Metformin Enhances The Anti-cancer Effect Of Androgen Deprivation Therapy By Inhibiting Tumor Associated Macrophage And EMT

Background: Androgen deprivation therapy(ADT)is the standard therapeutic treatment for advanced prostate cancer but with limited benefit due to the profound relapse and metastasis.The initiation of epithelial mesenchymal transition(EMT)and infiltration of tumor associated macrophage are closely related to drug resistance,tumor relapse as well as metastasis.Materials and Methods: Prostate cancer cell lines including PC-3,DU145,CWR22Rv1,LNCaP,C4-2(human),RM-1(mouse);macrophage cell lines including THP1(human)and RAW264.7(mouse)were used in this study.To investigate the in vivo effect of metformin,transgenic adenocarcinoma of the mouse prostate(TRAMP)mice model,nude mice xenograft model were used;the tissues of the mice were used for IHC staining.The prostate tissues of the patients with prostate cancer were collected and used for IHC staining.This study used diverse methods to demonstrate the effect of metformin on prostate cancer,including IHC staining,Western blot,Transwell assay,wound scratch assay,ELISA,clonogenic assay,immunofluerescence,macrophage recruitment assay,cell cycle,apoptosis and so on.Results: By using PC-3 and DU145 cell lines,we found that metformin could inhibit the proliferation,promote cell apoptosis,arrest cell cycle,as well as repress cell migration and invasion in prostate cancer.In TRAMP mice model,we found that metformin could inhibit the progression of prostate cancer from low-grade prostaticintraepithelial neoplasia(PIN)to high-grade PIN,undifferentiated to well-differentiated,and PIN to adenocarcinoma.Moreover,combined with enzalutamide,metformin could enhance its anti-cancer effect in vitro cell line model and in vivo xenograft model.Since castration treatment(bicalutamide and enzalutamide)could induce EMT,our results showed that metformin could reverse castration-induced EMT by using cell line model,mouse and human tissues.At the same time,we found that metformin could inhibit the infiltration of tumor-associated macrophages(TAM)during prostate cancer development and after castration induction.Mechanismly,we found that metformin could repress the expression of COX-2 and subsequent production of PGE2,which play important roles in castration-induced EMT and TAM infiltration.In addition,we found that metformin could also inhibit TGF-?/STAT3 axis,which promotes EMT.Conclusion: EMT and TAM infiltration plays crucial roles in cancer cell invasion,metastasis,and therapeutic resistance.We examined the effect of metformin on tumor-associated inflammation and cancer cell EMT in prostate cancer initiation and post androgen deprivation therapy(ADT)in both TRAMP mouse mode and human patient samples.We found that by downregulating COX2 and its enzymatic product PGE2,metformin is capable of inhibiting inflammatory infiltration and cancer cell EMT,and consequently represses prostate cancer progression.These findings suggest that combination of ADT with metformin could be a more efficacious therapy for prostate cancer.