Dual-targeted Inhibiting PI3K/mTOR Induces Apoptosis Of Ovarian Cancer Cells By Degradation Of Mcl-1 Through P62

In ovarian cancer chemotherapy,chemoresistance has become one of the bottlenecks in antitumor therapy.It is expected to explore new methods to overcome chemoresistance by increasing the anti-tumor effect of targeted drugs.It was found that PI3K/AKT/mTOR pathway which promotes cell survival can directly phosphorylate Bax,Bim and other proteins,leading to conformational changes and inhibit the activity of pro-apoptotic proteins,then induces apoptosis of cancer cells.Due to the cross-regulation mechanism among the key molecules of PI3K/AKT/mTOR pathway,the single-targeted PI3K or mTOR inhibitors had poor anti-tumor effect.Therefore,in order to increase the anti-tumor effect,the dual PI3K/mTOR inhibitors were invented.Thus,the research on the regulatory mechanism of PI3K/AKT/mTOR pathway onapoptosis-related proteins using dual PI3K/mTOR inhibitors may provide new clues for reversing chemotherapeutic resistance.With the deepening of research,it has been found that autophagy can be induced by inhibiting PI3K/mTOR pathway,but it's role is unclear.These suggest that it will help to clarify the mechanism of apoptosis activated by inhibiting PI3K/mOR through exploring the role of autophagy in cancer cells.It has been found that kinases such as ULK1,the downstream substrate of mTOR,can phosphorylate p62,promoting the binding of ubiquitin related domain(UBA)of p62 to the substrate protein,and realizing its precise regulation of autophagic degradation pathway of specific substrate protein.Previous researches have found that the anti-apoptotic protein Mcl-1 can be degraded through proteasome pathway,but whether autophagy is involved in the degradation of Mcl-1 remains unclear.Therefore,to explore the role of multifunctional protein p62/SQSTM1 as an autophagic receptor in promoting apoptosis by degradation of Mcl-1 through autophagy,may provide new theoretical support for improving the anti-tumor drug effect of targeting PI3K/mTOR pathway.In this study,ovarian cancer cell lines which have PIK3CA gene mutation and different sensitivities of cisplatin were selected as research objects,through targeting the PI3K/ATK/mTOR pathway,to study the degradation of Mcl-1 through autophagy regulated by the phosphorylation of p62,and to explore the mechanism of inhibiting PI3K/AKT/mTOR pathway to induce apoptosis in mitochondrial pathway.It will provide experimental basis for targeting inhibitors of PI3K/ATK/mTOR pathway to reverse drug resistance of ovarian cancer.Methods:(1)Sequencing and analysis the mutation sites of PIK3CA gene in human serous epithelial ovarian cancer SKOV3 and A2780 cells.(2)MTT assay was used to detect the effect of single and dual PI3K/AKT/mTOR signaling pathway inhibitors on the survival of ovarian cancer SKOV3 and A2780 cells,and the effect of PKI-402 combined with cisplatin on the survival of SKOV3 and A2780 cells.CompuSyn software was used to analyze the synergistic effect of dual PI3K/mTOR inhibitor PKI-402 combined with cisplatin on SKOV3 and A2780 cells.(3)To detect the effects of single and dual PI3K/mTOR inhibitors on the activity of PI3K/AKT/mTOR signaling pathway and the apoptosis of ovarian cancer cells.Western blot was used to detect the expression of p-AKT,P-P70S6K and P-4EBP1 which are the key downstream molecules of PI3K/AKT/mTOR signaling pathway.The expression of Cleaved caspase 3,Cleaved caspase 9 and Bcl-2 family proteins were detected by western blot.The apoptosis rate was measured by flow cytometry.(4)To detect the mitochondrial function of SKOV3 cells which treated by single and dual PUK/mTOR inhibitors;Mitochondrial membrane potential was detected by flow cytometry with JC-1 staining,intracellular ROS level was assessed by flow cytometry with DCFH-DA staining,the ROS level in mitochondria was observed by fluorescence microscopy with mito-sox staining.The ATP assay kit was used to detect the content of ATP in cells.(5)The mitochondria of ovarian cancer cells were isolated,and detected the effects of single and dual PI3K/mTOR inhibitors on Mcl-1,Bak in mitochondria.The expression of Mcl-1 was detected when SKOV3 cells were treated with dual PI3K/mTOR inhibitor alone or in combination with cisplatin.(6)To detect the autophagic flux of SKOV3 cells which treated by single and dual PI3K/mTOR inhibitors;The expressions of p62 and LC3 proteins were measured when cells were treated with single and dual PI3K/mTOR inhibitors,and detected the expressions of p62 and LC3 proteins when added the lysosomal inhibitor chloroquine to reflect the autophagic flux.Lysosomal inhibitor chloroquine and proteasome inhibitor MG-132 were used to block the degradation pathway of protein,and detected the expression of Mcl-1.(7)Immunofluorescence was used to detect the co-location of p62 and Mcl-1 protein.Cells were transfected with a truncated plasmid of p62 UBA domain,and the binding of P62 to Mcl-1 protein via ubiquitin-associated domain(UBA)was detected by immunoprecipitation.(8)The expression of p-p62(Ser4O3)was measured by specific p-p62(Ser403)antiboday.p62 Ser403 plasmids with inactivated mutation were transfected into cells,and the binding of p62 to Mcl-1 was observed by immunocoprecipitation.The cell survival was observed by MTT assay.Results:(1)The mutation site of human serous epithelial ovarian cancer SKOV3 cells was H1047R(A?G),located in the kinase domain,and the mutation site of A2780 cells was E365K(G?A),located in the C2 domain.The two domains had different functions.(2)MTT results indicated that dual PI3K/mTOR inhibitor PKI-402 decreased the cell viability of SKOV3 and A2780 cells.CompuSyn software showed that PKI-402 combined with cisplatin has synergistic effect in SKOV3 cells(Cl:0.41),and has no significant synergistic effect in A2780 cells(CI:0.99).It may be related to the difference of mutations of PIK3CA gene in the two cells.(3)Compared with the single inhibitors,dual PI3K/mTOR inhibitor PKI-402 decreased the expressions of p-AKT,p-p70s6k and p-4EBP1,inhibited the cell viability and the clony formation in ovarian cancer cell significantly.PKI-402 increased the ratio of Bax/Bcl-2,and the expressions of Cleaved caspase-3 and Cleaved caspase-9,inducing the cell apoptosis.(4)Compared with the single-targeted inhibitors,dual PI3K/mTOR inhibitor PKI-402 reduced the mitochondrial membrane potential,increased mitochondrial and intracellular ROS levels,and decreased intracellular ATP content,leading to mitochondrial dysfuction.(5)The expression of anti-apoptotic protein Mcl-1 was decreased gradually when treated with dual PI3K/mTOR inhibitor PKI-402.Compared with cisplatin,dual PI3K/mTOR inhibitor PKI-402 combined with cisplatin could significantly decrease the expression of Mcl-1 in SKOV3 cells.PKI-402 increased the ratio of Bak/Mcl-1 in mitochondria of the SKOV3 cells,suggesting that PKI-402 induced the apoptosis of SKOV3 cell by reducing the expression of anti-apoptotic protein Mcl-1.(6)The expression of LC3-?/? protein was increased significantly in a time-dependent manner when cells were treated with dual PI3K/mTOR inhibitor PKI-402,the expression of p62 increased first and then decreased.When cells were treated with PKI-402 and CQ,the ratio of LC3-?/? and the expression of p62 were increased compared with that cells treated with PKI-402.When interfering with the functions of proteasome and lysosome,the expression of Mcl-1 protein was increased compared with the control group,suggesting that PKI-402 can degrade Mcl-1 through the autophagy pathway and the proteasome pathway.(7)Immunofluorescence assay showed that p62 and Mcl-1 were colocalized around the nucleus when SKOV3 cells were treated with PKI-402.After inhibition of autophagy and proteasome pathway,the colocalized spots aggregation of p62 and Mcl-1 were increased.In the cells transfected with the truncated plasmid of p62 UBA domain,the binding of Mcl-1 to p62 protein was significantly decreased,indicating that p62 binds to Mcl-1 through the UBA domain and then degrades through autophagy and proteasome pathways.(8)Dual PI3K/mTOR inhibitor PKI-402 increased the phosphorylation of p62 Ser403.With the proceeds of autophagy,the phosphorylation of p62 Ser403 decreased with autophagy.In the cells transfected with the inactivated plasmid of p62 Ser403,the binding ability of p62 to Mcl-1 was decreased and the cell survival was partially increased,suggesting that PKI-402 could improve the binding ability of p62 to Mcl-1 by promoting the phosphorylation of p62 Ser403.Conclusions:(1)Considering that ovarian cancer SKOV3 cells with PIK3CA 1047 locus A?G mutation(the kinase domain encoding PI3K? protein)are more sensitive to dual PI3K/mTOR inhibitor PKI-402,mutation screening at this site is helpful for the clinical treatment of patients with cisplatin resistance of ovarian cancer.(2)Compared with single targeted PI3K or mTOR inhibitor,dual PI3K/mTOR inhibitor PKI-402 could inhibit the activity of PI3K/AKT/mTOR signaling pathway and promote the apoptosis of ovarian cancer cells more effectively.(3)Dual PI3K/mTOR inhibitor PKI-402 induced higher autophagic flux,and Mcl-1 degraded through autophagy is one of the mechanisms by which PKI-402 induces mitochondrial pathway apoptosis.PI3K/mTOR inhibitor PKI-402 increased phosphorylation of p62 Ser403 by kinase mTOR/ULK1 and promoted the degradation of anti-apoptotic protein Mcl-1 through autophagy,which is one of the mechanisms of apoptosis induced by dual-targeted inhibition of PI3K/mTOR.In summary,this study found that dual PI3K/mTOR inhibitor PKI-402 regulated the degradation of Mcl-1 through by changing the phosphorylation of multifunctional protein p62 Ser403,induced mitochondrial pathway apoptosis,and increased the sensitivity of resistant cells to cisplatin.It provides a new hope for the treatment of patients with cisplatin resistance and mutations of PI3K/AKT/mTOR signaling pathway in ovarian cancer,and a new idea for the development of targeted drugs.