The Roles Of Smad4 Involved In The Androgen Receptor SUMOylation And Transcriptional Activity Regulation

Androgen receptor(AR)signal plays core roles in prostate cancer development,and its abnormal activation activates the downstream target proliferative genes,to promoting cell growth and a directly result to hormone-dependent malignant transformation.Therefore,seeking and identifying the new AR regulatory factors,particularly inhibitory factors,is the key to prevent prostate cancer development and malignant transformation.TGF-?/Smads signal is the most important intracellular signal for cell differentiation,while Smad4 is the core protein for achieving TGF-p/Smads signal transduction.In a variety of tumors including prostate cancer,since the deletion or inactivation of Smad4,induced the disorder of TGF-p in cell differentiation,and cause EMT malignant cell transformation.Therefore,It is important to study Smad4 regulation in tumor associated signals.In this thesis we systematically studied the roles of Smad4 in AR sumoylation and transcriptional activity regulation,provided a novel molecular target to cross-link between proliferation and differentiation signals in prostate cancer.First,we studied the effects of Smad4 on AR transcriptional activity.In human embryonic kidney cell line HEK293 or prostate cancer cell line LNCaP,using the strategy of overexpression or knockdown Smad4,we checked AR-mediated downstream gene tanscriptional activity,including prostate-specific antigen(PSA)and ARE luciferase reporters.The overexpression of Smad4 can inhibit the AR mediated PSA or ARE transcriptional activity,whereas the knockdown of Smad4 can enhance AR signal activity.Then by western blot experiment,we demonstrated that Smad4 reduced AR downstream target proteins expression,including PSA and KLK2,whereas the knockdown of Smad4 can enhance their expression.These results suggest that Smad4 has inhibitory effects in AR transcriptional activity.AR transcriptional activity is regulated by its protein modification,wherein,sumoylation is a novel protein modification mode,by ligase system,adding small molecule SUMO protein to a target protein,thus to modulate the protein transcriptional activity and to alter its physiological functions.AR is a transcription factor,which can be sumoylated in 386 and 520 lysine residues.With Luciferase reporter assay,we examined the effects of Smad4 on either wild type AR and AR(K386/520R)mutant-mediated PSA reporter activity.Smad4 inhibition of AR transcriptional activity was disappear in with SUMO sites mutant AR,suggesting that SUMO system involved in the inhibition of AR Signal by Smad4 protein.We found the knockout of SUMO1,but not SUM03 can block the inhibitory effects of Smad4 on AR signal,and the overexpression of Smad4 can enhanced inhibition effects on AR signal by SUMO1.These results suggest that SUMO1 is involved in the process of AR signal inhibition by Smad4.Then,we analyzed the effects of Smad4 on AR symoylation.Immunoblotting experiments found that a modified AR band was shown by the co-expression ofSmad4 and further enhanced with the amount of Smad4 increased.This modification band can be recognized by SUMO1 antibody after immunoprecipitation.While Smad4 could not mediate SUMO sites mutant AR modification occurs,indicating that Smad4 promote AR sumoylation.Furthermore,we examined the effect of Smad4 on exogenous SUMO1 mediated AR modification.Smad4 can significantly enhance AR sumoylation,and after silencing Smad4,AR is difficult to be modified by SUMO1.Thus,we identified Smad4 as a new AR sumoylation regulator.AR(K386/520R)could not be modified by Smad4,further proved the regulation of Smad4 on AR Sumoylation.When using shRNA silencing Smad4 to significantly reduce SUMO1-mediated AR modification.These results illustrated Smad4 is a key regulatorer for AR sumoylation.Further,we investigated the modulation of Smad4 on AR cellular localization of the AR in immunofluorescence experiment.Wild-type AR evenly distributed within the nucleus;when the co-expressed both AR and Smad4,AR and Smad4 was condensed into a spot in the nucleus,indication Smad4 promote AR gathering in the nucleus,while the morphological changes are very similar to the images of co-expression of AR and SUMO1.We also detected the regulation of Smad4 on AR/SUMO1 complexes by immunofluorescence.SUMO1 with AR together formed points in the nuclei,and this effects could be enhanced by co-expression of Smad4,showing further aggregation in the nucleus.However,co-expression of Smad4-shRNA for silencing Smad4,AR separated from SUMO1 complex,and manifested as diffuse distribution.In contrast,Neither Smad4 nor SUMO1 could mediate SUMO sites mutant AR condensed dots in the nucleus.These results indicated that Smad4 facilitated SUMO1-mediated AR sumoylation.By GST pull down experiments we verified the key functional domains of Smad4 in AR regulation.We found both GST-Smad4-MH1 and GST-Smad4-MH2 possess binding capacity to AR in vitro,and K113 site mutation will reduce the binding capacity of GST-Smad4-MH1 binds to AR,suggesting K113 site involved in the interaction between Smad4-MH1 and AR binding.In addition,SUMO1 co-expression can be enhanced GST-Smad4-MH1 and AR in vitro binding capacity.So we proved that Smad4-MH1 has the ability to bind the AR,and this effect can be enhanced co-expressed by SUMO1,K113 of Smad4 is contributed to the interactions between the two proteins.We further confirmed the participatory role of Smad4 K113 site in AR sumoylation process.We compared the effects of each Smad4(wild type,K113R,K159R,or K113/159R)on AR sumoylation modulation,and wild type and K159R Smad4 can be induced AR sumoylation,but K113R and K113/159R Smad4 deficient in this function,suggesting Smad4 through its K113 site regulation AR sumoylation.Furthermore,we examined each Smad4 on AR transcriptional activity,and found wild type and K159R Smad4,but not K113R and K113/159R inhibited AR-mediated PSA promoter transcriptional activity,indicating the 113 lysine residue play a key role for Smad4 in AR transcriptional activity and sumoylation regulation.Furthermore,y GST pull down experiments we have compared the binding capacity between GST-Smad4-MH1 and each of wild-type AR or SUMO site mutants AR(K386R,K520R,K386/520R)in vitro.AR(K386R)or AR(K386/520R)and GST-Smad4-MH1 physical binding was weak,indicating K386 site is a key amino acid for AR and Smad4 binding.We further confirmed the participatory role of AR of K386 in AR sumoylation process.We compared the effects of Smad4 on each AR(wild type,K386R,K520R,or K386/520R)isoforms sumoylation modulation,and found Smad4 can be induced AR sumoylation in wild type or K520R type,but not in K386R or K386/520R type,suggesting Smad4 regulation AR K386 sumoylation site.Furthermore,we examined Smad4 on each AR transcriptional activity,and found that transcriptional activity of wild-type and K520R AR significantly decreased when overexpression of Smad4,while K386R and K386/520R AR had no significant change,indicating the Smad4 regulates AR transcriptional activity and sumoylation in its 386 lysine residue.We also discussed the possible mechanisms for Smad4 enhanced AR sumoylation.PIAS protein family is an important E3 ligase,which mediated core enzyme in sumoylation.So,we explored the possible role of Smad4 in the recruitment of PIAS proteins.In the co-precipitation experiment,we tested that Smad4 recruited three proteins from PIAS protein family,PIAS1,PIAS2,PIAS3,further to promote AR and SUMO1 complex formation.Further by over-expressing and knockout PIAS protein,we demonstrated the significance of the recruitment.PIAS1,PIAS2,PIAS3,but not PIAS4,can be further enhance Smad4 induced SUMO1-mediated AR sumoylation.The knockdown of PIAS 1,PIAS2,or PIAS3 can significantly inhibit Smad4 induced SUMO1-mediated AR sumoylation.All above indicate Smad4 recruited PIAS1/2/3 to promote AR sumoylation.Finally,we examined the association between Smad4 expression and AR sumoylation levels.When overexpressing AR and SUMO1 in the five lines of cancer cells(prostate cancer LNCaP,breast cancer MCF7 and MDA-MB-231,colon cancer SW480,andcolon cancer HT29),Smad4 expression cells,AR SUMO1 can be modified,and Smad4-deficient cells,AR was sumoylated by SUMO1 only in Smad4 positive cells,but not in Smad4 negative cells,indicating that Smad4 expression level determines the AR sumoylation by SUMO1.Luciferase reporter gene assay showed that Smad4 expression and transcriptional activity of AR is low in Smad4 positive cells,and high in Smad4 negative cells,proved Smad4 expression level affect AR sumoylation levels and transcriptional activity.In summary,this paper identified Smad4 as a new regulatory factor for AR modification and AR signal inhibition,elucidating the molecular mechanisms in Smad4 modulated AR transcriptional activity,and providing the cellular processes evidence that Smad4 promotes AR sumoylation and inhibit AR transcriptional activity.The present study provides new molecular target to cross-link between proliferation and differentiation signals,and new molecular targets and signals for tumor therapy.