Investigating The Role Of Serum And Glucocorticoid-regulated Protein Kinase 1(SGK1)in Redox Homeostasis In Cervical Cancer

Objective:Cervical cancer is one of the most widespread gynecological malignant tumors.Patients with cervical cancer often develop to the late stage at the time of diagnosis due to the lack of obvious symptoms at early stage.Importantly,patients with advanced or recurrent cervical cancer usually have poor prognosis and limited therapeutic options,it is therefore imperative to find new treatment strategies against this disease.Serum and glucocorticoid-induced protein kinase 1（SGK1）is a major downstream effector of PI3K signaling,and its deregulated activation has been implicated in the pathogenesis and progress of a variety of tumors.However,the role of SGK1 in cervical cancer has not been explored.Previous study has displayed that the lack of SGK1 activity in decidualizing cells enhances susceptibility to oxidative cell death,suggesting the possibility that SGK1 participates in redox regulation.The regulation of redox homeostasis is fundamental to maintaining normal cellular functions and ensuring cell survival.In this study,we aim to investigate the biological role of SGK1 in cervical cancer and evaluate its potential as a therapeutic target in cervical cancer.We found that SGK1functions as an anti-oxidative protein promoting cell survival via the c-JUN/NRF2 axis.This work opens new perspectives on exploring the potential of SGK1 inhibitors as sensitizing agents to enable the design of therapeutically redox-modulating strategies against cervical cancer.Methods:In vitro:（1）Inhibition of SGK1 was achieved by si RNA or its pharmacological inhibitor GSK650394 in four cervical cancer cell lines and H₂O₂ was used to stimulate cells to examine the biological role of SGK1.We detected cell viability and ROS levels of these cells treated as above described to verify whether SGK1exhibited antioxidant ability.Through using ROS scavenger GSH to check that SGK1relies on its antioxidant effect to enhance cervical cancer cell survival.（2）Comparing the transcriptomes of SGK1-knockdown cells with those of the control cells by RNA-Seq,and GSEA analysis was performed to investigate whether the SGK1 knockdown was accompanied by changes in redox-related signal pathways.q RT-PCR was used to examine the effects of SGK1 inhibition on the expression of NRF2 and its target genes.（3）We stably expressed constitutively activated mutant SGK1-S422D or kinase-dead mutant SGK1-K127M in cervical cancer cells.We then knock-downed NRF2 in these cells and conducted Western Blot analysis and q RT-PCR to confirm whether SGK1activity was relevant to NRF2 expression and activity.The effect of SGK1 knockdown on protein level of c-JUN was evaluated to examine if SGK1 regulates the stability of c-JUN in the presence or absence of MG132.Subsequently,we knock-downed c-JUN in SGK1-S422D cells and SGK1-K127M cells and examined the expression of NRF2 and ROS levels to investigate whether SGK1 functions through modulating SGK1-c-JUN-NRF2 signal pathway to exert its antioxidant activity.（4）Western Blot analysis,ROS detection,Annexin V/PI staining assays and 3D sphere culture assay were performed to explore whether the combined use of Melatonin and SGK1 inhibitor GSK650394 can cause redox dysregulation of cervical cancer cells and render cervical cancer cells sensitive to the treatment.In vivo:（5）We generated ME180 human cervical tumor xenografts to investigate whether melatonin potentiates the therapeutic effect of SGK1inhibition on cervical cancer in vivo.（6）We conducted IHC for proteins in ME180-xenograft tumors treated with GSK650394 or melatonin alone,or in combination for 4days to validate our in vitro findings and examine whether combining SGK1 inhibitor and melatonin is effective in the treatment of cervical tumors.Results:（1）Inhibiting SGK1 impaired the growth ability of cervical cancer cells;GSEA results indicated that the oxidative phosphorylation signaling pathway was negatively enriched in SGK1 knockdown cells,indicating a positive correlation between SGK1 and oxidative phosphorylation.The results of cellular ROS detection and cell viability assay exhibited that the inhibition of SGK1 result in the increase of ROS level in cells and rendered cells more sensitive to H₂O₂.However,the treatment of cervical cancer cells with GSH reversed these phenomena,indicating that SGK1 may participate in the regulation of redox homeostasis and enhance the survival of cervical cancer cells by exerting its antioxidant effect.（2）We analyzed RNA-seq results and clinical database and found that the expression of SGK1 exhibited significant positive correlation with the antioxidant transcription factor NRF2.Based on the results of Western Blot analysis and q RT-PCR in SGK1-S422D cells and SGK1-K127M cells,we discovered that the expression of NRF2 and its transcriptional target genes in SGK1-S422D cells were strikingly increased compared with that in SGK1-K127M cells.Furthermore,si RNA-mediated NRF2 silencing downregulated the expression of NRF2 transcriptional targets in SGK1-S422D cells but not in SGK1-K127M cells.These results indicated that SGK1kinase activity is essential for regulating NRF2 expression and transcriptional activity.（3）In the condition of using proteasome inhibitor MG132,SGK1 knockdown no longer down-regulated c-JUN protein level,suggesting that SGK1 positively regulates the stability of c-JUN;The knockdown of c-JUN induced the increase of ROS level and decrease the expression of NRF2 in S422D cells.These data suggested that SGK1 relies on its activity to positively regulate the expression of NRF2 by increasing c-JUN protein level.（4）GSK650394 treatment rendered the cells more sensitive to Melatonin,leading to excessive accumulation of ROS,which in turn increased cytotoxicity evidenced by Annexin V/PI staining assays and 3D sphere culture assays.（5）In line with in vitro observations,our in vivo xenograft experiments showed that the combined treatment of Melatonin and GSK650394 resulted in severe oxidative damage and dramatically reduced the tumor volumes.Conclusions:（1）SGK1 promotes cervical cancer cell survival by an anti-ROS mechanism（2）In cervical cancer cells,SGK1 may positively regulates NRF2 expression and activity by upregulating c-JUN protein level.（3）Inhibition of SGK1 combined with melatonin will give rise to ROS accumulation and increase cytotoxicity in cervical cancer cells.（4）Targeting the antioxidant SGK1-c-JUN-NRF2 axis may represent an efficacious and promising therapeutic strategy against cervical cancer.