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  • SCH 58261 br Fig C The GSEAs of TCGA prostate

    2022-05-19

    
    (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 SCH 58261 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) [55] 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 SCH 58261 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 
    MDV3100 and ZBTB46-expressing vector (Fig. 5I and J). We concluded that PTGS1 induction by ADT is dependent on ZBTB46-upregulated transcriptional activation. These data support a positive association between ZBTB46 and PTGS1 in prostate cancer cells after ADT.
    3.6. Ectopic ZBTB46 reduces the sensitivity of the combination of MDV3100 and a PTGS1 inhibitor
    To study the effect of the anti-inflammatory drug on prostate cancer after ADT, we assessed the contribution of a PTGS inhibitor to re-sensitized anti-AR therapy in prostate cancer cells. We used a PTGS inhibitor (NS-398) alone or in combination with MDV3100 to treat C4-2B and 22Rv1 cells. We found no significant reduction in the cell morphology and growth rate with NS-398 treatment alone specific to either PTGS1 (75 μM) or PTGS2 (1.77 μM) (Supplementary Figs. S5A