br Fig Reduced SPDEF is
Fig. 3. Reduced SPDEF is essential for ZBTB46 expression and involved in NEPC diﬀerentiation. (A) Western blotting of SPDEF and ZBTB46 in various prostate cancer cell lines. (B) SPDEF and ZBTB46 mRNA levels in LNCaP, C4-2B, and PC3 Galactose 1-phosphate after 24 h of treatment with DHT or MDV3100 relative to the controls (charcoal-stripped serum (CSS) or DMSO, respectively). (C) RNA levels of SPDEF and NE markers (CHGA, CHGB, and ENO2) in LNCaP and C4-2B cells stably expressing an SPDEF shRNA or control vector (shLuc). (D) SPDEF, ZBTB46, and ENO2 proteins as determined by immunoblotting in LNCaP and C4-2B cells following the control (NC) or SPDEF siRNA transfection. (E) SPDEF, ZBTB46, and ENO2 proteins in C4-2B cells stably expressing an SPDEF or Luc shRNA vector and cells treated with DHT in CSS-containing medium for 24 h. (F) Western blotting of SPDEF, ZBTB46, and ENO2 in C4-2B cells following stable empty vector (EV) or SPDEF expression and then treatment with MDV3100 in FBS-containing medium for 24 h (G) NE markers (CHGA, CHGB, and ENO2) and SPDEF in PC3 and RasB1 cells following stable transfection with an EV or SPDEF by qRT-PCR. (H) SPDEF, ZBTB46, and ENO2 proteins as determined by immunoblotting of PC3 and RasB1 cells following stable EV or SPDEF expression. Quantification of mRNA is presented as the mean ± SEM, n = 3 biological replicates. Significance was determined by Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
Because PTGS is key to the inflammatory response in cancers [23,24], we investigated the role of PTGS in ZBTB46-promoted malignant pro-gression. We measured the ZBTB46, PTGS1, and PTGS2 expressions in AR-positive cells relative to perturbations in AR signaling. We found that the DHT-treated C4-2B and 22Rv1 cells had lower ZBTB46 and PTGS1 expressions, whereas PTGS2 expression was not aﬀected by the DHT treatment (Fig. 5A). Moreover, the ZBTB46 and PTGS1 expressions increased when the cells were treated with MDV3100 (Fig. 5B). We further analyzed the PTGS1 and PTGS2 proteins in the ectopic ZBTB46-expressing AR-positive cells in response to DHT. Indeed, DHT reduced endogenous ZBTB46 and PTGS1 expressions, whereas ZBTB46 over-expression induced PTGS1, but not PTGS2, despite DHT treatment
(Fig. 5C). The GSEAs of TCGA prostate cancer dataset showed that ZBTB46 induction was positively associated with the expressions of gene sets that were activated by inflammatory response pathways (Gene Ontology and BioCarta) (Supplementary Fig. S4A). Significantly, the GSEAs showed that the tissues expressing low PTGS1 but not PTGS2 were associated with upregulated androgen-responsive signatures [51,52] (Supplementary Fig. S4B). These data suggest a negative as-sociation between PTGS1 expression and androgen-responsive signaling activation. We hypothesized that antagonized AR signaling-induced ZBTB46 acts as a transcriptional activator of PTGS1. Notably, ZBTB46-knockdown in RasB1 and PC3 cells reduced PTGS1 mRNA expression (Fig. 5D). In contrast, ZBTB46-overexpressing 22Rv1 and C4-2B cells
Fig. 4. ZBTB46 bypasses the tumor-suppressive eﬀect of SPDEF. (A) Proliferation of RasB1 cells transfected with an empty vector (EV), SPDEF, or SPDEF + ZBTB46; n = 8. (B) Quantification (n = 3, left) and selected images (right) of colony-formation assays of the stable RasB1 cell line containing an EV, SPDEF, or SPDEF + ZBTB46. (C) Western blotting of samples from cells assessed in 4A and 4B. (D–F) Growth (D), images (E), and weights (F) of tumor xenografts in male nude mice 4 weeks after subcutaneous inoculation with RasB1 cells stably expressing an EV, SPDEF, or SPDEF + ZBTB46. n = 4 mice per group. (G and H) IHC staining (G) and analysis (H) of subcutaneous tumors with antibodies specific for ZBTB46, SYP, and SPDEF in tumor-bearing mice from 4E. Scale bars represent 50 μm. (I–K) Survival analysis by a log-rank test (I) and bioluminescence imaging analysis (J and K) of prostate tumor cell lesions in the bone of mice 45 days after receiving an intracardiac injection of RasB1 cells stably transfected with the EV (n = 5), SPDEF (n = 4), or SPDEF + ZBTB46 (n = 5). Quantification of the proliferation and colony formation assays is presented as the mean ± SEM from three biological replicates. Significance was determined by Student's t-test. *p < 0.05, **p < 0.01,
had significantly higher PTGS1 mRNA expressions (Fig. 5E). The ana-lysis of putative ZBTB46 response elements (ZREs)  identified a candidate ZRE in the PTGS1 promoter (Fig. 5F). The ChIP assays in-dicated that the ZRE site was enriched with antibodies against ZBTB46 and a positive control H3K4me3 (Fig. 5G). We also found significantly decreased ZBTB46 binding at the putative ZRE after the DHT treatment (Fig. 5H, left), whereas endogenous binding of ZBTB46 was induced after the MDV3100 treatment (Fig. 5H, right). Using a reporter assay, we found that DHT decreased WT reporter activity, but the MDV3100 treatment induced reporter activity (Fig. 5I). Moreover, increased re-porter activity was detected in the cells with ZBTB46 overexpression, whereas ZBTB46-knockdown reduced reporter activity (Fig. 5J). We mutated the putative ZRE in the PTGS1 promoter (Fig. 5F) and found that mutating ZRE disrupted reporter activity by either the inhibitory eﬀects of DHT and ZBTB46 siRNA or the stimulating eﬀects of