Study On The Role And Mechanism Of USP22 In Controlling Melanoma Metastasis And Vulnerability To Ferroptosis

Background: Cutaneous melanoma is the deadliest type of skin cancer with a highly aggressive phenotype.Metastasis is a major contributed factor that affects the prognosis of melanoma patients.Therefore,it is of great significance to elucidate the molecular mechanisms of melanoma metastasis and find novel therapeutic targets and avenues to improve the survival and prognosis of melanoma patients.Ubiquitin-Specific Proteases(USP)family is the largest and most diverse subfamily of deubiquitinases.More than fourty USPs have been reported to modulate cancer biological processes.However,potential pro-oncogenic USPs in melanoma progression and its underlying mechanisms still need further investigations.Ferroptosis is an iron-dependent form of regulated cell death,which is closely related to the progression and drug resistance of melanoma.Targeting ferroptosis becomes a new strategy for melanoma treatment.Previous studies revealed the role of the USP family in the regulation of ferroptosis.However,whether the USP family can affect melanoma progression by controlling the vulnerability to ferroptosis still needs further exploration.Objectives: This study aims to unravel the functional role and regulatory mechanisms of USP22 in melanoma metastasis and explores the sensitization effect of targeting USP22 in melanoma ferroptosis,thus providing a potential therapeutic intervention of advanced melanoma.Methods:1.Univariate COX regression analysis was performed on fifty-two USP genes survival profiles based on the TCGA-SKCM database.GEPIA2 was used to analyze USP22,USP35,USP36 and USP43 expression in melanoma tissues compared with the normal skin.The prognosis value of USP22 on melanoma was evaluated by survival analysis and multivariate COX regression analysis.The expression of USP22 in melanoma cells and tissues was detected by Western blot and immunohistochemistry.2.CRISPR/Cas9 was performed to generate USP22 knockout plasmid,and lentivirus were used to construct stable USP22 knockout and overexpression melanoma cell lines.3D Matrigel drop invasion assays and Transwell assays were carried out to determine migration and invasion abilities of melanoma cells.Lung metastasis model was further established to test the regulation of USP22 on melanoma metastasis in vivo.Bioinformatics analysis,q RT-PCR and WB were utilized to verify the effect of USP22 on melanoma EMT.3.Transcriptome sequencing was performed to elucidate the regulatory mechanism of USP22 in melanoma EMT and metastasis.GSEA analysis and WB were used to validate the relationship between USP22 and PI3K/AKT pathway.Mass spectrometry and PPI analysis were performed to explore the target protein of USP22.CHX assays and Co-IP were conducted to verify the interaction between USP22 and SIRT1.4.Potential USP22 inhibitor was identified through the FDA-approved drug library screening.Functional and rescue experiments were carried out to validate the inhibition effect of Topotecan on melanoma metastasis in vitro and vivo.The combination efficiency of USP22 silencing and ferroptosis was further evaluated by CCK-8 and Lipid ROS assaysResults:1.USP22 is tightly associated with poor clinical outcomes accompanied with a significantly upregulated expression in melanoma.Univariate and multivariate COX regression analysis confirmed USP22 is significantly associated with poor overall survival and disease specific survival,and USP22 expression is an independent prognostic factor of melanoma.Compared with normal skin tissue,the expression of USP22 is significantly upregulated in melanoma tissues and USP22 expression is positively correlated with melanoma pathological stage.2.USP22 promotes melanoma metastasis in vitro and vivo.Functional experiments confirmed that USP22 knockout remarkably attenuates melanoma migration and invasion in vitro;while USP22 overexpression enhances melanoma migration and invasion abilities.By establishing mice lung metastasis model,H&E images confirmed that USP22 loss significantly impairs the melanoma lung metastasis with less metastatic foci.3.USP22 controls melanoma metastasis through inducing epithelial-mesenchymal transition.EMT-related pathways and EMT-related genes are significantly activated in USP22 high expression group by analyzing public database.q RT-PCR confirmed that EMT-TFs are downregulated when USP22 is knocked down,and upregulated when USP22 is overexpressed.WB showed that the expression of epithelial marker E-cadherin increases in USP22 knockout melanoma cells,while the expressions of mesenchymal markers Vimentin and SNAL1 decrease.4.USP22 promotes melanoma metastasis and EMT by activating PI3K/AKT/m TOR pathway.Transcriptome sequencing and GSEA analysis confirmed that USP22 significantly activates the PI3K/AKT/m TOR pathway.USP22 knockout reduces the phosphorylation of AKT and m TOR.Rescue experiments confirmed that USP22 inhibition could not further enhance the inhibition of melanoma migration in the presence of PI3K/AKT/m TOR inhibitors.5.USP22 interacts with SIRT1 and regulates PI3K/AKT/m TOR signaling pathway through the SIRT1/PTEN axis.USP22 directly interacts with SIRT1.USP22 overexpression significantly prolongs the half-life of SIRT1.USP22 protects SIRT1 from proteasome-mediated degradation via deubiquitination.USP22 overexpression increases the expression of SIRT1,while inhibiting the acetylation of PTEN,thereby activating the PI3K/AKT/m TOR pathway.SIRT1 knockdown could partially eliminate this effect.Finally,SIRT1 expression is positively correlated with the EMT marker Vimentin.6.Topotecan is identified as a potential clinically applicable USP22 inhibitor by screening the FDA-approved drug library.Screening FDA-approved drug library by WB assay determined that Topotecan inhibits USP22 and its downstream SIRT1 expression.Topotecan treatment decreases phosphorylation of AKT and m TOR and EMT activity,which is attenuated in USP22 knockout melanoma cells.Topotecan has an equal effect with USP22 knockout on inhibiting melanoma lung metastasis where no synergy is between USP22 knockout and topotecan treatment.7.Both pharmacological inhibition and genetic knockout of USP22 sensitize RSL3-induced ferroptosis.Transcriptomics sequencing combined with GSEA analysis confirmed that ferroptosis pathway is significantly enriched in the RSL3 and Topotecan combination group.Ferroptosis biomarkers,PTGS2,CHAC1 and TFRC are highly expressed in combination group.The lipid ROS levels of combination group significantly increase.Further,USP22 knockout melanoma cells have increased susceptibility to RSL3,and this vulnerability is inhibited by PI3 K pathway inhibitors.Conclusions:1.USP22 controls melanoma EMT and metastasis through SIRT1/PTEN/PI3 K axis.2.Topotecan is a potential USP22 specific inhibitor and targeting USP22 can sensitize melanoma cells to ferroptosis.