinresistant ovarian cancer cells,which proved that the activity of anti-ROS system increased.The above results show that the high sensitivity of cisplatin-resistant ovarian cancer cells to Erastin does not seem to depend on the inactivation of the Ferroptosis defense system.(2)The down-regulation of ferritin FTH1 in cisplatin-resistant ovarian cancer cellsWestern blot analysis of transferrin FTL1 and FTH1 showed that compared with parent cells,the level of FTH1 protein in SKOV3-Res and A2780-Res cells increased,but the level of FTL1 protein did not increase.The protein expression of FTH1 and FTL1 was inhibited after Erastin treatment,but the expression level of FTH1 in cisplatin-resistant ovarian cancer cells was still lower than its parent cells.RT-PCR results showed that there was no significant difference in the m RNA level of FTH1 in cisplatin-resistant cells compared with the parent cells.These data indicate that FTH1 may be subject to post transcriptional regulation.(3)FTH1 degradation increased in cisplatin-resistant ovarian cancer cells(1)Blocking the synthesis of new protein with 10μ/m L CHX treatment cells,and taking the expression of FTH1 protein in cells without CHX treatment as the starting standard value,it was found that the degradation rate of FTH1 protein in cisplatinresistant ovarian cancer cells was significantly higher than that of their parent cells.(2)After recovering the expression of FTH1 protein in cisplatin-resistant ovarian cancer cells,the sensitivity of cisplatin-resistant ovarian cancer cells to Erastin-induced Ferroptosis decreased.It is suggested that cisplatin-resistant ovarian cancer cells may increase the level of free iron ions in LIP by promoting the degradation of FTH1,which may be the reason for the increased sensitivity to Ferroptosis.(4)Increased autophagy level of cisplatin-resistant ovarian cancer cells(1)The autophagic bodies of cisplatin-resistant ovarian cancer cells and their parent cells were observed by transmission electron microscopy.It was found that the autophagic bodies of SKOV3-Res and A2780-Res cells were increased compared with the corresponding parent cells.The expression of autophagy marker LC3 b in cisplatinresistant ovarian cancer cells and their parent cells was detected by immunofluorescence staining.It was found that the expression of LC3 b in SKOV3-Res and A2780-Res cells was higher than that of the corresponding parent cells.It showed that cisplatin-resistant ovarian cancer cells maintained high autophagy level.(2)After treatment with autophagy inhibitor 3-MA,the expression of FTH1 protein was detected by Western blot,and it was found that blocking autophagy restored the expression of FTH1 protein.It is suggested that the high autophagy level of cisplatin-resistant ovarian cancer cells leads to the degradation of FTH1 protein.(3)3MA treatment reduced the sensitivity of SKOV3-Res and A2780-Res cells to Erastin.These results suggest that the degradation of FTH1 caused by high autophagy level mediates the sensitivity of cisplatin-resistant ovarian cancer cells to Ferroptosis.4.AKT1 deletion mediates FTH1 autophagy degradation in cisplatinresistant ovarian cancer cells(1)Screening of cisplatin-resistant genes in ovarian cancer based on GEO databaseBy comparing the sequencing results of 17 CR patients and 10 IR patients in the GEO database GSE23603,it was found that the autophagy inhibitor AKT1 in the sequencing results of IR patients was significantly reduced.KEGG enrichment analysis showed that the differential genes of CR and IR patients were enriched in MAPK and PI3K-AKT pathways.(2)Association between AKT1 deletion and prognosis of ovarian cancerTCGA database analyzed the relationship between the expression level of AKT1 and the prognosis of ovarian cancer,and found that the low expression of AKT1 may indicate a shorter survival time and poor prognosis.Survival analysis showed that the loss of AKT1 expression in ovarian cancer patients was associated with low overall survival rate,low disease-specific survival rate and shorter progression-free interval.These results suggested that the loss of AKT1 is a poor prognostic factor for ovarian cancer.(3)AKT1 deletion up-regulates autophagy level of cisplatin-resistant ovarian cancer cellsWhen exogenous AKT1 is transduced in SKOV3-Res and A2780-Res cells,p ULK1 decreases and LC3 b expression increases,indicating that cisplatin-resistant cells maintain autophagy by down-regulating AKT1.After restoring the expression of AKT1,the expression of FTH1 in drug-resistant ovarian cancer cells was saved.After restoring the expression of AKT1 in cisplatin-resistant ovarian cancer cells,the ferroptosis induced by Erastin decreased.It is suggested that the loss of AKT1 may be the reason why cisplatin-resistant ovarian cancer cells are highly sensitive to ferroptosis inducers,which is caused by the degradation of FTH1 autophagy mediated by p-ULK.Conclusion:1.The deletion of AKT1 causes cisplatin-resistant ovarian cancer cells to maintain high autophagy level;2.AKT1 deletion-mediated FTH1 autophagy degradation results in cisplatinresistant ovarian cancer cells sensitive to iron death;3.Targeted iron death may be a potential way to reverse cisplatin resistance in ovarian cancer.