The Mechanism Of Survivin Promoting Prostate Cancer Metastasis By Regulating SMAD2 Phosphorylation

ObjectiveInvasion and metastasis are the decisive factors leading to the death of patients with prostate cancer(PCa).Understanding the molecular mechanism leading to distant metastasis is helpful to improve the treatment of metastatic PCa and formulate strategies to prevent metastasis of primary PCa.As a member of the inhibitors of apoptosis(IAP)family,Survivin plays an important role in cell cycle progression and apoptosis.Previous studies have confirmed that Survivin is overexpressed in PCa tissue,and its expression level is positively related to the degree of malignancy and poor prognosis.However,the role of Survivin in the metastasis progress of PCa is not clear.This study aimed to investigate the role and mechanism of abnormal expression of Survivin in the metastasis of PCa.MethodsWe used GEO and TCGA databases to predict the differential expression of Survivin in normal prostate tissues and PCa tissues,and its expression was statistically analyzed in different grades of PCa(Gleason=6,7,8,9,10).Fifteen specimen of benign prostatic hyperplasia(BPH)and 60 samples of PCa tissue were collected from the Second Hospital of Tianjin Medical University.The protein expression of Survivin in BPH and PCa with different grades was measured by immunohistochemistry(IHC),and the correlation between Survivin expression and various clinicopathological factors was estimated.Western blot and real-time quantitative polymerase chain reaction were used to detect the differential expression of Survivin in benign prostatic hyperplasia cell line BPH,hormone-dependent PCa cell line LNCa P,hormoneindependent PCa cell lines Du-145 and PC-3.Furthermore,Survivin stably knockdown,overexpression and control cells were constructed,and YM155,a small molecule inhibitor of Survivin,was used to treat PC-3 cells.The changes of cell invasion was detected by Transwell test,and the changes of cell proliferation were assessed by CCK-8 test.The correlation between the expression of Survivin and epithelial-mesenchymal transformation(EMT)related markers in PCa was predicted by bioinformatics analysis,and the changes of m RNA and protein levels of the EMT molecular markers in control group and treatment group were evaluated.Finally,we examined the TGF-β pathway in different treatment groups by detecting phosphoSMAD2 levels using Western blot.Result 1.Survivin was frequently up-regulated in PCa tissue and cells.TCGA database analysis showed that compared with normal prostate tissue,the expression of Survivin m RNA was abnormally higher in PCa tissue,and there was a positive correlation between Survivin expression and Gleason score.We was also examined Survivin expression in BPH and PCa tissue by IHC.Survivin was strongly up-regulated in PCa samples.Western blotting and real-time quantitative polymerase chain reaction confirmed that Survivin was low-expressed in RWPE-1,but overexpressed in LNCa P,Du-145 and PC-3 gradually.2.The overexpression of Survivin is positively related to tumor grade and clinical stage.The expression of Survivin increased successively in BPH,low-risk PCa and highrisk prostate cancer.The abnormal expression of Survivin was positively correlated with clinical factors such as lymph node metastasis and distant metastasis.3.Survivin promotes cell proliferation,migration and invasion in PCa.Decreasing Survivin inhibited cell invasion and proliferation in PC-3 cells.And the growth of PC-3 cells was also suppressed after treating with Survivin inhibitor YM155.Conversely,overexpression of Survivin in LNCa P could significantly promote cell migration,invasion and proliferation.4.Survivin promotes EMT by activating TGF-β / SMAD2 signaling pathway in PCa.Western blotting and real-time quantitative polymerase chain reaction confirmed that knocking down Survivin increased the expression of epithelial marker E-cadherin and reduced the expression of mesenchymal marker N-cadherin in PC-3 cells.In contrast,E-cadherin was inhibited and the expression of interstitial markers N-cadherin was induced in LNCa P cells conducted with Survivin overexpression vector.We further determined that knocking down or overexpressing Survivin can lead to changes in the level of phospho-SMAD2.ConclusionSurvivin is abnormally overexpressed in prostate adenocarcinoma,and it is positively correlated with the malignant degree and invasiveness of PCa.Survivin promotes EMT progress by activating TGF-β/SMAD2 pathway,and improve the metastatic and invasive ability of PCa.The drug YM155 can inhibit the expression of Survivin and reduce the growth of PCa.To sum up,our findings highlight that Survivin is a potential biomarker of PCa metastasis,and targeting Survivin/TGF-β/SMAD2 pathway provides a novel clinical therapeutic strategy for metastatic PCa.