Characterization Of RacGAP1 In Prostate Cancer Progression

BackgroundProstate cancer is among the most threatening tumors worldwide,with which incidence ranks second in male malignant tumors.Meanwhile,it ranks fifth in the mortality among all tumors worldwide.The incidence of prostate cancer is relatively lower in China,however,with the aging of the population,obesity,lifestyle westernization and the improvement of diagnosis technology in recent years,the incidence in China has grown rapidly,with an annual increasing rate of 5%.Androgen Receptor(AR)is an important molecule in the development of prostate and prostate cancer.It functions as a transcriptional regulator by bind to ligands such as Dihydrotestosterone(DHT),and then bind to the androgen-response elements(ARE)of androgen-dependent genes' promotor,plays a role in transcriptional regulation with the aid of transcriptional co-regulatory factors.AR regulates the proliferation,migration,anti-apoptosis and epithelial-mesenchymal transition of prostate cancer cells by regulating a series of downstream genes expression.The first-line treatment for advanced prostate cancer is androgen deprivation therapy(ADT)in view of the key role of AR in the development of prostate cancer.In early stages of treatment,tumor cells tend to be highly sensitive to treatment.However,most tumors recur after 12-18 months of ADT treatment,and produce resistance to androgen deprivation,which is converted to castration resistance called castration-resistant prostate cancer(CRPC).AR signaling reactivation is present in most CRPC cases.By analyzing differentiate expressed genes during prostate cancer progression,we found a key differentiate expressed gene in prostate cancer progression,Rac GTPase activating protein 1,or RacGAP1.RacGAP1 belongs to the Rho small G protein family,functions to activate the GTPase hydrolytic activity of Rho protein and hydrolyzes the binding GTP,inactivating Rho protein.RacGAP1 is also an essential component of the central spindle,controlling the formation of the cytokinesis contraction ring and the anchorage to plasma membrane,mediating cell division.Additionally,RacGAP1 is also involved in STAT3 activation and its nucleus entry,plays a key role in Jak-STAT3 pathway transcriptional activity,while STAT3.can affect the expression of RacGAP1 at the transcriptional level;RacGAP1 negatively regulates HIF-1? and is involved in the regulation of hypoxia via HIF-1?pathway.RacGAP1 is associated with diseases such as teratozoospermia,infertility,and virus infection.In the same time,RacGAP1 expression dysregulation is also associated with tumors.RacGAPl promotes cytokinesis by inhibiting Hippo/YAP signaling pathway,thereby promoting tumor cell proliferation;RacGAP1 promotes tumor cell metastasis by regulating Rho protein activity;RacGAP1 and STAT3 regulatory loops also play an important role in tumors,promote tumor progression such as bladder cancer and liver cancer.RacGAP1 high expression is also associated with malignancy,lymph node metastasis,vascular invasion,and poor prognosis of many cancers such as colorectal cancer,ovarian cancer,breast cancer,lung cancer,gastric cancer,and meningioma.RacGAPl expression is also dysregulated in prostate cancer,but the underlying mechanism is still unclear.Therefore,we focus on whether RacGAP1 is related to the biological behavior of prostate cancer,and the underlying mechanism of RacGAPl in prostate cancer progression,wishing to have a deeper understanding of this disease.Research purposes1.Searching for differentiate expressed genes in prostate cancer progression;2.Exploring the role of RacGAP1 in prostate cancer progression;3.Exploring the relationship between RacGAPl and androgen receptors;4.Exploring the role of RacGAPl plays in prostate cancer progression mediated by AR signaling pathways;5.Exploring the regulatory of downstream molecules by RacGAP1.Research method1.Screening for differentiate expressed genes in prostate cancer progression1.1.GEO databases and GO function analysis were used to screen for differentiate expressed genes between metastatic prostate cancer and primary prostate cancer;1.2.TCGA and GEO databases were used to explore the relationship between RacGAP1 and clinicopathological characteristics of prostate cancer;1.3.Prostate cancer specimens were used for immunohistochemistry assay,to explore the relationship between RacGAP1 and clinicopathological characteristics of prostate cancer.2.The role of RacGAP1 in prostate cancer progression2.1.Western blot and RT-qPCR were used to detect the expression of RacGAPl in prostate cancer cell-lines;2.2.Western blot and RT-qPCR to detect RacGAP1 siRNA and overexpression plasmid transfection efficiency;2.3.DU145,VCaP cell were used for interference,while LNCaP cell were used for RacGAPl overexpressing,carrying out functional experiments to detect the alteration of tumor cell proliferation and metastasis;3.Relationship between RacGAP1 and androgen receptor3.1.Analysis of GEO database to explore the effect of androgen on RacGAP1 expression;3.2.Detect whether RacGAP1 has androgen reactivity(dose/time);effect of androgen deprivation on RacGAP1 expression.3.3.Effect of AR on RacGAP1 expression;3.4.Prediction the Androgen Responding Elements(ARE)in the promoter region of the RACGAP1 gene;3.5,ChIP assay to explore whether AR have a transcriptional regulation of RacGAP1.4.The characterization of RacGAP1 plays in the progression of prostate cancer mediated by AR signaling pathway4.1.Changes in RacGAP1 expression as overexpressing AR expression with/without interfere RacGAP1;4.2.Overexpression of AR and interfere RacGAPl expression,detecting the ability of proliferation of tumor cells;4.3.Interfering with AR and overexpressing RacGAP1,detecting whether RacGAPl can compensate for the decreased proliferation of tumor cells caused by decreased AR expression.5.Exploring the regulatory of downstream molecules by RacGAPl5.1.DU145 cells transiently interfering RacGAPl expression,microarray was used to detect differentially expressed genes;5.2.GSEA was used to predict possible downstream genes of RacGAP1;5.3.After interference/overexpression of RacGAP1,RT-qPCR and Western blot to detect expression changes of related genes and protein;Research result1.RacGAP1 is significantly up-regulated in metastatic prostate cancer.253 differentially expressed genes were discovered by comparing two GEO databases about prostate cancer progression;molecules about cytoskeleton and cell cycle play an essential role in tumor proliferation and metastasis,so a GO analysis was carried out to identify genes correlated with these functions.41 and 71 genes were found separately from 253 genes through GO functional analysis.35 genes involved both in the cytoskeleton and cell cycle,and RacGAP1 is the most significantly differentiate expressed genes among those genes.Consequently,we chose RacGAP1 as the target for our research.RacGAP1 is closely related to clinical/pathological T stage,lymph node metastasis,distant metastasis,Gleason score in TCGA and GEO database data;RacGAP1 is associated with Gleason score in our prostate cancer samples in immunohistochemistry assay;moreover,RacGAPl overexpression is closely related to biochemical recurrence of prostate cancer;2.RacGAPl functions to promote prostate cancer cell proliferation and migrationRacGAPl is highly expressed in VCaP and DU145 cells,and downregulated in LNCaP cells among prostate cancer cell lines;RacGAP1 plays a role in promoting cell proliferation,migration in vitro.3.RacGAP1 is an Androgen induced gene and is transcriptional up-regulated by ARAR plays an important role in the development,progression and metastasis of prostate cancer.Therefore,we explored the relationship between RacGAPl and AR.We first used GEO database for analysis,data shows that RacGAP1 is up-regulated by androgen and AR;After androgen deprivation,RacGAP1 expression first decreased significantly and then increased gradually,which is also convinced by our cellular experiment results.RacGAPl showed a time-and dose-dependent dependence on androgen in LNCaP cells.AR could positively regulated RacGAPl expression.AR is a transcriptional regulator,so we hypothesized that AR could play a direct regulatory role in RacGAPl at the transcriptional level.Software predictions showed that there are AR binding sites in RacGAP1 promoter region;ChIP assay confirmed that AR binds on RacGAPl promoter.RacGAPl could act as a molecular chaperone and promote the nucleus entry of some nuclear proteins.Therefore,we hypothesized that RacGAPl may also promote AR nucleation and activation.However,CoIP experiments confirmed that RacGAPl could not exert molecular chaperone function on AR by binding to AR.The above experimental results indicate that RacGAP1 is an androgen-inducible gene that can be activated by AR transcription.4.RacGAPl plays a role in the proliferation of prostate cancer cells mediated by AR signaling pathwayIn the previous part of the experiment,we found that RacGAPl is activated by AR,so we hypothesized that RacGAPl may act as a downstream molecule of AR and play a role in the progression of prostate cancer.After interfering AR expression in LNCaP cells,RacGAP1 expression was down-regulated,in the meanwhile,cell proliferation ability were decreased.Interfering AR and then adding RacGAP1 overexpression vector can reverse the decrease of RacGAPl and the decrease of cell proliferation ability.AR overexpression lead to an up-regulation of RacGAP1 expression,and an increase of cell proliferation capacity;overexpression of AR and interference with RacGAPl expression may lead to a decrease in cell proliferation ability.From this part of the experiment,we concluded that RacGAPl is involved in AR-regulated prostate cancer progression.5.RacGAPl regulates downstream moleculesNext,we explored the mechanism of action of RacGAPl in prostate cancer progression.After interference with RacGAP1 expression in DU145 cells,genome-wide expression profiling was performed.Gene set enrichment analysis(GSEA)was used to screen the results.Differentially expressed genes were mainly enriched in tumor-related pathways such as BMI1,MYC,mTOR and E2F.Next,Western blot confirmed that RacGAPl can positively regulate C-MYC expression at the protein level,but not in transcriptional level.RacGAPl also regulated cell-cycle related genes expression,which was confirmed by GSEA,co-expression analysis and cellular experiment.Conclusion1.RacGAP1 expression level is significantly increased as prostate cancer progression;2.RacGAP1 high expression in prostate cancer is closely associated with higher clinical stage,metastasis,Gleason score and biochemical recurrence;3.RacGAP1 is an androgen-inducible gene regulated by AR at transcriptional level;4,RacGAPl plays an essential role in the proliferation and metastasis of prostate cancer cells,and this effect is partially regulated by AR;5,RacGAP1 may play a role in promoting cancer by regulating cell cycle and c-Myc and other pathway activities.