Study On The Role And Molecular Mechanism Of GOLM1 And MIIP In Prostate Cancer

BackgroundProstate cancer(PCa)is the most common malignancy in men and a major cause of cancer deaths.With an estimated 220000 American men diagnosed yearly,nearly 30000 men in the United States die from PCa each year,and its incidence continues to rise in the last 10years in China as well,due to the westernization of life style and the aging of the population.Early detection of PCa depends on prostate-specific antigen(PSA)testing,but it is estimated that there is often a lag-time of 15 years or more from initially detectable PSA elevation to clinically manifested PCa.There has been considerable debate on PSA testing because this could lead to over-treatment for some patients.In addition,therapies for PCa usually involve either reducing or blocking the production of androgen and most PCa patients receive primary androgen deprivation therapy(ADT).However,nearly all men with metastatic PCa develop resistance to primary ADT,a stage of disease known as metastatic castration-resistant PCa(CRPC),which represents the final stage of PCa,with a median survival of less than 2 years.The occurrence and progression of PCa is a complex regulatory process involving multiple genes and signal pathways.Therefore,it is one of the research hotspots that the understanding of molecular mechanism of PCa progression and the discovery of novel biomarkers and therapy targets.PI3K-AKT-mTOR signaling pathway plays a key role in the development of a variety of malignant tumors.It is a key oncogenic signaling pathway that has been linked to tumorigenesis and resistance to both conventional and targeted anticancer therapies in PCa.It has been reported in 42%of primary prostate tumors and 100%of metastatic CRPC tumors.In prostate cancer,alterations of components of the PI3K-AKT-mTOR signaling pathway,including mutation,altered expression,and copy number alterations.These abnormal activation of the PI3K-AKT-mTOR signal pathway is an important reason for promoting the progression of PCa.Therefore,it is important to elucidate the regulatory molecular mechanism of PI3K-AKT-mTOR signaling pathway in PCa progression.PI3K-AKT-mTOR signaling pathway inhibitors could be as single agents or in combination with other agents in the currently evolving treatment landscape of CRPC.Golgi membrane protein 1(GOLM1),also named Golgi phosphoprotein 2(GOLPH2)or Gogi protein 73(GP73),was identified as a novel biomarker for localized PCa.but its biological function and molecular mechanisms remain poorly understood in PCa progression.GOLM1 is up-regulated and consistently overexpressed in PCa tissues.Aberrant overexpression of GOLM1 has also been reported to correlate with many other cancers,such as hepatocellular carcinoma and oesophageal cancer.Currently,the knowledge on the function of GOLM1 is limited.It was reported that GOLM1 acts as a key oncogene by promoting tumor cell invasion,migration,growth,and metastasis in hepatocellular carcinoma and oesophageal cancer.GOLM1 augments hepatocellular carcinoma invasion through activation of the CREB-MMP-13 signaling pathway.The most recent study revealed that GOLM1 promotes hepatocellular carcinoma growth and metastasis through modulating EGFR/RTK cell-surface recycling.Some studies demonstrated GOLM1 as a novel marker for PCa,but its functional role and molecular mechanism are unclear.In part?,we will systematically study the biological function and molecular regulation mechanism of GOLM1 in PCa,especially in regulatory of PI3K-AKT-mTOR signaling pathway,from three levels,including PCa specimen histology,PCa cells and PCa xenograft models.The migration and invasion inhibitory protein(MIIP)is a new tumor suppressor gene.MIIP expression is down-regulated in glioma and lung cancer,and is negatively correlated with the pathological stage and prognosis.MIIP can regulate multiple important intracellular processes by interaction with other molecules,such as IGFBP-2,HDAC6,Cdc20 and so on.MIIP gene is located on chromosome 1p36,which is deleted in prostate cancer(40%).Role and molecular mechanism of MIIP are pool understood.In part?,we will systematically study the biological function and molecular regulation mechanism of GOLM1 in PCa.Elucidates the regulatory relationship between MIIP and AR/PI3K-AKT-mTOR signaling pathway on PCa.In addition,identifies MIIP expression in different Gleason score stages in PCa specimens.Part?Study on the role and molecular mechanism of GOLM1 in prostate cancer1.ObjectivesGolgi membrane protein 1(GOLM1)was identified as a novel biomarker for localized PCa,but its biological function and molecular mechanisms remain poorly understood in PCa progression.In this study,we further identified that GOLM1 expression in all stages and grades of PCa,GOLM1 oncogenic function in PCa cell and xenograft model,and molecular mechanism of PCa progression.2.MethodsGOLM1 expression was determined in PCa by tissue microarrays(TMAs)and real-time RT-PCR,Western blot,and immunohistochemistry(IHC)analyses.To investigate GOLM1 functions in vitro and in vivo,we overexpressed and knocked down GOLM1 in PCa cell lines and established xenograft mice models.Signaling pathway downstream of GOLM1 was detected by Western blot and IHC analyses.3.Results(1)GOLM1 expression was up-regulated in PCa tissues whereas there was no difference across different Gleason score stages.GOLM1 expression was significantly up-regulated in PCa tissues compared with paracancer tissues,and consistently overexpressed in Gleason score 3,4,and 5 stages.GOLM1 m RNA and protein expression was increased compared with paracancer tissues.(2)GOLM1promotes PCa cell proliferation,migration and invasion,and inhibits cell apoptosis.Exogenous overexpresion of GOLM1 significantly accelerated proliferation,migration and invasion and inhibits cell apoptosis in DU145 and PC3.GOLM1 down-regulation induced significant suppression on cell proliferation migration and invasion as well as promotion on cell apoptosis in DU145 and CWR22Rv1.(3)GOLM1 activates PI3K-AKT-mTOR signaling.PI3K(p110),p-AKT(Ser473),and p-mTOR(Ser2448)were significantly up-regulated by GOLM1 overexpression in DU145 and PC3.Strikingly,the expression of PI3K(p110),p-AKT(Ser473),and p-mTOR(Ser2448)were significantly decreased when GOLM1 was knocked down by si GOLM1s in DU145 and CWR22Rv1.(4)GOLM1 promotes tumor growth in xenograft mice models of PCa.The nude mice injected with GOLM1-DU145 cells developed tumors faster than those injected with vector-DU145 cells.As a result,the final tumor sizes of nude mice injected with GOLM1-DU145 cells were much bigger than those injected with Vector-DU145 cells.Ki67 staining and TUNEL analysis also confirmed that GOLM1 overexpression promoted PCa cells proliferation and inhibits PCa cells apoptosis.(5)GOLM1 promotes PCa progression through activating PI3K-AKT-mTOR signaling.PI3K inhibitor BKM120 at a concentration of 500n M significantly abrogated GOLM1's effect on proliferation,invasion and apoptosis in DU145 cells.4.ConclusionsIn this study,we identified that GOLM1 expression is up-regulated in all stages and grades of PCa.GOLM1 promotes cell proliferation,migration and invasion,and inhibits cell apoptosis.GOLM1 plays oncogenic functions mainly through activating PI3K-AKT-mTOR signaling pathway.GOLM1 not only becomes a novel biomarker for PCa diagnosis,but also is able to guide individualized treatment.Particularly,agents that block PI3KAKT-mTOR signaling pathway could be used in PCa patients with GOLM1up-regulation.Part?Study on the role and molecular mechanism of MIIP in prostate cancer1.ObjectivesMIIP(migration and invasion inhibitory protein)was first identified as a new tumor suppressor gene in gliomas.Many studies have reported MIIP was a key negative regulator of cell migration and invasion.In this study,we mainly focus on the proliferation inhibition function and regulation mechanism of MIIP in PCa,and clarify the regulatory mechanism of MIIP on AR signaling pathway and PI3K-AKT-mTOR signaling pathway.In addition,we investigate the relationship between MIIP expression and PCa in different stages and grades of PCa.2.MethodsTo investigate MIIP functions in vitro and in vivo,we overexpressed a MIIP in PCa cell lines and established xenograft mice models.Signaling pathway downstream of MIIP was detected by Western blot and IHC analyses.The interaction between MIIP and serine/threonine phosphatase PP1?was identified by Immunoprecipitation(co-IP)and Immunofluorescence co localization analyses.MIIP expression was determined in PCa in different stages by tissue microarrays(TMAs)analyses.3.Results(1)MIIP inhibits the proliferation of PCa cells.CCK8 and colony formation assay revealed MIIP overexpression inhibits the proliferation in AR~+PCa cell line LNCa P and C4-2.(2)MIIP has no effect on transcriptional activity and nuclear translocation of AR.The expression of AR target genes PSA and TMPRSS2 were both similarly increased in MIIP-LNCa P compared with control cells treated by dihydrotestosterone with dose dependent.After enzalutamide treatment,AR target genes PSA and TMPRSS2 were similarly down-regulated in MIIP-LNCa P and MIIP-C4-2 compared with control cells,and AR expression of nucleoprotein was similarly decreased in MIIP-LNCa P compared with control cells with dose dependent.(3)MIIP inhibits PI3K-AKT-mTOR signaling.p-AKT(Ser473),p-AKT(Thr308),p-mTOR(Ser2448)and p-mTOR(Ser2481)were down-regulated by MIIP overexpression in LNCa P and C4-2,but PI3K(p110),AKT and mTOR were no changing by MIIP overexpression.After PI3K inhibitor BKM120treatment,p-AKT(Ser473),p-AKT(Thr308),p-mTOR(Ser2448)and p-mTOR(Ser2481)were more significantly decreased in MIIP-C4-2 and MIIP-PC3 compared with control cells.(4)MIIP interacts with Ser/Thr phosphatase PP1?in PCa cell cytoplasm,especially in the endoplasmic reticulum.The interactions of both exogenous and endogenous MIIP with PP1?were confirmed by co-immunoprecipitation(co-IP)in MIIP-LNCa P and MIIP-PC3.The co-localization of MIIP with PP1?was detected in Hela and LNCa P by Immunofluorescence co localization analyses.(5)PP1?silencing can abrogate MIIP's effect on AKT-mTOR signaling pathway and cell proliferation.The down-regulation of p-AKT(Ser473)and p-AKT(Thr308)expression and inhibition of proliferation can be recovered in MIIP-C4-2 and MIIP-PC3 transfected with si PP1?s.(6)MIIP inhibits tumor growth in xenograft mice models of PCa.The nude mice injected with MIIP-C4-2 and MIIP-PC3 cells developed tumors faster than those injected with Vector-C4-2 and Vector-PC3 cells.As a result,the final tumor sizes of nude mice injected with MIIP-C4-2 and MIIP-PC3 cells were much bigger than those injected with Vector-C4-2 and Vector-PC3 cells.Ki67 staining also confirmed that MIIP overexpression inhibits PCa cells proliferation in vivo.(7)The expression level of MIIP was negatively correlated with the PCa stages.We detected the MIIP expression in PCa tissues of Gleason score 3,4,and 5 stages by TMAs analyses.The expression level of MIIP gradually decreased with the increasing of Gleason scores.4.ConclusionsIn this study,we identified that MIIP inhibits the proliferation of PCa cells,and has no effect on transcriptional activity and nuclear translocation of AR.We further demonstrated MIIP interacts with Ser/Thr phosphatase PP1?and promotes its activity to down-regulate AKT-mTOR signal pathway and inhibit the proliferation of PCa cells.In addition,we also revealed the expression level of MIIP was negatively correlated with the PCa stages.Therefore,MIIP can be used as a novel biomarker for PCa diagnosis and a potential target of molecular therapy.All in all,we systematically studied that the biological function and molecular regulation mechanism of GOLM1 and MIIP in PCa progression in this paper.It is found that GOLM1and MIIP both participate in the regulation of PI3K-AKT-mTOR signaling pathway at different levels,which are key reasons to result in abnormal activation of PI3K-AKT-mTOR signaling pathway in promoting PCa progression.GOLM1 promotes cancer function in PCa progression through activating the upstream of PI3K-AKT-mTOR signaling pathway.MIIP inhibits the phosphorylation level of AKT by interacting with Ser/Thr phosphatase PP1?.MIIP plays tumor suppressor function in PCa progression by negatively regulatory of AKT-mTOR signaling pathway.