Valproic Acid Inhibits Epithelial-Mesenchymal Transition In Prostate Cancer Via TIF1?

BackgroundProstate cancer is the second-highest morbidity malignancies worldwide,the incidence of prostate cancer in China increased year by year in nearly a decade.More than 40%of patients diagnosed with prostate cancer in our country has entered the middle-late stage period,far more than Europe and the United States.Androgen deprivation therapy(ADT)is the main treatment of advanced prostate cancer at home and abroad,but its validity in general can only maintain 18 to 24 months.After failure of ADT therapy,disease will develop in castration resistant prostate cancer(CRPC),part of the patient's tumor even further transferred to the outside of the prostate other organs.For those prostate tumor,new type of Androgen biological synthetase inhibitors treatment will be a probable candidate,but the effect is limited.EMT and mesenchymal-epithelial transition(MET)play important roles in the regulation of cancer metastasis.During EMT in cancer,transformed epithelial cells lose their characteristic of epithelial membrane adhesions,invade through the epithelium basement membrane and acquire the ability to move freely in neighbouring tissue or to metastasize to distant organs by means of vascular routes.In addition to losing inter-cellular junctions,cells undergoing EMT lose epithelial cell polarity,decrease epithelial markers,such as E-cadherin,and increase mesenchymal markers,such as N-cadherin.Therefore,cells with these characteristics are more prone to invasion and distant metastasis.Tumor related EMT is not a simple process that is needed only to acquire migration and invasion ability but a complicated and comprehensive recombination that is involved in epigenetics,metabolism and differentiation.In prostate cancer patients,EMT plays a major role in the in situ transformation and progression of tumor cells into metastatic cells.Such cancer cells lose their epithelial capabilities and gain mesenchymal abilities.EMT also stimulates the process of CRPC,which is considered a drug-resistence stage of PCa.EMT is controlled by several signal transduction pathways,including the TGF-? signalling pathway.The transforming growth factor ?(TGF?)signalling pathway plays a paradoxical role in cancer development and outcome.SMAD4 is a major tumour promoter that is currently thought to function constitutively in the TGF-? signalling pathway.As a key promoter of the TGF-? signalling pathway,SMAD4 plays an crucial role in reversing the EMT process in PCa.The E3 ubiquitin-protein ligase transcriptional intermediary factor1?(TIF1?)is thought to interact with TGF?/SMAD signalling by mono-ubiquitinating SMAD4 and then inhibiting the association of SMAD4 with phospho-SMAD3.Our previous studies indicate that Valproic acid(VPA)regulates EMT by suppressing SMAD4.In the present study,twenty patients with prostate cancer from Shandong Provincial Hospital affiliated with Shandong University were selected randomly and subjected to laparoscopic radical prostatectomy.The pathologic results of the patients were definitively diagnosed as prostate cancer.This study evaluated the different expression of prostate cancer related proteins and the association of those proteins and clinicopathological factors between colon cancer patients and normal cases using immunohistochemical staining.Experiments in vivo and in vitro,we have confirmed molecular mechanism of VPA inhibits epithelial-mesenchymal transition in prostate cancer via the mono-ubiquitination of SMAD4 by TIFly using immunohistochemical staining?Co-IP and western blotting.Objective1.This study evaluated the different expression of prostate cancer related proteins and the association of those proteins and clinicopathological factors between prostate cancer patients and normal cases using bioinformatics.2.To study the expression of TIF1? and SMAD4 in colon cancer and the corresponding adjacent non-cancerous tissues using immunohistochemical staining.3.Experiments in vivo and in vitro,we cultured PC3 cells and established prostate carcinoma xenografts,treated with VP A,to measure the expression level of TIF1??SMAD4 and EMT related marker proteins,study explored the association between the process of VP A inhibiting EMT via TIF1? and the mono-ubiquitination of SMAD4.Methods1.The prostate cancer tissues and para-carcinoma tissues from twenty patients with prostate cancer were harvested,fixed in 10-15%neutral buffered formalin solution,embedded in paraffin,cut into 5-mm slices and then deparaffinized and rehydrated.To study the different expression of TIF1? and SMAD4 using immunohistochemical staining.Combined with clinical data,To study explored the association between prostate related proteins and clinicopathological variables and patient outcome to determine whether SMAD4 or TIF1 ? expression was an independent prognostic factor and a potential therapeutic target for colon cancer.2.To measur the expression of TIF1? and SMAD4 in PC3 and LNCaP cells and prostate carcinoma xenografts both of VPA treatment group and control group,study the association between the process of VPA inhibiting EMT via TIF1 ? and the mono-ubiquitination of SMAD4.Results1.VPA increases the levels of SMAD4 mono-ubiquitination in wild-type PC3 cells but has no effect on PC3 cells expressing a Smad4 point mutant;2.VPA regulates EMT via TIF1?;3.VPA suppresses the growth of PCa xenografts;4.TIF1? expression correlates negatively with SMAD4 expression in prostate cancer tissues;5.VPA exerts opposite effects on SMAD4 and TIF1? nuclear expression in vivo.ConclusionsVPA inhibits prostate cancer metastasis by decreasing nuclear retention of SMAD3/SMAD4.The mono-ubiquitination of SMAD4 by TIF1? can occur "in solution" before nuclear SMAD4 and phosphorylation SMAD3 form a complex on DNA or while SMAD3/SMAD4 complexes are engaged in transcription.Our results establish the existence of crosstalk between TIFly and the TGF-?/SMAD4 pathways.Aims to prove that valproic acid as clinical medicine in the treatment of advanced prostate cancer,enriched the adanced prostate cancer treatment strategies,and provides more detailed statistical basis for clinical application.