Metformin Inhibits Androgen-associated Epithelial-to-Mesenchymal Transition Via Upregulating ?Klotho In Prostate Cancer

BackgroundProstate cancer(PCa)is the second most common cause of cancer among men worldwide,the incidence of PCa is higher than lung caner,becoming the first health hazard of cancer in the United States.Also,the incidence in China has been showed a rising trend year by year.The disease in early stage can be cured by radical surgery or radical radiotherapy.For advanced prostate cancer,androgen deprivation therapy(ADT)is still the main treatment.Although ADT can initially inhibit tumor growth,more than half of patients will develop to the stage of drug resistance in 2-3 years,which is usually known as castrated-resistant prostate cancer(CRPC).The median survival time of CRPC is only 12 to 18 months.The continued activation of AR signaling pathways remains a key factor in the pathogenesis and development of CRPC.Recent studies have shown that the crosstalk between epithelial-to-mesenchymal transition(EMT)and AR signaling pathways has played an important role in the development and progression of CRPC.Although there is a close relationship between EMT and AR signaling pathways,different researchers have came to different or even contradictory conclusions,the specific mechanism remains unclear which needs further elucidated.In addition to the AR signaling pathway,other signaling pathways are also involved in prostate cancer EMT,including:RTK,Pten,STAT3,Wnt,Notch-hedgehog and so on.Reversing EMT by interfering with the signaling pathways above may provide new prospects for the treatment of prostate cancer in the future.Fibroblast growth factor(FGF)and its receptors FGFRs are commonly found in normal cells,which together form FGF signaling pathway,play an important role in cell growth,differentiation,migration,angiogenesis and tissue wound repair.Recent studies have shown that FGF signaling also involved in the prostate cancer.Its mechanism is mainly through the activation of a series of downstream pathways,including Ras-Raf-Mapk,PI3k-Akt,Stats and PLCy and so on.Most of the FGFs play physiological role by autocrine or paracrine.FGF 19 subfamily is a special class of the FGFs family,including FGF19,FGF21,FGF23,which regulate physiological functions,including energy and bile acid metabolism,glycolipid metabolism,and maintenance of phosphorus,vitamin D dynamic balance,by endocrine.Some scholars have reported that FGF 19,FGF23 can promote the progression of prostate cancer.And activation of FGFR1 can lead to irreversible prostate cancer EMT,leading to disease progression.Although plenty of studies have linked FGFR signaling activation to tumor progression,FGFR signaling have tumor suppressive functions in certain contexts.At present,a series of FGFs and FGFRs inhibitors have been used in the experimental models and clinical trials in prostate cancer,showing a broad application prospect.The activation of the FGF/FGFR signaling pathway requires a key molecule:?klotho(KLB).?klotho act as FGF19 and FGF21 coreceptor that can enhance the affinity of FGF 19,FGF21 and FGFRs(especially FGFR1 and FGFR4),Forming?klotho-FGF19/FGF21-FGFR4 complex,which can regulate blood glucose,bile acid and energy metabolism.The literatures concerning association between ?klotho and tumor were rare and controversial.Although the FGF 19/FGFR4 signaling pathway was reported to promote progression of prostate cancer,the relationship between the expression of ?klotho and the biological behavior of prostate cancer has not been reported.Metformin is the most widely used drug for the treatment of type 2 diabetes worldwide.Epidemiological studies have shown that metformin can reduce the incidence of multiple tumors.A large number of in vivo,in vitro studies have shown that metformin has a wide range of anti-tumor effect,mainly attributed to activation of AMPK(AMP-activated protein kinase)pathway and increase of insulin sensitivity.It was reported that there was a negative feedback between AMPK and AR regulation.Metformin can down-regulated AR expression,inhibiting the growth of prostate cancer.In addition,metformin has been shown anti-EMT activity in a variety of tumors(including prostate cancer).Although there are plenty of researches concering the metformin anti-tumor role,new mechnisms continue emerging.?Klotho-FGF19-FGFRs complex can lowering serum glucose,which has the similar effect with metformin.However,whether there is a potential link between the metformin and?Klotho has not been reported.Part ? ?Klotho inhibits androgen-associated epithelial-to-mesenchymal transition in prostate cancerObjectivesStudying the relationship between AR signaling pathway and EMT in prostate cancer,investigating the relationship between the ?klotho expression and the clinical parameters and prognosis of prostate cancer,and then explore the relationship between?klotho and prostate cancer EMT in vitro and in vivo.Methods1.The effect of dihydrotestosterone(DHT)on EMT in AR-negative PC3 cells and AR-positive LNCaP cells was detected by Western Blot.2.The expression of ?Klotho in different prostate cancer cell lines was detected by Western Blot.The expression of ?Klotho in benign prostatic hyperplasia and prostate cancer tissues was detected by immunohistochemistry.Using The Human protein altas database to analyzes the expression of PKIotho in normal prostate tissue and prostate cancer.3.The relationship between the ?Klotho expression and the age,tumor stage and grade of prostate cancer were detected by chi-square test.The relationship between the PKlotho expression and the survival was detected by Kaplan-Meier single factor survival analysis.4.The effect of ?Klotho on the proliferation of prostate cancer cells were detected by CCK-8 and clone formation assay.The effects of ?Klotho on cell migration and invasion were detected by transwell and cell scratch test.Using Annexin V-PE/7-AAD staining to assess early cell apoptosis.5.The effect of over-expression and knockdown of ?Klotho on EMT in prostate cancer cells was detected by Western Blot and Immunofluorescence assays.6.Investigating the effect of ?Klotho on the growth of prostate cancer in vivo by using nude mice and PC3 cell line,and the effect of ?Klotho on EMT in nude mice was detected by immunohistochemistry.Results1.The expression of E-cadherin was significantly increased in AR-negative PC3 cells after DHT treatment,whereas the expression of N-cadherin,vimentin,slug,snail were upregulated.DHT was able to activate ERK1/2.and MAPK/ERK1/2 inhibitor U0126 was able to reverse DHT-induced EMT in PC3 cells.However,in AR-positive LNCaP cells,DHT had no significant effect on EMT.2.All three cell lines,PC3,LNCaP and C4-2B had ?Klotho expression.Amomg them,expression level in LNCaP cells was highest,which was 2.24 times of PC3 and 1.67 times of C4-2B.PKlotho staining score of 30 benign prostatic hyperplasia cases was 10.15 ± 0.29,30 cases of prostate cancer was 7.47 ± 0.41,the expression difference between them was statistically significant,higher grade(Gleason score>7)patients had lower levels of ?Klotho expression,whereas lower grade(Gleason score<6)patients had higher ?Klotho expression.By analyzing the The Human protein altas dataset,it was found that the expression of ?Klotho in prostate cancer was lower than that in normal prostate tissue,which was consistent with our findings.3 · The chi-square test showed that the lower expression of ?Klotho was associated with higher grade of prostate cancer.The expression level of ?Klotho was not related to the age and clinical stage of the patients.Kaplan-Meier survival analysis,log-rank test showed that patients with low expression of ?Klotho has lower survival rate than that with high expriession of ?Klotho,There was no significant difference between high grade and low grade patients concerning the survival rate.4.The over-expression of ?Klotho in PC3 cells significantly reduced the cell proliferation,increased the early apoptosis,inhibited the cell migration and invasion.Knocking down ?Klotho in LNCaP cells promoted cell proliferation,inhibited early cell apoptosis,promoted cell migration and invasion.5.?PKlotho over-expression reversed DHT-induced E-cadherin downregulation and DHT-induced N-cadherin,vimentin,slug and snail upregulation in PC3 cells.Knocking know ?Klotho decreased E-cadherin expression,increased N-cadherin,vimentin expression in LNCaP cells.And these effects verified in PC3 and LNCaP cells were further confirmed by using immunofluorescence assay.6.The tumor volume of the control group was 2436±790(mm3)and the tumor volume of the ?Klotho over-expression group was 1590±504(mm3)after 4 weeks of tumor formation.The difference between two groups was statistically significant(p<0.01).Over-expression of ?Klotho significantly inhibited the growth of PC3 cells in subcutaneous xenografts.Immunohistochemistry assay showed that the expression of E-cadherin was increased by 2.68-fold(p<0.01),and the expression of N-cadherin was reduced by 69.7%(p<0.01),and the expression of snail was decreased by 46.1%(P<0.01)in ?Klotho over-expression xenografts compared to the control group.Conclusions1.The effect of androgen in the EMT phenotype of prostate caner results from the combined effects of AR-dependent and AR-independent pathways.2.The expression of ?Klotho in prostate cancer is lower than that in benign prostatic hyperplasia,and the low expression of ?Klotho is related to the high grade of prostate cancer.Low ?Klotho expression is a unfavorable factor of prostate cancer survival.3.?Klotho inhibits androgen/AR-associated EMT,acts as a tumor suppressor in prostate cancer.4.?Klotho acts as an inhibitory factor of EMT,could be a potential target for advanced prostate cancer treatment.Part II Metformin inhibits epithelial-to-mesenchymal transition via upregulating ?Klotho in prostate cancerObjectivesStudying the link between metformin and EMT,metformin and AR signaling pathwayin prostate cancer.In view of both metformin and ?Klotho have the effect of lowering blood glucose,to further study the potential relationship between metformin and ?Klotho,and make a preliminary mechanism investigation.Methods1.The effect of metformin on the proliferation of PC3 and LNCaP cells was detected by CCK8 and clone formation assay.2.The effect of metformin on ?Tlotho and EMT was detected by Western Blot.3.The effect of metformin on EMT was detected by tissue culture.4.The effect of metformin on AR transcription activity was detected by RT-PCR.5.The effect of metformin on AR translocation was detected by immunofluorescence assay.6.Explore potential binding site between AR protein and ?Klotho DNA by using the GEO database.Results1.Metformin inhibited PC3 and LNCaP cells proliferation in dose-and time-dependent way.2.In PC3 cells,metformin significantly activated AMPK signaling pathway,reduced AR expression,up-regulated E-cadherin expression,and down-regulated N-cadherin expression,which was confirmed in tissue culture.Interestingly,metformin was able to significantly upregulate the expression of ?Klotho in a concentration-dependent manner.Knocking down ?Klotho in PC3 cells reversed metformin-induced E-cadherin upregulation and N-cadherin down regulation.3.Metformin inhibited DHT-induced AR transcriptional activation in LNCaP cells,decreased AR and its'downstream PSA,NKX3.1 mRNA levels.Moreover,metformin inhibited AR translocation from cytoplasm to the nucleus.4.We found a binding site between AR protein and ?KlothoDNA by analysing GEO database.This binding site was located between exon 1 and exon 2 of ?Klotho.DHT treatment can promote AR protein and ?Klotho DNA binding,whereas anti-andorgen drug enzalutamide reversed this binding increase.Conclusions1.Metformin inhibits the proliferation of prostate cancer cells and upregulates the expression of ?Klotho.Metformin inhibits the EMT,which depends on the expression of ?Klotho in prostate cancer.2.Metformin inhibits the activity of AR signaling pathway.3.Hypothetical mechanism concerning metformin upregulating ?Klotho expression:AR protein binds to ?Klotho promoter region,inhibits the transcription and translation of ?Klotho.Metformin upregulates ?Klotho by inhibiting AR activity.