Research On The Mechanism Of ADT Resistance And Bone Metastasis Of Prostate Cancer Regulated By The Feedback Loop Between Tumor Cells And Tumor Microenvironment

Purpose:Increasing evidence revealed that therapy resistance was the most common reason for the recurrence and progression of prostate cancer(PCa).Our previous study demonstrated that tumor microenvironment,specifically tumor associated macrophages(TAMs),played a critical role in the drug resistance,tumor progression and heterogeneity differentiation of PCa.Nevertheless,the interaction between tumor and tumor microenvironment and the underlying mechanism to facilitate the PCa ADT resistance and progression remain to be elucidated.PCa stem-like cells,which contributed to the intratumor heterogeneity,also exert a significant regulating effect on the tumor microenvironment.However,we know little about the influence of PCa stem-like cells on bone microenvironment.Thus,this study aims to reveal the influence of interaction between tumor and tumor microenvironment on PCa ADT resistance,tumor progression and bone metastasis and the underlying mechanism,which could provide new ideas to improve the therapy response of PCa and the prognosis of PCa patients.Methods:Immunohistochemistry(IHC)was applied to investigate the correlation between gankyrin expression and PCa ADT resistance or patient’s prognosis.Meanwhile,the relevance between PCa stem-like cell mark OV6 and bone metastasis was also assessed by IHC.In vitro,a co-culture system was designed to examine the interaction between PCa cells and tumor microenvironment(including prostatecancer-bone microenvironment).The colonization of PCa cells in bone microenvironment was detected by cell adhesion assay.Moreover,western blotting,migration and invation assay,ELISA,flow cytometry and immunofluorescence analysis was employed to find the relationship between PCa malignant phenotype and tumor microenvironment.Furthermore,coimmunoprecipitation analysis,mass spectrometry,antibody microarray assay and CHIP was used to reveal the key signaling pathway that mediated the interaction between PCa cells and the tumor microenvironment.In vivo,subcutaneous xenograft assay and intra-tibial xenograft model were designed to explore the communication between PCa cells and tumor microenvironment or bone microenvironment respectively.Results:(1)Gankyrin was positively correlated with PCa ADT resistance and progression,predicting unfavourable prognosis of PCa patients.Firstly,higher gankyrin expression was detected in PCa tissues than normal ones in online tumor database.Pos-ADT PCa samples also exhibited higher gankyrin expression than na?ve ones.Meanwhile,gankyrin appeared to be an excellent prognostic indicator of BCR and DFS for PCa patients.Secondly,overexpression of gankyrin facilitated the ADT resistance of PCa,while gankyrin knockdown exerted a contrary effect.At last,the expression and secretion of HMGB1,the expression of which also elevated in Pos-ADT PCa tissues,were regulated by gankyrin.In addition,HMGB1 knockdown could reverse the ADT resistance of PCa induced by gankyrin overexpression.(2)The network between PCa gankyrin signaling and TAMs within tumor microenvironment facilitated the development of PCa ADT resistance.Firstly,co-IP and mass spectrometry demonstrated that NONO could bind gankyrin and HMGB1 in PCa cells,and the expression of NONO was regulated by gankyrin.Furthermore,the transcription level of HMGB1 was regulated by AR,a downstream transcription factor of NONO.Secondly,gankyrin facilitated TAMs recruitment and polarization in PCa,which reciprocally promoted ADT resistance and progression of PCa cells.Thirdly,HMGB1-activated TAMs facilitated the secretion of IL6 and the activation of STAT3,which in turn promoted the transcription of gankyrin.At last,combined inhibition of both gankyrin signaling and TAMs in vivo was an effective way to prolong the duration of response to ADT for PCa.(3)The network between PCa gankyrin signaling and TAMs within tumor microenvironment facilitated the development of PCa ADT resistance.Firstly,higher positive rate of PCa stem-like cell marker OV6 was detected in PCa bone metastasis tissues.In animal model,OV6~+PCa cells shown a higher bone metastasis possibility and increased proliferation ability.Secondly,more OV6~+cells were found in PCa cells after the co-culture with pre-osteoblast.In addition,co-cultured OV6~+PCa cells had higher expression of PCa stem-like cell marker(such as c-Myc,OCT4 and CD44),improved sphere-formation ability and increased bone colonization ability than OV6~-PCa cells.Besides,more expression of osteogenic differentiation marker in pre-osteoblast after the co-culture with PCa cells.Thirdly,antibody microarray assay results demonstrated than IL11 was the key signaling molecule that mediating the interaction between OV6~+PCa cells and bone microenvironment.The binding of IL11 to IL11R activated the STAT3signaling,which further regulated the expression of PCa stem-like cell marker c-Myc.Meanwhile,activated c-Myc,as a transcription factor,could bind to the promoter of IL11and improve the transcription level of IL11.Conclusion:Gankyrin expression was positively correlated with PCa ADT resistance and poor prognosis for PCa patients.Furthermore,NONO/AR signaling,which activated by gankyrin,facilitated the expression and secretion of HMGB1.Moreover,HMGB1promoted TAMs recruitment and polarization in PCa,which further activated IL6/STAT3pathway.Gankyrin transcription was improved by activated STAT3.Accordingly,the positive feedback loop between PCa cells and tumor microenvironment was formed,blocking of which could effectively reverse the ADT resistance of PCa.Tumor microenvironment not only facilitated the ADT resistance of PCa,but also played a critical role in the development of PCa bone metastasis as the form of PCa-bone microenvironment.Increased expression of PCa stem-like cell marker OV6 was detected in PCa bone metastasis tissues and animal bone metastasis samples.The interaction between PCa cells and bone microenvironment,on one hand,increased the expression of stem-like cell marker and the bone colonization ability,and,on the other hand,facilitate the differentiation of pre-osteoblast.Blocking the IL11/STAT3/c-Myc positive feedback loop,which mediated the interaction between PCa cells and bone microenvironment,could be served as a potential therapeutic target for the treatment of PCa bone metastasis.