| BackgroundProstate cancer is the most common male malignant tumor in western countries,and it is also the second leading cause of cancer death in male.Inflammation is closely related to the pathogenesis of prostate cancer.More and more evidence shows that inflammatory factors produced by inflammatory environment and tumor cells,such as tumor necrosis factor-α(TNFα),can promote the downstream carcinogenic target nuclear factor κB(NF-κb)and cancer cell survival.In terms of the importance of inflammation in the development and progression of prostate cancer,the regulation of NF-κB activated signaling pathway has been an attractive target in the chemoprevention and treatment of prostate cancer.At present,a number of tumor treatment schemes based on inflammation inhibition are in clinical trials,but the results are not perfect,which also suggests the importance of further exploring the mechanism of inflammation in prostate cancer reserach.MAP3K7 is a protein serine/threonine kinase,also known as TNFα activated kinase 1(TAK1).MAP3K7 is an important part of MAP kinase signaling pathway and is considered to be one of the key regulatory molecules in inflammatory signaling pathway.MAP3K7 can be activated by cytokines including TNFα,TGF-β and lipopolysaccharide(LPS).Under the stimulation of these cytokines,MAP3K7 can form a complex with TAK1 binding protein 1(TAB1),TAK1 binding protein 2(TAB2)and TNF receptor associated factor 6(TRAF6)to activate IκB kinase(IKK)and its downstream NF-κB pathway.IKK complex have two typical catalytic subunits(IKKa and IKKβ),and a regulatory subunit IKKy(which also known as NEMO).Under the stimulation of inflammatory cytokines(such as TNFα),IKK is activated by phosphorylation and mediates the polyubiquitination and degradation of phosphorylation dependent substrate IκB.The degradation of IκB is a key step in the activation of NF-κB pathway.Recent studies have shown that in addition to IκB,IKK can also phosphorylate other cancer-related proteins,including BAD,FOXO3,BCL-10 and A20,and promote ubiquitination and degradation of these proteins.Therefore,MAP3K7 pathway not only plays an important role in mediating inflammation,but also plays an important role in tumor regulation.It is worth noting that MAP3K7 gene is also closely related to human prostate cancer.MAP3K7 gene is significantly deleted in more than 10%of prostate cancer patients.However,the exact role of MAP3K7 deletion in the pathogenesis of prostate cancer remins largely unclear.Therefore,exploring the mechanism of MAP3K7 in the occurrence and development of prostate cancer is of great significance for the prevention and treatment of prostate cancer.Androgen receptor(AR),a member of steroid nuclear receptor family,plays an important role in the initiation and progression of prostate cancer.At present,AR targeted therapy has always been the first-line minstream treatment for prostate cancer.As a key regulatory target of prostate cancer,all possible mechanisms of regulating AR level are important research fields of prostate cancer treatment.AR protein level and activity are precisely regulated by post-translational modifications,including phosphorylation,acetylation,SUMOylation,methylation and ubiquitination.Similar to other transcription factors,AR protein is strictly regulated by ubiquitin proteasome pathway.The known E3 ubiquitin ligases include MDM2,chIP and Siah,which are involved in regulating the stability and activity of AR under different conditions.In this study,we found a significant decrease in AR level in the inflammatory region when analyzing the IHC results of prostate tissue in patients with prostate cancer.Combined with the relevant background and previous research of our group,it was further confirmed that pro-inflammatory cytokine TNFa induced AR protein polyubiquitination and proteasome degradation.In order to further study the mechanism of TNFα inhibiting AR,we constructed prostate-specific Map3k7 gene knockout mice,which proved that Map3k7 deletion increased the level and activity of AR protein in prostate of mice.At the same time,a series of molecular experiments showed that MAP3K7 was the key target of TNFa inhibiting AR.In order to further clarify the related mechanism,we found that IKKβ,a downstream molecule of MAP3K7,can directly bind to and affect the ubiquitination degradation of AR protein.In view of the fact that IKKβ can phosphorylate the substrate and mediate the ubiquitination of the substrate,we found the classical substrate binding domin ofβ-TRCP in the AR protein sequence by analyzing the AR protein sequence,and confirmed that β-TRCP2 is the direct ubiquitination ligase of AR.At the same time,we also confirmed that the deletion of MAP3K7 and the mutation of AR proteinβ-TRCP binding domin can significantly promote the formation of transplanted tumor in animal models.Blocking AR degradation can enhance AR activity and the growth of prostate cancer cells in vitro and under inflammatory conditions.In this study,we found a new mechanism of MAP3K7 inhibiting prostate cancer by exploring MAP3K7-IKKβ inflammatory signaling pathway,and identified the degradation process of AR by β-TRCP2 mediated by IKKβ for the first time.At the same time,this study also enriched our understanding of the relationship between prostatitis and prostate cancer,and provided new ideas and programs for the prevention and treatment of prostate cancer.Section I:The inhibition of TNFa on AR expressionObjectives1.To explore the effect of prostatitis on AR expression;2.To clarify the inhibitory effect of TNFaon AR protein expression.Methods1.Prostate cancer tissue specimen collection:with the approval of Medical Ethics Committee,prostate cancer tissue specimens of parts of patients with prostate cancer who visited Medical Center from 2010 to 2018 were collected.2.Immunohistochemistry(IHC)was used to detect the expression of AR protein in non inflammation,inflammation adj and inflammation;3.Western blot was used to detect the expression of AR protein after treatment with different concentrations of TNFα or without treatment,and after treatment with MG132 or without treatment;4.Co-IP was used to detect the changes of ubiquitination level of AR protein with different concentrations of TNFα or without TNFα treatment;5.Western blot was used to detect the half-life changes of AR protein with or without TNFαtreatment.Results1.The results of IHC showed that the expression level of AR protein in inflammatory region was significantly lower than that in non-inflammatory and peri-inflammatory regions(P<0.05);2.In LNCaP and LAPC-4 cells,the level of AR protein decreased with the increase of TNFa concentration.The inhibition of AR protein expression by TNFαwas blocked by MG132(P<0.05);3.The ubiquitination level of AR protein increased with the increase of TNFαconcentration(P<0.05);4.TNFα treatment could shorten the half-life of AR protein(P<0.05).SummaryTNFα inhibits the stability of AR protein by promoting the ubiquitination of AR protein.Section Ⅱ:MAP3K7 deletion promotes AR expression and tumor progressionObjectives1.To explore the expression level of AR and its downstream molecules in prostate tissue-specific Map3k7 deficient transgenic mice model;2.To explore the relationship between MAP3K7 and AR in prostate cancer tissues;3.To clarify the regulation of MAP3K7 on AR expression.Methods1.The prostate tissue-specific Pb-Cre+;Map3k7(TAK1)loxP/loxP(Map3k7P/P)mouse model was constructed.The expression of AR protein between Map3k7 deficient mice and control mice was compared by IHC,and the expression of AR and AR downstream genes between Map3k7 deficient mice and control mice was compared by qRT-PCR;2.Construct the organoids of Pb-Cre+;Map3k7P/P transgenic mice and control mice,and compare the expression of AR protein between Map3k7 deficient mice and control mice by immunofluorescence method.3.Western blot was used to detect the expression of AR protein after MAP3K7 knockdown with or without TNFαtreatment;4.IHC was used to detect the expression of MAP3K7 and AR protein in prostate cancer tissues;5.Construct C4-2 cell lines with MAP3K7/AR knockout alone or with MAP3K7 and AR knockout together,and inject them subcutaneously into immune deficient mice to construct xenografts in vitro.QRT-PCR was used to detect the expression of AR and its downstream genes.ResultsThe results were as follows:1.Although there was no significant tumor or precancerous lesion(PIN)in the prostate tissue of Map3k7 deficient mice,significant hyperplastic lesions could be seen.At the same time,the protein level of AR in the prostate tissue of Map3k7 deficient mice decreased significantly,and the expression of AR downstream genes also decreased significantly(P<0.05);2.Compared with the control group,the AR protein level of Map3k7 deficient mice was also significantly decreased(P<0.05);3.After MAP3K7 knock down,the expression level of AR protein increased significantly,and MAP3K7 knockout could block the effect of TNFa on AR protein level(P<0.05);4.There was a significant negative correlation between MAP3K7 protein and AR protein expression in prostate cancer tissues of patients with prostate cancer(P<0.05);5.Knocking down of MAP3K7 can significantly promote the growth of prostate cancer cells,and knockout of AR can reverse the effect of promoting the growth of tumor cells(P<0.05);similarly,knockout of MAP3K7 in vivo can also promote the growth of transplanted tumor in vitro,and this effect can also be reversed by knockout of AR(P<0.05);6.The expression levels of KLK3,NKX3.1 and TMPRSS2 were significantly increased in MAP3K7 knocking down group,which could be reversed by MAP3K7 knocking down(P<0.05);SummaryMAP3K7 can inhibit the expression of AR protein,and the deletion of MAP3K7 can block the inhibition of TNFaon the expression of AR protein.Section Ⅲ:IKKβ promotes the ubiquitination of AR protein and inhibits the expression of AR proteinObjectives1.To explore whether IKKβ can inhibit the expression of AR protein by regulating the ubiquitination level of AR protein;2.To explore whether IKKβ is involved in TNFα regulating AR pathway.Methods1.Western blot was used to detect the expression of AR protein in MG132 treated with or without IKKβ knockdown;2.The effect of IKKβ mutation on AR protein expression was detected by Western blot;3.Western blot was used to detect the effect of wedelolactone on TNFαinhibiting AR protein expression;4.Co-IP was used to detect IKKβ deficiency mutation,and the effect of knockdown of IKKβ and wedelolactone on ubiquitination of AR protein;5.The effect of IKKβ knockdown on the half-life of AR protein was detected by Western blot;Results1.After knockdown of IKKβ,the level of AR protein increased significantly,and this inhibitory effect could be blocked by MG 132(P<0.05);2.Compared with IKKβ wild type,IKKβ functional deficient mutant could not inhibit the expression of AR protein(P<0.05);3.Wedelolactone,an inhibitor of IKKβ,can block the inhibitory effect of TNFαon AR protein;4.Compared with IKKβ wild type,IKKβ functional deficient mutant could not increase the ubiquitination level of AR protein,while knockdown of IKKβ and wedelolactone,an inhibitor of IKKβ,significantly decreased the ubiquitination level of AR protein(P<0.05);5.Knockdown of IKKβ could significantly prolong the half-life of AR protein(P<0.05).SummaryIKKβ can inhibit the ubiquitination of AR and the expression of AR protein.Section Ⅳ:IKKβ-mediated ubiquitination of AR by β-TRCP2Objectives1.To explore the detailed mechanism of ubiquitination degradation of AR by βTRCP2;2.To explore the mechanism of IKKβ involved in the regulation of AR ubiquitination by β-TRCP2.Methods1.Compare and find out whether there is a classical recognition domain ofβ-TRCP substrate in AR protein sequence;2.Western blot was used to detect the effect of wild type β-TRCP1 andβ-TRCP2 and their F-box domain deletion mutants on AR protein level;3.The effects of TNFa and IKKβ on the regulation of AR protein level byβ-TRCP2 were detected by Western blot;4.Co-IP was used to detect the effect of knockdown of β-TRCP2 and IKK β on ubiquitination of AR protein;5.The effect of Co-IP on ubiquitination of AR protein was compared with that ofβ-TRCP2 wild type and its F-box domain deletion mutant;6.The effect of knockdown of β-TRCP2 on the half-life of AR was detected by Western blot;7.According to the prediction,S158A/S162A and S298A/S302A mutations of AR protein were constructed,and the effects of S158A/S162A and S298A/S302A mutations on the degradation of AR by β-TRCP2 were detected by Western blot;the effects of S158A/S162A and S298A/S302A mutations on the ubiquitination of AR protein by β-TRCP2 were detected by Co-IP;8.The effects of S158A/S162A and S298A/S302A mutations on the half-life of AR protein were detected by Western blot;9.Luciferase assay was used to verify the effect of IKKβ and/or β-TRCP2 knockdown on downstream target genes of AR;10.The expression of KLK3,NKX3.1,KLK2 and TMPRSS2 were detected by qRT-PCR;11.MTS test verified that AR wild type and S158A/S162A and S298A/S302A mutants could promote the growth of prostate cancer cells under the action of TNFa;12.Control group,wild-type AR overexpression group and S298A/S302A mutant AR overexpression group stable cell line were constructed and subcutaneously injected into immune-deficient mice and treat with TNFa at the same time,sacrificed different groups mice to compare the tumor size after 27 days;Results1.There are two significant classical recognition domains of β-TRCP substrate in AR sequence;2.Overexpression of wild-type β-TRCP1 and β-TRCP2 could inhibit the expression of AR protein(P<0.05).The deletion mutation of F-box domain ofβ-TRCP1 and β-TRCP2 had no significant effect on the level of AR protein;3.Knockdown of β-TRCP2 blocked the inhibitory effect of TNFa and IKKβ on AR(P<0.05);4.Knockdown of β-TRCP2 could significantly reduce the ubiquitination level of AR protein(P<0.05);5.Compared with β-TRCP2 wild type,β-TRCP2 F-box domain deletion mutant could not inhibit the ubiquitination level of AR protein(P<0.05);6.After knockdown of β-TRCP2,the half-life of AR protein was significantly prolonged(P<0.05);7.AR S298A/S302A mutation could inhibit the effect of β-TRCP2 on the ubiquitination of AR protein and inhibit the expression of AR protein,but S158A/S162A had no effect(P<0.05);8.AR S298A/S302A mutation but not S158A/S162A mutation could prolong the half-life of AR(P<0.05);9.Knockdown of IKKβ and β-TRCP2 could promote the expression of AR downstream genes(P<0.05);10.AR S298A/S302A mutation,but not S158A/S162A mutation,could resist the killing effect of TNFαon prostate cancer cells;under the effect of TNFα,AR S298A/S302A mutant stable cell line had faster tumorigenesis in mice(P<0.05).Summaryβ-TRCP2 is an ubiquitin ligase of AR,and participates in the TNFα-MAP3K7IKKβ pathway to regulate AR,AR DSAGKS region is the specific target of IKKβ andβ-TRCP2 on AR. |
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