Effect And Mechanism Of TR4 Nuclear Receptor On Invasion Of CD133~+ Prostate Cancer Cells

Background and Objective:Prostate cancer(PCa) continues to be the most common malignant tumor in men. Androgen deprivation therapy(ADT) is the standard therapy for advanced PCa. However, most ADT eventually fails and PCa progresses into castration resistant PCa(CRPC) which is often accompanied with metastasis. The detailed mechanisms, however, remain unclear.One key factor that may contribute to the metastasis development, despite the ongoing ADT treatment, is the increasing population of stem/progenitor(S/P) cells. Testicular nuclear receptor 4(TR4) plays key roles to influence stem cell pluripotency. Previous studies found that TR4 plays important role in the initiation, progression, and metastasis of PCa. The purpose of this study was to evaluate the effect and detailed mechanism of TR4 on invasion ability of CD133+ PCa cells.Methods:Magnetic beads conjugated with CD133 antibody were used to sort the PCa S/P cells from PCa C4-2 and CWR22Rv1 cells. Then the stem cell markers and TR4 mRNA expression of the sorted cells were evaluated. Lentivirus-mediated shRNA targeting TR4 was used to inhibit TR4 expression. Transwell invasion and 3D invasion assays were used to investigate TR4 effect on the invasion ability of CD133+ PCa cells. Thirty PCa metastasis-related genes were screened to find the most significantly changed genes upon knockdown of TR4. Western blot assay was used to evaluate the protein expression change of EZH2 and its downstream metastasis-related genes. Ch IP assay was performed to check the binding of TR4 on the predicted TR4 response elements. Luciferase reporter assay was used to evaluate TR4 effect on EZH2 promoter activity. Rescue assays were performed to logically validate whether TR4 affect PCa S/P cell invasion via regulating EZH2. Orthotopic-injected PCa mice model were used to check TR4 effect on invasion and metastatic ability of PCa S/P cellsin vivo.Results:CD133+ sorted S/P cells have higher expression of the S/P markers include CD133 and Nanogas compared to parental cells. Higher expression of TR4 was found in CD133+ S/P cells at protein and mRNA level. Transwell assay as well as 3D invasion assay showed that knocking down of TR4 led to the suppression ofinvasion ability of CD133+ S/P cells.Five key genes include KAI1 and TIMP2 increased and IGF, HIF2α, and EZH2 decreased upon knocking down of TR4. Knocking-down TR4 suppressed the expression of both EZH2 and its downstreammetastasis-related genes include NOTCH1, SLUG, TGBβ1, and MMP9. ChIP and Luciferase reporter assay showed that TR4 transactivates EZH2 via binding TR4 RE of EZH2 promoter. Western blot results revealed that adding EZH2 could reversethe suppression effect of TR4-shRNA on EZH2 and its downstream metastasis-related genes. Adding EZH2 also partially reversed the TR4 ability to influence the CD133+ S/P cell invasion ability. Orthotopically xenografted PCa S/P cells mice model showed that the control group had higher incidence of metastasisas compared to the shTR4 group.IHC staining of the xenograft tumor found that EZH2 expression is much lower in the TR4 knocked-down tumors as compared to vehicle control.Conclusions:This study demonstrates that TR4 is overexpressed in PCa CD133+ cells. TR4 might be able to function through modulation of EZH2 expression to alter its downstream signals to enhance the PCa S/P cell mediated invasion. Targeting TR4 may become a new potential therapeutic approach to better suppress PCa S/P cell invasion.