A Preliminary Investigation On The Mechanism Of Exosome-carrying HSP90α Inhibiting Autophagy-dependent Ferroptosis In Angiosarcoma Cells By Regulating The MTOR-GPX4 Signaling Axis

Objective:To investigate the mechanism by which exosomal delivery of HSP90αfrom angiosarcoma stem cell-like cells（Sphere cells）promotes tumor progression by inhibiting autophagy-dependent ferroptosis in angiosarcoma cells（ISOHAS cells）.To clarify the molecular mechanism by which HSP90αaffects autophagy-dependent ferroptosis in ISOHAS cells.Methods:（1）Exosomes were extracted and isolated from cell culture supernatants using an exosome extraction kit,and the morphology and size of the exosomes were identified by transmission electron microscopy.Western blot was used to detect the expression of exosome markers CD63,CD81 and HSP90α.（2）ISOHAS cells and Sphere cells-derived exosomes were incubated with ISOHAS cells for 24h,then western blot was used to detect the expression of HSP90αas well as the expression of SLC7A11 and GPX4,the key molecules of ferroptosis.The kit was used to detect the changes of Fe²⁺,MDA and GSH levels,the indicators of ferroptosis in ISOHAS cells.（3）GV141-HSP90αand siRNA-HSP90αwere used to overexpress and silence HSP90αin ISOHAS cells,respectively.Western blot was used to detect the expression of SLC7A11 and GPX4;the kit was used to detect the changes of Fe²⁺,MDA and GSH levels.（4）ISOHAS cells were treated with the ferroptosis inducer Erastin,and the effect of Erastin on cell migration was detected by scratch assay and Transwell assay.The expression of SLC7A11,GPX4 and the expression of autophagy-related molecules P62 and LC3 were detected by western blot.Erastin treatment of ISOHAS cells after incubation with Sphere cell-derived exosomes and ISOHAS cells after overexpression of HSP90αby GV141-HSP90α.Western blot to detect the expression of GPX4.The kits to detect changes in Fe²⁺,MDA,and GSH levels.（5）Detection of the molecular mechanism of mTOR affecting ferroptosis with the help of Ferr db V2 database.（6）After applying the mTOR inhibitor RAPA to ISOHAS cells,the effect of RAPA on migration was detected by scratch assay and Transwell assay.Autophagic vesicle was observed by MDC staining and AO staining.The expression of SLC7A11,GPX4,mTOR,P62,LC3 was detected by Western blot.RAPA treatment of ISOHAS cells incubated with exosomes and overexpression of HSP90α.Western blot to detect GPX4 expression.The kits to detect changes in Fe²⁺,MDA,GSH levels.（7）After applying autophagy inhibitor 3-MA to ISOHAS cells,the effect of 3-MA on migration was detected by scratch assay and Transwell assay.Autophagic vesicle was observed by MDC staining and AO staining.The expression of SLC7A11,GPX4,P62,LC3 was detected by Western blot.ISOHAS cells were treated with 3-MA after induced ferroptosis,CCK8 was applied to detect the change of cell activity,Western blot to detect the expression of GPX4.ISOHAS cells were treated with 3-MA after silencing HSP90α,Western blot to detect the expression of GPX4.The kit was used to detect the change of Fe²⁺,MDA,GSH level.Results:（1）ISOHAS cells and Sphere cell-derived exosomes upregulated the expression of HSP90αand GPX4 after incubation with ISOHAS cells.Exosomes inhibited the development of ferroptosis in ISOHAS cells by suppressing MDA,Fe²⁺accumulation and GSH depletion.Sphere cell-derived exosomes inhibited ferroptosis more significantly than ISOHA cell-derived exosomes.（2）Overexpression of HSP90αupregulated GPX4 expression.Overexpression of HSP90αinhibited MDA,Fe²⁺accumulation and GSH depletion,suggesting that HSP90αinhibited the occurrence of ferroptosis in ISOHAS cells by activating GPX4.Silencing of HSP90αinhibited GPX4 expression.Silencing of HSP90αpromoted MDA,Fe²⁺accumulation and GSH depletion,suggesting that HSP90αactivates the occurrence of ferroptosis in ISOHAS cells by inhibiting GPX4.（3）Erastin reduced the expression of SLC7A11,GPX4 in ISOHAS cells and could re-reduce the expression of GPX4 that was elevated by exosomes as well as overexpression of HSP90α.Erastin reversed the inhibited MDA,Fe²⁺accumulation and GSH depletion.It is suggested that Erastin can re-induce ferroptosis inhibited by Sphere cell-derived exosomes and overexpression of HSP90α.Erastin inhibits the migratory capacity of ISOHAS cells by activating ferroptosis.Erastin treatment of ISOHAS cells elevated the LC3Ⅱ/LC3Ⅰ ratio,suggesting that Erastin induced ferroptosis in ISOHAS cells while activating their autophagy.（4）Ferr db V2 database results suggest that mTOR can inhibit ferroptosis by interfering with GPX4 expression.（5）RAPA treatment of ISOHAS cells increased the number of autophagic vesicles as indicated by MDC and AO fluorescence staining.RAPA inhibited the expression of mTOR,P62 and GPX4,increased the LC3Ⅱ/LC3Ⅰ ratio.RAPA increased MDA,Fe²⁺accumulation and GSH depletion.RAPA inhibited ISOHAS cell migration.The results suggest that RAPA activates autophagy-dependent ferroptosis in ISOHAS cells by inhibiting the expression of mTOR and GPX4.RAPA could re-reduce the expression of GPX4 that was elevated by exosomes and overexpression of HSP90α.RAPA reversed the inhibited MDA,Fe²⁺accumulation and GSH depletion.It is suggested that RAPA can re-induce ferroptosis inhibited by exosomes and overexpression of HSP90α.Exosomes and overexpression of HSP90αinhibit autophagy-dependent ferroptosis.（6）After the treatment of ISOHAS cells with 3-MA,MDC and AO fluorescence staining results suggested that the number of autophagic vesicles was reduced.3-MA upregulated the expression of P62 and GPX4,decreased the LC3Ⅱ/LC3Ⅰ ratio.3-MA inhibited MDA,Fe²⁺accumulation and GSH depletion.3-MA promoted the migration of ISOHAS cells.After co-treatment of Erastin with 3-MA in ISOHAS cells,3-MA could significantly inhibit cell death caused by Erastin.3-MA could re-elevate the expression of GPX4 inhibited by Erastin.3-MA can re-regulate GPX4 expression inhibited by silencing HSP90α.3-MA could reverse the promoted MDA,Fe²⁺accumulation and GSH depletion.The results suggest that 3-MA can re-inhibit ferroptosis activated by silencing HSP90α.Silencing of HSP90αactivates autophagy-dependent ferroptosis.Conclusion:（1）Angiosarcoma stem-like cell-derived exosomes inhibit autophagy-dependent ferroptosis in angiosarcoma cells through upregulation of HSP90α.（2）HSP90αmay inhibit autophagy-dependent ferroptosis through activation of the mTOR-GPX4 signaling axis.（3）The ferroptosis inducer Erastin may induce autophagy in angiosarcoma cells.