Autophagy Induced By Topotecan Through ATM-mediated Phosphorylation Of PTEN And Its Mechanism In Cancer Cells

Objective: PTEN is a protein that has lipid phosphatase activity and anti-tumour activity. It isa tumor suppressor gene with a dual-specificity phosphatased activity, which can maintain thestability of intracellular environment and the normal metabolism. Its low or lacking expressionis closely related to tumor development and progression. Autophagic level of tumor wascorrelated with the expression of PTEN, but how PTEN regulates autophagy is not clear yet.Topotecan (Topotecan, TPT) is a semisynthetic, water-soluble analog of the alkaloidcamptothecin, which is a specific inhibitor of topoisomerase I in the nucleus. ATM is activatedin response to DNA double-strand breaks(DSBs). The previous studies found that, topotecancould induced phosphorylation of ATM and PTEN nuclear translocation in tumor cells. Theseindicated that PTEN was related to ATM. The purpose of this study is to explore the autophagyinduced by topotecan through PTEN and its mechanism in cancer cells, and to aselucidate theeffect of PTEN on autophagy in cancer cells.Methods: Human lung cancer cell line A549and cancer of the cervix line cell HeLa werecultured in vitro. The stably transfected cells or transiently transfected cells were established bymolecular cloning technology. The nuclear translocation of PTEN, the punctuate dots ofYFP-LC3and histone variant H2AX (γ-H2AX) foci were detected by confocal microscopy. Theexpression of phospho-ATM, LC3, P62, ATM, phospho-PTENS113, γ-H2AX and Flag were detected using Western blot analysis. ATM specific inhibitor Ku55933and siRNA-ATMtransient transfection were used to inhibit ATM activity. Immunoprecipitation was employed todetermine that Flag binded to phospho-PTENS113.Results:1Topotecan induced phosphorylation of ATM in A549and HeLa cells.ATM was activated in A549and HeLa cells which was exposed to topotecan, and itsexpression in these cells was detected by immunoblotting. And ATM kinase phosphorylationlevel could be enhanced in a concentration-dependent and time-dependent manner.2ATM regulated Topotecan-induced PTEN nuclear translocation in A549and HeLa cells.A549and HeLa cells were first treated with TPT, and then observed under confocalmicroscopy, endogenous PTEN translocation was from cytoplasm to nucleus. When ATMspecific inhibitor Ku55933was used to inhibit ATM activity, the translocation of PTEN wasclearly suppressed. HeLa cells were transient transfected with3×Flag-PTEN-WT plasmid, andconfocal microscopy detected the exogenous PTEN translocation from cytoplasm to nucleus,which was consist with endogenous PTEN translocation. When ATM specific inhibitor Ku55933or siRNA-ATM transient transfection was used to inhibit ATM activity, the translocation ofPTEN was clearly suppressed. Cytoplasm protein and nuclear protein were extracted from A549cells and HeLa cells, respectively. The cytoplasmic PTEN was reduced and the PTEN in nuclearwas increased. When ATM specific inhibitor Ku55933or siRNA-ATM transient transfectionwas used to inhibit ATM activity, the translocation of PTEN was clearly suppressed.3ATM regulated TPT-induced PTEN phopshorylation.Based on sequence analysis of PTEN was analyzed by Scansite software, we generatedphospho-specific antibody with PTENS113and checked its effectiveness. The expression ofp-PTENS113level could be enhanced with a concentration-dependent manner in HeLa cells. A549and HeLa cells were first treated with TPT, the p-PTENS113was detected byimmunoprecipitation assay. When the ATM activity was inhibited by siRNA-ATM transient, thephosphorylation of PTEN at S113was significantly inhibited. Collectively, ATM regulatedTPT-induced PTEN phopshorylation. 4Topotecan induced autophagy and the overexpression or knockdown of PTEN couldregulate autophagy in A549and HeLa cells.4.1Topotecan induced autophagy in A549and HeLa cells.After treatment of TPT, A549and HeLa cells were transient transfectly with YFP-LC3plasmids. A significant accumulation of YFP-LC3dots was observed. Using Western blotanalysis, we observed a notable increase of LC3-II and decrease of P62inconcentration-dependent and time-dependent manner,which indicated induction of autophagy.4.2The overexpression or knockdown of PTEN could regulate autophagy in A549andHeLa cells.A549and HeLa cells were stably transfected with shRNA–PTEN plasmids. Western blotanalysis indicated that the autophagy of these cells was suppressed. Then A549and HeLa cellswere transient transfectly with3×Flag-PTEN-WT plasmids, the autophagy of these cells wasenhanced.5PTEN translocation and autophagy depended on PTENS113phosphorylation in A549andHeLa cells.5.1PTEN translocation was dependent of PTENS113phosphorylation in cancer cells.PTEN S113was further mutated by a site-directed mutagenesis, then the3×Flag-PTEN-S113A plasmids were constructed. HeLa cells were transiently transfected with3×Flag-PTEN-S113A plasmids. After treatment of TPT, we found that PTEN did not shownuclear translocation under the confocal microscopy and PTENS113was not phopshorylated byimmunoblotting assay. But the cells were transfected with3×Flag-PTEN-WT plasmids, whichshowed PTEN translocation that was from cytoplasm to nucleus. And PTENS113wasphopshorylated by immunoblotting assay. Therefor, PTEN translocation was dependent ofPTENS113phosphorylation in cancer cells.5.2Autophagy was dependent of PTENS113phosphorylation in A549and HeLa cells.After treatment of TPT, A549and HeLa cells were transiently transfected with3×Flag-PTEN-WT plasmids and3×Flag-PTEN-S113A plasmids. Western blot analysis indicatedthat LC3-II was markedly decreased with transfection of the3×Flag-PTEN-S113A plasmids. Theresults suggest that p-PTENS113could promote autophagy. 6Topotecan induced DNA damage and PTEN-S113A mutant enhanced TPT-induced DNAdamage in A549and HeLa cells.6.1Topotecan induced DNA damage in A549and HeLa cellsImmunofluorescence assay was used to detect γ-H2AX foci in A549and HeLa cellsexposed to TPT. Notably, γ-H2AX foci was observed in time-dependently manner by confocalmicroscopy. And γ-H2AX protein was detected in time-dependently manner by immunoblotting.6.2PTEN-S113A mutant enhanced TPT-induced DNA damage in A549and HeLa cells.A549and HeLa cells were transiently transfected with3×Flag-PTEN-WT or3×Flag-PTEN-S113A plasmids. Notably, γ-H2AX foci was observed after treatment of TPT, but γ-H2AX fociwas mostly in cells which were transiently transfected with3×Flag-PTEN-S113A plasmids.Conclusions:1Topotecan induced phosphorylation of ATM, autophagy and DNA damage concentration-dependently and time-dependently in A549and HeLa cells.2ATM mediated TPT-induced PTENS113phopshorylation and PTEN nuclear translocation,which promoted autophagy and inhibited DNA damage induction in A549and Hela cells.3The silence of PTEN could inhibit TPT-mediated autophagy, overexpression of PTENcould promote autophagy in A549cells and Hela cells.