MDSCs Drive Metastasis And Castration Resistance In Prostate Cancer By Mobilizing Autocrine Wnt/PCP

Objective:Androgen deprivation therapy(ADT)is an important part of the first-line treatment strategy for prostate cancer(PCa).However,a large proportion of advanced prostate cancer patients treated with ADT will relapse and continue to develop fatal metastatic castration-resistant prostate cancer(m CRPC).Therefore,early identification and blocking of this undesirable progress is currently a clinical problem that needs to be resolved urgently.Studies have shown that myeloid-derived suppressor cells(MDSCs)that infiltrate the tumor microenvironment contributed to the severity of the malignant progression of PCa,but the specific molecular mechanisms involved in the regulation of CRPC need to be further studied.This project explores the specific participation mechanism of Wnt/PCP in the process of castration resistance and metastasis of PCa driven by abnormally aggregated MDSCs in the m CRPC tumor microenvironment from the perspective of immunology,to provide a new molecular marker and treatment ideas for clinical diagnosis and control of such refractory diseases.Methods:(1)To analyze the effects of the copy number variation(CNV)of chemokine CXCL5 and receptor CXCR2 in PCa tissue on the survival rates of patients and the relationship between their expressions and tumor hormone sensitivity use the TCGA database.Tissue specimens from patients with prostate hyperplasia(BPH),primary prostate cancer(PPC)and castration-resistant prostate cancer(CRPC)were collected to detect the expression levels of CXCL5 and CXCR2 by immunohistochemistry(IHC)and to detect MDSCs infiltration by tissue immunofluorescence technology.(2)The conditioned media from culturing MDSCs,hereafter referred to as activated conditioned medium(ACM),is used to cultivate PCa cells,observing the changes of cell protrusions under a phase contrast microscope.Cell migration ability was tested by wound healing assay and Transwell assay.After 3-D culture,the changes in cell morphology was observed with phase contrast microscope.Establish the blood tract metastasis model in nude mice,observe the metastasis of each group using in vivo imaging technology,and then observe and analyze the number of lung nodules and the average area of nodules by IHC.Changes in proliferation capacity were detected by clone formation experiment and Ki-67.The activation status of AR signal pathway was detected with RT-PCR.The cell derived xenograft(CDX)models in nude mouse were established;observed and recorded the tumor size,and detected Ki-67 in tumor tissues to analyze the proliferation activity.(3)Smurf1/2 is the protrusions regulator and key regulator of PCP.To verify whether the activation of ACM is mediated by core PCP,knocked down Smurf1/2 and the core components of PCP and observed the changes of protrusive activity and migration capacity.(4)The TCGA database was used to analyze the CNV of the main receptors in the Wnt/PCP axis,clarify their impact on survival,and explore the relationship with antiandrogen therapy in PCa.The GEO database was used to analyze the m RNA expression of FZD6 in CRPC and PPC tissues and the correlation with AR.The effect of receptor FZD on the activation of AR pathway and the expression of AR-V7 was analyzed by RT-PCR.After the dominant receptor molecule anchored,its relative expression in BPH,PPC and CRPC tissues was analyzed with IHC.And its correlation with clinical parameters was Analyzed.(5)PCa cells were infected with lentivirus that targeted to knock down the expression of FZD6 and the stable strains were screened.Under the premise of in vitro castration culture,Ki-67,clone formation,flow cytometry,3-D culture,and CDX assays were used to detect the effect of FZD6 knockdown on cell proliferation in vivo and in vitro.Wound healing experiment,Transwell assay,Phalloidin staining,in vivo imaging after the establishment of blood tract metastasis model,and lung tissue IHC experiment were applied to explain the migration ability in vivo and in vitro.(6)MDSCs and ACM induced 22RV1 cells were treated with inhibitor LGK-974 to detect the changes in cell migration and proliferation.Knockdown of Wnt5 a or Wnt11 in 22RV1 cell to observe the changes in cell migration and detect activation status of AR pathway.HA-labeled Wnt5 a recombinant protein was added to 22RV1 cells,collecting the supernatant.The wild-type 22RV1 cells were cultured with the collected supernatant,and the cell lysate was collected for IP experiment to analyze whether Wnt autocrine occurred.The laser scanning confocal microscope(LSCM)was used to observe the arrangement of F-actin and the binding of Wnt5 a and FZD6.(7)22RV1 cells with stable knockdown of FZD6 were induced by ACM,then the protein levels of CAMK?/STAT3/ROR?/AR and Rho A were detected by western blot(wb)experiment,to verify their participation in the regulation of m CRPC triggered by MDSC.MDSCs or 22RV1 cells were treated with MDSCs,Cabozantinib,recombinant IL-23 and LGK-974,respectively and in combination under the premise of castration culture.The protein expressions of Rho A and CAMK?/STAT3/ROR?/AR were tested to indicate that IL-23 secreted by MDSCs play a regulatory role in the driving of m CRPC.Nude mice blood tract metastasis and CDX model were randomly divided into control group,enzalutamide(Enza)oral gavage group,LGK-974 intraperitoneal injection group and combination group,and tumor metastasis,proliferation,and apoptosis were detected.(8)Collected clinical anticoagulated blood samples,separated the plasma,detected the concentration of IL23 by enzyme-linked immunosorbent assay(ELISA),drew the receiver operating curve(ROC),analyzed the area under the curve(AUC)and determined the cut-off value of IL-23 as a CRPC diagnostic marker.Analyze the relationship between IL-23 and clinical parameters,and further confirm its propelling role in this regulation at the cellular level.IL-23 may be defined as a potential biomarkers for the diagnosis and prognosis monitoring of CRPC.Results:(1)Analysis of the TCGA database found that the copy numbers of CXCL5 and CXCR2 in PCa tissues were both increased and accompanied by multiple organ metastasis and decreased overall patient survival(OS)and disease-free survival(DFS),but the copy numbers of the two not changed in hormone-sensitive PCa.The results of IHC illustrated that the expressions of CXCL5 and CXCR2 in CRPC tissues were both significantly higher than that in BPH and PPC.The tissue immunofluorescence suggested that a large number of MDSCs were infiltrated in CRPC tissues.(2)The protrusion numbers of ACM induced PCa cells under conventional or matrigel 3-D culture conditions were notably increased;the cell migration ability was also significantly enhanced.In vivo imaging results recommended that femoral metastasis occurred in each group in the third week after the establishment of the blood tract metastasis model.In the sixth week,the number of metastases in the co-injection group increased markedly,and multiple organ metastases occurred.Subsequent IHC indicated that the number of pulmonary nodules in the co-injection group increased distinctly and the lesions area increased significantly.The results of clone formation and Ki-67 demonstrated that the proliferation ability of PCa cells,especially22RV1 cells,in the ACM-induced group was enhanced.In 3-D culture and CDX model,MDSCs were also proved to promote the growth and proliferation of PCa cells.RT-PCR showed that MDSCs could induce and activate the AR signaling in 22RV1 cells.(3)Knockdown of Smurf1/2 in 22RV1 cells can inhibit the formation of protrusions caused by ACM,and the cell migration ability will be correspondingly weakened.After knocking down the core components of PCP,the cell protrusions activity and migration ability were significantly inhibited,and the difference in the FZD6 knockdown group was the most significant.(4)Analysis of TCGA database found that the copy number of the receptor FZD6 was the most significantly increased receptors in PCa.Compared with DFS,OS was more significantly affected by the change in FZD6.And the copy number of FZD6 increased in the anti-androgen exposure group.GEO database analysis also revealed that the m RNA expression of FZD6 in CRPC tissues was significantly increased and positively correlated with AR amplification.RT-PCR clarified that knocking down of FZD3/6/7 in 22RV1 cells can affect the activation of AR signaling and the expression of AR-V7,and FZD6 has the most significant effect.IHC demonstrated that the expression of FZD6 in PPC and CRPC tissues increased,but it was more significant in CRPC tissues.The level of FZD6 was positively correlated with CRPC patients' Gleason score and bone metastasis.(5)Ki-67,clone formation experiments,and flow cytometry confirmed that knockdown of FZD6 can inhibit cell proliferation and promote their apoptosis.The nude mouse PDX model shows that stable knockdown of FZD6 can inhibit PCa proliferation in vivo.Wound healing assay,Transwell migration test and Phalloidin staining show that knocking down FZD6 can significantly inhibit the migration of PCa cells.Nude mouse live imaging and lung tissue IHC results show that FZD6 can inhibit the migration of PCa in vivo.(6)Wnt5a and Wnt11 were almost not expressed in MDSCs.When MDSCs were treated with LGK-974,the migration and proliferation ability of ACM induced 22RV1 cells did not change significantly.Directly adding LGK-974 to 22RV1 cells induced by ACM can notably inhibit the migration and proliferation of cancer cells.Knockdown of Wnt5 a inhibited cell migration activity and AR pathway activation,while knockdown of Wnt11 was not obvious.IP experiments illustrated that the autocrine phenomenon of Wnt5 a enhanced in ACM-induced 22RV1 cells.LSCM indicated that ACM induction can change the distribution of cytoskeleton proteins and promote the co-localization of Wnt5 a and FZD6.(7)The expressions of CAMK?/p-STAT3Y705/ROR?/AR and Rho A were down-regulated in ACM-induced 22RV1 cells after FZD6 was knocked down,which clarified the role of this pathway in the regulation of m CRPC by Wnt/PCP triggered by MDSCs.After treating cells with MDSCs,Cabozantinib,recombinant IL-23 and LGK-974,the wb results suggested that IL-23 secreted by MDSCs played a regulatory role in the driving of m CRPC.Animal experiments illustrated that compared with Enza or LGK-974 alone,the combination of the two significantly inhibited the metastasis and proliferation of PCa and accelerated tumor apoptosis.(8)The results of ELISA indicated that the plasma concerntration of IL-23 in CRPC patients were evidently increased and the cut-off value was higher than that in PPC patients.Statistical analysis testified that plasma IL-23 levels in CRPC patients were positively correlated with the Gleason score and bone metastasis.In vitro experiments further clarified that IL-23,as an intermediate mediator,lead to accelerated proliferation and enhanced migration of 22RV1 cells by affecting the abnormal activation of the AR pathway and rearrangement of skeletal proteins.Conclusion:From an immunological point of view,this topic explored that the abnormally aggregated immunosuppressive cells MDSCs in CRPC promoted the autocrine of Wnt5 a in PCa cells by secreting IL-23.The combination of Wnt5 a and FZD6 could promote the abnormal activation of AR signaling through the CAMK?/p-STAT3/ROR? axis,and could also mediate Rho A-related cytoskeletal rearrangement,leading to the further development of tumors into m CRPC.