br MDV and ZBTB expressing vector Fig I and J
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
Fig. 5. Positive association between ZBTB46 and PTGS1 in prostate cancer cells in response to AR signaling. (A and B) ZBTB46, PTGS1, and PTGS2 mRNA levels in C4-2B and 22Rv1 cells treated with DHT in charcoal-stripped serum (CSS)-containing medium (A) or MDV3100 in FBS-containing medium (B) for 24 h. (C) Western blotting of ZBTB46, PTGS1, and PTGS2 in C4-2B and 22Rv1 cells transiently transfected with an empty vector (EV) or ZBTB46 expression vector for 24 h and then treated with DHT in CSS-containing medium for 24 h (D) RNA levels of ZBTB46 and PTGS1 in RasB1 and PC3 cells stably expressing a ZBTB46 shRNA or control vector (shLuc). (E) ZBTB46 and PTGS1 mRNAs as determined by qRT-PCR in 22Rv1 and C4-2B cells following control (EV) or ZBTB46 expression. (F) Schematic of the predicted ZBTB46 responsive element (ZRE), the non-ZBTB46 responsive element (Non-ZRE), and the ZRE wild-type (WT) and mutant (M) promoter green fluorescent protein (GFP)-reporter constructs of human PTGS1. (G) ChIP assays of PC3 cells with CY7-SE against ZBTB46, H3K4me3, and GAPDH. Enrichment is given as a percentage of the total input and then normalized to IgG. * vs. non-ZRE. (H) ChIP assays in C4-2B cells treated with DHT (left) or MDV3100 (right) with antibodies against ZBTB46 and GAPDH. (I and J) Relative mean fluorescent intensities (MFIs) of PTGS1 reporters (ZRE WT and ZRE M) in C4-2B cells treated with DHT or MDV3100 (I) or transfected with the ZBTB46 expression vector or siRNA (J). The quantification of mRNA, ChIP data, and MFIs is presented as the mean ± SEM from three independent experiments. Significance was determined by Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
and B and Fig. 6A). Interestingly, the combination of the MDV3100 and NS-398 treatment with only concentration-specific PTGS1 reduced tumor growth (Fig. 6A). We also found that this combined treatment reduced ZBTB46 expression but not in the C4-2B and 22Rv1 cells treated with MDV3100 or NS-398 alone (Fig. 6B). The AR-negative RasB1 cells did not respond to this combined treatment (Supplementary Figs. S5C and D). These data suggest that specific inhibition of PTGS1 can induce sensitivity to MDV3100 and reduce ZBTB46 expression. Moreover, with ectopic ZBTB46 expression, the C4-2B and 22Rv1 cells showed increased cell proliferation (Supplementary Fig. S5E) and colony formation (Fig. 6C and Supplementary Fig. S5F) despite treat-ment with a combination of MDV3100 and NS-398. We also found that the ZBTB46-overexpressing C4-2B cells induced PTGS1 and ENO2 abundance despite this combined treatment (Fig. 6D), demonstrating
that high ZBTB46 expression led to PTGS1 induction and NE diﬀer-entiation of the prostate cancer cells. We further explored the tumor growth eﬀect of ZBTB46 in the NS-398-treated cells. We found that the PC3 and RasB1 cells with ZBTB46-knockdown (Supplementary Fig. S4G) showed reduced cell proliferation and colony formation and fur-ther reductions following NS-398 treatment (Fig. 6E and F and Supplementary Figs. S5H and I). The tumor-formation capability of RasB1 cells with ZBTB46-knockdown was further tested by a sub-cutaneous injection. The results show that the mice injected with the RasB1 cells expressing control shRNA did not exhibit reduced tumor growth rates after the NS-398 treatment; however, the mice injected with the ZBTB46-knockdown cells and treated with NS-398 exhibited reduced tumor sizes and weights (Fig. 6G–I). These results indicate that the ZBTB46 levels may have aﬀected the sensitivity to the anti-
Fig. 6. ZBTB46 regulates the sensitivity of the PTGS1 inhibitor and induces tumor growth. (A) Proliferation assay in C4-2B and 22Rv1 cells treated with enzalutamide (MDV3100, 10 μM) and a PTGS inhibitor (NS-398, 1.77 and 75 μM), n = 8. DMSO, control. * vs. DMSO. (B) Monitoring of ZBTB46 mRNAs in C4-2B and 22Rv1 cells following treatment with MDV3100 and NS-398. * vs. DMSO. (C) Quantification of the colony formation of C4-2B and 22Rv1 cells with combined MDV3100 and NS-398 treatment following ZBTB46 or empty vector (EV) overexpression. (D) Western blotting of ZBTB46, PTGS1, and ENO2 in C4-2B cells treated with MDV3100 and NS-398 following stable EV or ZBTB46-expressing vector transfection. (E) Proliferation assays in RasB1 cells treated with a PTGS1 inhibitor (NS-398, 75 μM) following stable ZBTB46 or control (Luc) shRNA vector expression, n = 8. DMSO, control. (F) Quantification and images of the colony formation of PC3 cells treated with NS-398 following ZBTB46 or Luc shRNA vector overexpression. (G–I) Tumor growth analysis of stable RasB1 cell lines (shLuc vs. shZBTB46) subcutaneously inoculated into male nude mice followed by treatment with NS-398. Tumor sizes were monitored weekly (G), and images (H) and tumor weights (I) were also measured at the end. n = 5 mice per group. Quantification of the mRNA, 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, ***p < 0.001.