| ObjectiveAt present,the main treatment of prostate cancer is surgical and Androgen Deprivation Treatment(ADT).Androgen deprivation therapy is according to that grow of prostate cancer based on the androgen.Although these two treatments can be very effective in most patients,a considerable proportion of patients will develop to Castration Resistant Prostate Cancer(CRPC).However,there is no effective treatment plan for those patients.Clinical studies have shown that immunotherapy is efficiency in some cancers,and also have well effect in CRPC,so immunotherapy has become a hot research direction.The most important foundation of immunotherapy is to find the effective target and the regulatory pathway.Immunotherapy,especially for programmed cell death protein-1(PD-1)and programmed cell death protein ligand-1(PD-L1),has been applied in many kinds of tumors,and has achieved very gratifying therapeutic effect.At the same time,indoleamine-2,3-dioxygenase-1(IDO-1)has also attracted more and more attention.Although immunotherapy for PD-L1/IDO-1 has attracted more and more attention in recent years.But there are not enough reports about the treatment of PD-L1/IDO-1on prostate cancer,and the basic research is rarely.At present,a considerable number of clinical data show that the expression level of PD-L1/IDO-1 is closely related to the prognosis of prostate cancer patients.Moreover,its expression level is significantly higher in the small cell neuroendocrine prostate cancer than that of other early tumors.Small cell neuroendocrine prostate cancer is usually accompanied by Epithelial Mesenchymal Transition(EMT).In some studies about other tumors,EMT process has an important impact on the expression of PD-L1/IDO-1,according with the relationship between EMT and Neuroendocrine transformation,we propose this hypothesis that EMT may be able to regulate the expression of PD-L1/IDO-1 in prostate cancer.However,there are few reports about this phenomenon and mechanism in prostate cancer.Our research will start from exploring the relationship between the expression level of PD-L1/IDO-1 on prostate cancer and prostate tissue type.In order to further exploration about the mechanism.According to the published reports,IFN gamma receptor and the Janus kinase/signal transducer and activator of tranions(IFNGR-JAK-STAT)pathway plays an important role in the expression of PD-L1/IDO-1.Therefore,we will conduct in-depth study on this path way.Materials and Methods1.1 In the clinical research part,we collected the pathological samples of the patients who had undergone radical prostatectomy or puncture biopsy in the Urology of Duke Hospital.According to the pathological identification,the patients were divided into four different groups: Benign Prostatic Hyperplasia,adenocarcinoma,CRPC and small cell neuroendocrine tumor of prostate.Then,the immunohistochemistry(IHC)was used to determine whether there was more than1% PD-L1 expression on the pathological samples.If there was more than 1%PD-L1 expression,it would be considered as positive.More than 90% of IDO-1 was positive.More than 60% of N-cadherin was positive.At the same time,the IHC scores of each sample were calculated and analyzed.And the liner relationship between PD-L1/IDO-1 and N-cadherin also studied;1.2 Different cell lines were divided into four groups,corresponding to the group of patients.BPH-1 cell line representing benign prostate tissue,LNCap cell line representing adenocarcinoma,CWRR-1 cell line and C4-2 cell line representing CRPC cell line,PC3 cell line,LASCPC cell line and NCI-H660 cell line representing neuroendocrine tumor.Different cell lines were treated with IFN-g.Western blot and PCR were used to detect whether the expression of PD-L1/IDO-1 could be induced and the different expression of PD-L1/IDO-1 among several groups.At the same time,EMT status of these cell lines was determined,EMT positive and EMT negative cell lines were distinguished,and the expression levels of PD-L1/IDO-1 were compared;1.3 In order to further improve the relationship between PD-L1/IDO-1 expression and different types of prostate cancer,we also studied the expression of PD-L1/IDO-1 in NSG mice.Animal models were made by subcutaneous implantation of human prostate cancer tumor cells.In order to make the animal model more representative,we also chose two different groups: in early prostate cancer group,we chose LNCap,while in neuroendocrine group,we chose PC3.After subcutaneous implantation of tumor,we cultured the mice under the same culture conditions.When the tumor size reached 1.5cm-2cm,IFN-g reagent was given by intraperitoneal injection.DMSO was used in the control group.The mice were killed and the tumor was removed after48 hours.Then the specimen was cut into two parts,one was fixed with formalin,and embedded in paraffin for immunohistochemistry detection,the other was separated into single cell state by single cell separation technology for RNA extraction and flow cytometry.Compare the data from mice model in two different groups with the data obtained in patients and cells.2.1 When we study the specific regulatory pathway of PD-L1/IDO-1,we blocked JAK/STAT pathway to detect whether PD-L1/IDO-1 expression could still be induced.In order to completely detect IFNGR1-JAK1/STAT1 signaling pathway,we detected the expression and activation of IFNGR1 at the patient level,cell level and animal experiment.Then we compared the expression levels of IFNGR1,p-IFNGR1 with PD-L1/IDO-1,and EMT status;2.2 In addition,as far as we know,not every factor in JAK/STAT pathway can be detected in all cell lines.Different cell lines representing different tumor types may be regulated by different JAK/STAT pathways.Therefore,we first need to detect the expression and phosphorylation of JAK1/STAT1 and JAK2/STAT3 in cell lines,and then compare the expression of PD-L1/IDO-1 in different cell lines to determine the specific regulatory pathway.At the same time,we also blocked STAT1 and STAT3 pathways to compare their effects on the expression of PD-L1/IDO-1.3.1 In order to determine the relationship between EMT and PD-L1/IDO-1,we first detected the relationship between the EMT status and tumor types in animal samples.And compared with the expression of PD-L1/IDO-1;3.2 We used PC3N-cadherin knockout cell model(PC3-N-cad KO)and EMT activated LNCa P cell model(LNCap C1,C2,C3)to detect the expression of PD-L1/IDO-1.Then use those models in animal to detect the expression of PD-L1/IDO-1.At the same time,we also applied PD-L1 blocking antibodies or stimulate cells with tyrosine to study its effect on EMT process.PC3-N-cad KO model and LNCap C1 C2 C3 models were used in studied the effect of EMT on the expression and activation of JAK/STAT pathway to determine the mechanism of EMT regulating PD-L1/IDO-1 expression.Results1.1 Our study showed that the expression of PD-L1/IDO-1 in small cell neuroendocrine tumor was significantly higher than that in other types of prostate cancer,moreover,the expression level of PD-L1/IDO-1 was significantly increased in EMT positive specimens;1.2 The expression of protein level is consistent with that of RNA level.Only on neuroendocrine tumor cell lines,PD-L1 can be induced under IFN-g stimulation.Although the expression of IDO-1 can also be found in CRPC group,the level is lower than that of small cell neuroendocrine group.This result is consistent with the clinical data;1.3 The results of animal experiments showed that PD-L1 and PD-L1/IDO-1 could be expressed only in PC3 model stimulated by IFN-g,but there was no expression in LNCap group.This result further proves that only neuroendocrine tumors have IFN-g reactivity and can express PD-L1/IDO-1.2.1 By detecting the expression of IFNGR1 and p-IFNGR1 in patients,we found that although IFNGR1 was expressed in both CRPC patients and small cell neuroendocrine tumor patients,p-IFNGR1 was only found in small cell neuroendocrine tumor patients.It is well known that p-IFNGR1 is the activation state of IFNGR1.Only IFNGR1 is activated can further activate the downstream JAK/STAT signal pathway.In addition,the expression level of IFNGR1/p-IFNGR1 in EMT positive group was significantly higher than that in EMT negative group.In vivo experiment,the results also prove that only neuroendocrine prostate cancer has IFN reactivity;2.2 Then we detected the activation of different JAK/STAT pathways.The results showed that STAT1 was expressed in most tumor cells,but JAK1 was only expressed in several neuroendocrine tumors.At the same time,STAT1 can only be activated in neuroendocrine tumors.However,STAT3 can be activated on CRPC,which provides the evidence that STAT1 can only induce the expression of IDO-1instead of PD-L1.While the PC3 do not express STAT3,so JAK1/STAT1 pathway can regulate both PD-L1 and IDO-1.Above all,we also clarified that JAK1-STAT1 regulates the expression of PD-L1 and IDO-1,while JAK2-STAT3 can only regulate the expression of IDO-1.The discovery was also reported for the first time.3.1 The effect of PD-L1/IDO-1 expression during EMT.Our study shows that EMT activation only occurs in the neuroendocrine prostate group(patients,cells and animals),and PD-L1 expression only be induced in EMT activated mice model;3.2The expression of N-cadherin was found only in the small cell neuroendocrine group(PC3).PC3-N-cad KO cell line has become EMT negative after N-cadherin knockout.After using these two models,we found that the expression levels of PD-L1 and IDO-1 significantly decreased after inhibited N-cadherin.Meanwhile,PD-L1/IDO-1 can be induced in EMT activated LNCap cell line(LNCa P C1 C2).However,PD-L1/IDO-1 could not be induced in primary LNCap cells.But in LNCap C3,PD-L1/IDO-1 could not be expressed.Although there was a small amount of N-cadherin expression in this cell line,the expression level was lower than that of LNCap C1 C2,but the expression level of E-cadherin was higher,and the overall EMT level was lower.The results of animal model experiments suggest that PD-L1 and IDO-1 can be induced in LNCap C1 tumor model after EMT activation.However,PD-L1 and IDO-1 could not be induced in PC3-N-cad KO model after N-cadherin knockout;3.3 We used tyrosine to stimulate the cells and detected the expression level of EMT markers.The results showed that the expression level of EMT markers was significantly up-regulated.The results proved IDO-1 can also regulate EMT process with positive feedback.After blocking PD-L1,we found that the expression level of EMT markers dramatically decreased;3.4Further study found that EMT can affect the activation of JAK-STAT pathway.This is the mechanism by which EMT regulates the expression of PD-L1/IDO-1.ConclusionThis project is composed of clinical research,cell level research and animal experiment.We found that the expression of PD-L1/IDO-1 significantly increased in EMT positive prostate cancer.The EMT process is accompanied by the neuroendocrine transformation.Blocking EMT can significantly reduce the expression of PD-L1/IDO-1,and PD-L1/IDO-1 can in turn increase the level of EMT activation.This suggests that immunotherapy(especially target to PD-L1/IDO-1)for prostate neuroendocrine tumors may be better than other stages of prostate tumors.More importantly,since EMT process is crucial to the expression of PD-L1/IDO-1,we can try to combine EMT related drugs with PD-L1/IDO-1 to improve the effect of immunotherapy of prostate cancer. |
PDF Full Text Request