| Objective:Poly(ADP-ribose)polymerase 1(PARP1)is a ADP-ribosyl transferase enzyme,which creates the posttranslational modification PARylation from substrate NAD+on different proteins to regulate multiple cellular processes.In addition to its well-known role in DNA repair,PARP1 can also plays a role in regulating gene transcription.And PARP inhibitors for clinical use in cancer therapy are developed based on their DNA repair function Studies have pointed to a role of PARP 1 in regulating gene expression through poly(ADP-ribosyl)ating,sequence-specific,DNA-binding transcription factors.Thus,it plays an important role in regulating various life processes such as immune cell differentiation and adipogenesis.However,due to the weak characteristics of PARylation modification sites and the lack of efficient screening techniques,only few studies reported on PARP1 regulation of gene transcriptionThere are multiple mechanisms for tumor cells to escape recognition and attack by the body's immune system,including immunosuppressive proteins expression,low immunogenicity,antigen modulation,and physical barrier generation,etc.,leading to tumor cell survival and proliferation in the body.The immunosuppressive molecule PD-L1 expressed by tumor cells plays a key role in tumor immune escape.Normal tissue also expresses PD-L1,which inhibits T cell activation after binding to the PD-1 receptor onthe surface of T cells to avoid autoimmune diseases caused by excessive activation of T cells.Therefore,PD-L1/PD-1 pathway plays an important role in maintaining the body's protective immunity and immune tolerance balance.The expression of PD-L1 on tumor cells due to oncogene activation,microenvironmental cytokine stimulation,and continuous activation of signaling pathways mediates tumor immunosuppression,thereby promoting the malignant proliferation of tumors.Therefore,in-depth study of the regulatory mechanism of PD-L1 is very important for the development of PD-L1-based tumor intervention strategiesPARP inhibitors,as small molecule inhibitors targeting PARP1 protein,exert synthetic lethal effect by inhibiting DNA repair of tumor cells,and have been applied to the treatment of ovarian cancer.However,clinical data suggest that PARP inhibitors could not significantly improve the overall survival(OS)of ovarian cancer patients.How to further improve the clinical treatment effect of PARP inhibitors has become a focus issue in this field.One of the current research speculations about the poor clinical use of PARP inhibitors is that the compensatory activation of other DNA repair proteins during the use of PARP inhibitors,therefore its tumor-killing effect is weakened.But this has only been confirmed in few patients.Overall,the reasons for the poor clinical efficacy of PARP inhibitors are far from clearIn summary,this study aims to investigate the regulatory effect of PARP 1 on immune checkpoint protein PD-L1 and its related molecular mechanisms:(1)The regulation of PARP1 on PD-L1 transcription.(2)PARP1 inhibits the transcription of PD-L1 through PARylation of STAT3.(3)The crosstalk between PARylation and Phosphorylation of STAT3.This article will take this as point,through studying in detail the expression regulation and molecular mechanism of PARP1/PD-L1 pathway,thus reveal a new biological function of PARP 1 in tumor immune escape,further perfects the theory of PARP1 in regulating gene expression,discovers a new regulatory mechanism of PD-L1 and a new biological function of STAT3.It is possible to form a new perspective of immune escape and reveal the reason for the poor clinical efficacy of PARP inhibitors Section 1 The function of PARP1 in the transcriptional regulation of PD-L1MethodsHuman ovarian cancer cell lines SKOV3 and OVCAR8,human colon cancer cell lines SW620 and Colo205,human lung cancer cell lines A549 and PC9,human breast cancer cell line MCF-7,human primary ovarian cancer cells,and human primary lung cancer cells were used in this study.Co-incubation models of T cells and tumor cells in vitro were used to evaluate the effects of PARP1 inhibition on PD-L1 regulation and T cell activity inhibition(1)Small-interfering RNA was used against PARP 1,and qRT-PCR was used to investigate the effect of PARP1 silencing on PD-L1 transcription;(2)Dual luciferase reporter assay was used to examine the PD-L1 transcriptional activity;(3)The effects of PARP1 inhibition on PD-L1 membrane proteins and total proteins were investigated by flow cytometry and Western blotting;(4)Single cell isolation assay was used to get primary tumor cells.Western blotting was used to investigate the regulatory effect of PARP 1 on PD-L1 protein.(5)PARP 1 plasmid was constructed to investigate the effect of PARP1 protein on PD-L1 expression.(6)Peripheral blood mononuclear cells(PBMCs)were used to construct a co-incubation model of tumor cells and T cells,and the effect of PD-L1 regulated by PARP1 on T cell activity was investigated;(7)Immunohistochemical method was used to investigate the expression relationship of PARP1 and PD-L1 in humuan tissue sections;(8)The effects of PARP inhibitors on cell proliferation and apoptosis were investigated by flow cytometry and sulforhodamine B(SRB)experiments;(9)Through T cell co-incubation experiments,Elisa assay was used to detect the effect of PARP 1 intervention on T cell secretion of IL-2 and Granzyme BResults(1)PARP1 suppresses the transcription of PD-L1 in cancer cellsTo investigate whether PARP1 is involved in cancer immune evasion,the ovarian cancer cells were transiently transfected with siRNA against PARP1,PARP1-depleted cells exhibited an enhanced transcription of PD-L1 mRNA.In parallel,the total PD-L1 protein expression and cell-surface PD-L1 expression were upregulated with PARP1 silencing The impact of individual PARP1 siRNA on PD-L1 expression was associated with the gene-silencing efficiency of the siRNA,suggesting a PD-L1-associated defect.The increased PD-L1 expression in PARP1-depleted cells was also recapitulated in the breast cancer cell line MCF-7,the non-small cell lung carcinoma cell lines A549 and PC9,and the colon cancer cell lines Colo205 and SW620.Taken together,these data suggest that PARP1 suppresses PD-L1 transcription in various cancer cells(2)PARP1 inhibits the transcription of PD-L1 in a catalytic-dependent mannerConsidering that PARP1 controls the transcription of target genes,either with both catalytic-dependent and catalytic-independent mechanisms,PARP inhibitors that could efficiently suppress the catalytic activity of PARP1 were used to confirm whether PARP1-mediated PD-L1 expression was correlated to its catalytic activity.A significant elevation of PD-L1 mRNA expression was observed when OVCAR8 and SKOV3 cells were treated with Olaparib.To exclude the impact of PARP1 on the degradation of PD-L1,cells were treated with Olaparib,Cycloheximide,or both for the indicated time.No significant difference in the kinetics of PD-L1 protein expression was observed between these two groups.To further test whether PARP inhibitors enhanced the transcription of PD-L1,we cloned the promoter region of PD-L1 downstream of a luciferase reporter gene(pGL4.14-PD-L1).The luciferase activity of the cells transfected with pGL4.14-PD-L1 was significantly increased when PARP1 was inhibited,and the protein expression of PD-L1 was upregulated by multiple PARP inhibitors in a dose-dependent manner.We also evaluated the expression of CD47,another cell-surface immunosuppressive factor,which was not altered at the mRNA level upon PARP1 silencing,and the cell-surface expression of CD47 was not significantly affected by the PARP inhibitor Olaparib.These data suggest that the inhibition of PD-L1 transcription by PARP1 depends on its catalytic activity(3)The regulation of PARP1 on PD-L1 expression influences the activity of T cellsWe constructed a model of co-incubation of tumor cells and T cells to investigate the effects of changes in PD-L1 protein levels caused by inhibition of PARP1,silence of PARP1,and overexpression of PARP1 protein on T cell function.Overexpression of PARP1 protein can restore the killing effect of T cells and promote the secretion of IL-2 and Granzyme B.While inhibition of PARP 1 and PARP1 silencing both inhibited T cell activity and reduced the secretion of IL-2 and Granzyme B.To further validate our findings in human cancer patient samples,we analyzed the correlation between the expression of PARP1 and PD-L1 in human ovarian cancer specimens using IHC.The Pearson test further showed an inverse correlation between PARP1 and PD-L1 expression in human cancer patient specimens.These results suggest a connection between high PARP1 expression and low PD-L1 expression in primary ovarian cancer tissuesSection 2 PARP1 Suppresses the Transcription of PD-L1 by PARylation of STAT3 MethodsIn this study,ovarian cancer cell lines SKOV3 and OVCAR8 were used to screen the transcription factors of PD-L1,and STAT3 was discoverd as a transcription factor involved in the regulation PARP1 on PD-L1.The role of STAT3 in regulation was clarified by a model of protein binding and RARylation in vitro(1)Small-interfering RNA was used against STAT3,STAT1 and MYC,and then flow cytometry was used to examine the up-regulation effect of PD-L1 caused by PARP1 inhibition;(2)Small-interfering RNA was used against STAT3,and qRT-PCR was used to investigate the transcription regulation of PARP inhibitor Olaparib on PD-L1 in ovarian cancer cells;(3)Dual luciferase reporter assay was used to examine the effect of the presence of PARP1 and STAT3 proteins on the transcription of PD-L1;(4)Chromatin immunoprecipitation(ChIP)was used to investigate the binding status of STAT3 and PD-L1 promotor region when PARP1 was inhibited by Olaparib;(5)The correlation between target gene expression of STAT3 and PD-L1 expression was analyzed through data analysis of 563 ovarian cancer patients samples in TCGA;(6)Co-immunoprecipitation method was used to investigate the binding between STAT3 and PARP1,and PARylation of STAT3;(7)The effect of plasmids lacking PA-P1 activity on STAT3-mediated PD-L1 transcriptional regulation was examined by dual luciferase reporter assay;(8)Flow cytometry method was used to investigate the effect of PARP1 PARylation dysfunctional plasmid on expression of membrane PD-L1Results(1)STAT3 is required for PARP1-mediated PD-L1 expressionWe hypothesized that PARylation of transcriptional factors by PARP1 might also apply to PARP1-modulated PD-L1 transcription.Thus,we silenced the transcription factors STAT1,STAT3,and MYC,which have been reported to regulate PD-L1 transcription by directly binding to its promoter region.We found that PD-L1 expression induced by PARP1 inhibition was not affected by STAT1 and MYC deficiency but was abolished with the silencing of STAT3.In parallel,PARP1 inhibition promoted PD-L1 transcription,which was attenuated by STAT3 silencing.Similar results were obtained with the OVCAR8 and MCF-7 cells.These data suggest a major contribution of STAT3 to PARP1-mediated PD-L1 transcription.To further confirm the involvement of STAT3 in PARP1-mediated PD-L1 transcription,SKOV3 cells were transfected with pGL4.14-PD-L1,Flag-PARP1,and HA-STAT3.The PD-L1 luciferase activity was upregulated compared with the vector when transfected with HA-STAT3,whereas it was significantly downregulated when cotransfected with Flag-PARP1.We also performed a ChIP assay using a PCR primer set capable of amplifying the promoter region of PD-L1 containing the STAT3 binding sites.The direct binding of STAT3 to the PD-L1 promoter was observed in SKOV3 cells,and this binding was significantly enhanced by PARP1 inhibition.We analyzed an ovarian serous cystadeno-carcinoma data set containing 563 samples from TCGA,based on the large-scale cancer genomics data sets provided by cBio-Portal.The mRNA expression of MCLl,CCL5,IFNG,IL4R,and IL2RA,five well-known STAT3 target genes,were correlated with mRNA expression of CD274,further suggesting that the expression of PD-L1 is positively associated with STAT3 activity These data collectively revealed that PARP1 regulates PD-L1 expression through STAT3(2)STAT3 is PARylated by PARP1We then asked whether PARP1 directly poly(ADP-ribosyl)ated STAT3 to inhibit its transcriptional activity.To test this hypothesis,we first determined if PARP1 could form complexes with STAT3 in cancer cells.Pull-down assays validated the direct endogenous interaction of PARP1 with endogenous STAT3.This interaction was further confirmed with the ectopic expression of PARP1 or STAT3.PARP1 inhibition also disrupted the interaction between PARP1 and STAT3.We used an ectopic expression system to study the ADP-ribosylation of STAT3.As expected,PAR was pulled down with STAT3 protein,and PARylation of STAT3 was observed,as the molecular weight smear signals began with 92 KD,indicating that PAR signal came directly from STAT3.The PARylation of STAT3 was also observed in STAT3-containing immunoprecipitates from HEK 293T cells ectopically expressing HA-STAT3 and Flag-PARP1,which was abolished by PARP1 inhibition.Taken together,these results demonstrated that STAT3 is PARylated by PARP1(3)PARP1 inhibits the transcription of PD-L1 by PARylating STAT3It remains to be determined if the role of PARP1-mediated PD-L1 transcription is due to the PARylation of STAT3.Previous studies have identified that the glutamate(E988)and histidine(H862)were the residues responsible for the polymerase activity of PARP1.We generated point mutations in the PARP1 gene at positions histidine(H862)and glutamate(E988),respectively.Compared with the group transfected with wild-type PARP1,the PARylation of STAT3 was not observed when the cells were transfected with the H862A or E988K mutants.We then analyzed the effect of mutated PARP1 on the transcription of PD-L1.Mutated PARP1 could not suppress STAT3-mediated PD-L1 promoter activity due to loss of its polymerase activity,and PARP1 inactivation did not downregulate PD-L1 protein expression.These data indicated that PARP1 inhibits PD-L1 transcription through the PARylation of STAT3.Section 3 The cross-talk between PARylation and PhosphorylationMethodsIn this study,human ovarian cancer cell lines SKOV3 and OVCAR8 cells,human lung cancer cell lines A549 and PC9,human colon cancer cell lines SW620 and Colo205,and human breast cancer cell line MCF-7 were used to examine the effects of PARP1 on p-STAT3.The plasmids with different Phosphorylation levels of STAT3 were constructed to investigate the changes in its PARylation level.The relationship between PARylation and Phosphorylation of STAT3 was further clarified by means of flow sorting,double staining,and immunohistochemistry.(1)Western blotting was used to investigate the changes of p-STAT3 levels in various tumor cells after PARP1 inhibiton and silencing;(2)Intra-and extra-nuclear distribution of STAT3 were investigated by immunofluorescence and nuclear fractionations separation assay after PARP1 activity was inhibited or silenced;(3)Flow cytometry double staining of membrane surface PD-L1 and nuclear p-STAT3 was used to examine changes in the proportion of cells expressing both proteins after PARP1 silencing;(4)Western blotting was used to investigate the effects of wild-type PARP1 and PARP1 lacking PARylation sites on the level of p-STAT3;(5)The plasmids of STAT3C(continuous Phosphorylation),STAT3(wild type)and STAT3Y705F(without Phosphorylation)were constructed by molecular cloning,then co-immunoprecipitation experiments were performed to investigate changes in STAT3 PARylation levels;(6)Cells with PD-L1 upregulation and cells with low levels of PD-L1 after Olaparib treatment were isolated by cell sorting,and investigated their p-STAT3 level change After the two groups of selected cells were cultured for a period of time to return to baseline levels,the two groups were analyzed by Western blotting.The level of PARylation was clarified;(7)The relationship between p-STAT3 levels and PARylation levels in different tumor cells were compared by Western blotting;(8)Immunohistochemistry was used to investigate the relationship between PARylation and Phosphorylation in tumor patients' tissue samples;(9)Western blotting was used to investigate the pathway of STAT3 upstream kinases and downstream phosphatases;(10)Co-immunoprecipitation method was used in screening for the invovled phosphorylases,which was participated in STAT3 de-phosphorylation and investigate the effect of PARR1 on the binding of STAT3 and its phosphataseResults(1)PARP1 regulates the Phosphorylation of STAT3We assessed whether PARP1 affected the tyrosine Phosphorylation of STAT3.The silencing or pharmacologic inhibition of PARP1 enhanced the Phosphorylation of STAT3 in SKOV3 and OVCAR8 cell lines.In parallel,a significant increase in the nucleus localization of STAT3 was observed,further suggesting the activation of STAT3,and the constitution of wild-type PARP1 significantly attenuated the Phosphorylation of STAT3 caused by PARP1 silencing.Using two-color flow cytometry,we found that PARP1-silenced cells exhibited a significant increase in the proportion cells positive for both phosphorylated(p)STAT3 and PD-L1.The increased STAT3 Phosphorylation of PARP1-deficient cells was also recapitulated in the breast cancer cell line MCF-7,the non-small cell lung carcinoma cell lines A549 and PC9,and the colon cancer cell lines Colo205 and SW620.These data suggest that PARP1 acts as a suppressor of STAT3 Phosphorylation in various cancer cells(2)The cross-talk between PARylation and PhosphorylationWe then generated STAT3 with a Y705F mutation(mutation of tyrosine 705 into phenylalanine,leading to activation deficiency)and a constitutively active STAT3(STAT3C).This mutant has STAT3 constitutively active as a result of substituting the cysteine residues(C661A and C663N),allowing STAT3 dimerization and activation.The PARylation signal could not be detected when transfected with STAT3 Y705F.However,a PARylation signal was detected in STAT3C transfected cells.The smear signal of STAT3 was consistent with its Phosphorylation.Alternatively,we used another assay,in which the cells were transfected with Flag-PARP1 and HA-STAT3 followed by treatment with AZD1480(JAK2 inhibitor).The Phosphorylation of STAT3 was efficiently inhibited by AZD1480,while the PARylation of STAT3 was significantly reduced.Taken together,these results indicated that PARP1 was prone to bind the active form of STAT3 To explore the correlation between PARylation and p-STAT3,cell sorting was used to separate low and high PD-L1 expression cells after PARP1 inhibitor administration.High PD-L1 expression associated with higher p-STAT3.After culturing for another 72 h(to return to baseline),a stronger smear band was observed in high PD-L1 expressing cells,suggesting that the response by cells to PARP1 inhibition might be due to their high basal PARP1 catalytic activity.IHC of ovarian cancer patient tissues(n=53)was used to define PAR and p-STAT3 levels.An inverse correlation was found between PARylation and p-STAT3 expression.Our results suggest that PARP1-mediated PARylation might affect PD-L1 by suppressing STAT3 activity(3)PARP1 regulates STAT3 Phosphorylation through PTPN9STATs are phosphorylated by JAKs,and they then dimerize and translocate to the nucleus,where they activate gene transcription.Our data showed that the Phosphorylation of JAK2 was not affected by PARP1 silencing.Thus,we inferred that PARP1-mediated PARylation might affect the de-phosphorylation process of STAT3.Cells were first transfected with Flag-PARP1 for 24 h and then Vanadate,a general inhibitor for tyrosine phosphatases,was added.Overexpression of PARP1 could not reduce the Phosphorylation of STAT3 in the presence of Vanadate.These results indicated that PARylation increases de-phosphorylation of STAT3.Therefore,we investigated the possible reported phosphatase of STAT3,and co-immunoprecipitation results showed that PTPN9 can form a complex with STAT3 and PARP1,and overexpression of PARP1 and inhibition of PARP1 can increase or decrease the binding of PTPN9 and STAT3 From this we could conclude that PARP 1 exerts an inhibitory effect on STAT3 Phosphorylation through PTPN9Conclusion:In this study,we showed that PARP1 silencing or pharmacologic inhibition enhanced the transcription of PD-L1 in cancer cells,which was accompanied by the upregulation of PD-L1 protein expression,both in the cyto-plasm and on the cell surface.This induction of PD-L1 was attenuated in the absence of the transcription factor STAT3.Cell-based studies indicated that PARP1 interacted directly with STAT3 and caused STAT3 poly(ADP-ribosyl)ation.STAT3's activation of PD-L1 transcription was abolished by the overexpression of wild-type PARP1 but not mutant PARP1,which lacks catalytic activity PARP1 downregulation or catalytic inhibition enhanced the Phosphorylation of STAT3,which was reversed by the ectopic expression of wild-type PARP1 but not by mutated PARP1.An inverse correlation between PARP1 and PD-L1 was also observed in clinical ovarian cancer samples.Overall,our study revealed PARP1-mediated poly(ADP-ribosyl)ation of STAT3 as a key step in inhibiting the transcription of PD-L1,and this mechanism exists in a variety of cancer cells. |
PDF Full Text Request