br treated with MDV Fig
treated with MDV3100 (Fig. 3F). We then analyzed the ZBTB46 and NE markers after SPDEF overexpression in PC3 and RasB1 cells and mouse TRAMP-C1 cells. We found that ectopic SPDEF significantly decreased the levels of ZBTB46- and NEPC-associated genes (Fig. 3G and H and Supplementary Fig. S3A). These results verified a model whereby in-hibition of the AR mediates a decrease in SPDEF, leading to the in-duction of ZBTB46- and NEPC-associated genes of prostate cancer cells.
3.4. ZBTB46 overcomes the tumor suppressor eﬀects of SPDEF in prostate cancer
To assess the contribution of SPDEF to the antitumor eﬀect of prostate tumorigenesis, we found that SPDEF overexpression reduced RasB1 cell proliferation and colony formation compared with the cells carrying an empty vector (EV) (Fig. 4A and B). We next examined the
functional relevance of ZBTB46 and found that ZBTB46 overexpression induced the cell proliferation rate of SPDEF-expressing RasB1 cells (Fig. 4A and B). Because C4-2B expressed abundant SPDEF (Fig. 3A), the results showed no significantly reduced cell proliferation rate or invasion potential in the SPDEF-overexpressing C4-2B cells (Supplementary Figs. S3B and C). In addition, we rescued ZBTB46 in the SPDEF-overexpressing LNCaP cells. As expected, SPDEF did not decrease the cell growth rates in the LNCaP cells; however, the ZBTB46-rescued cells exhibited increased cell proliferation (Supplementary Fig. S3D). We also found that ZBTB46 promoted SPDEF-overexpressing LNCaP cell proliferation regardless of MDV3100 treatment (Supplementary Fig. S3E), demonstrating that ZBTB46 bypasses the tumor-suppressive eﬀect of SPDEF. The SPDEF and ZBTB46 levels were confirmed by immunoblotting or qRT-PCR from these cells by manip-ulating the SPDEF and ZBTB46 expressions (Fig. 4C and Supplementary
Fig. 2. Repression of ZBTB46 by SPDEF-dependent transcriptional regulation. (A) SPDEF and ZBTB46 mRNA levels in LNCaP-AR and 22Rv1 cells stably expressing an SPDEF shRNA or control vector (shLuc). (B) SPDEF and ZBTB46 levels in RasB1 and PC3 cells following stable transfection with SPDEF or a control vector (empty vector (EV)) by qRT-PCR. (C) Schematic of the predicted SPDEF responsive elements (SREs) in serially deleted promoter green fluorescent protein (GFP)-reporter constructs of human ZBTB46. (D) ChIP assays of LNCaP-AR cells with 1806553 against SPDEF, H3K4me3, and GAPDH. * vs. SRE1. (E) ChIP assays in LNCaP-AR cells treated with DHT. Enrichment is given as a percentage of the total input and then normalized to IgG. (F) Relative median fluorescent intensities (MFIs) of ZBTB46 reporters (E1-E8) in LNCaP cells. (G) The same assay as in 2F in LNCaP cells cultured in charcoal-stripped serum (CSS)-containing medium and cells treated with DHT. (H) Relative MFIs of wild-type and mutant-ZBTB46 reporters (E2 and E3) in response to DHT in LNCaP cells. (I) Relative MFIs of wild-type and mutant-ZBTB46 reporters (E2 and E3) in response to SPDEF overexpression in RasB1 cells. Quantification of mRNA, ChIP data, and MFIs are given as the mean ± SEM of three independent experiments. Significance was determined by Student's t-test. **p < 0.01, ***p < 0.001.
Fig. S3F). Moreover, the mice administered a subcutaneous injection of SPDEF-expressing RasB1 cells showed significantly decreased tumor sizes and weights compared with the mice injected with cells harboring the EV (Fig. 4D–F), confirming the tumor-suppressive role of SPDEF. However, the mice injected with ZBTB46-expressing RasB1/SPDEF cells showed increased tumor growth and tumor weights (Fig. 4D–F). We further tested whether ZBTB46 aﬀects NEPC diﬀerentiation in these injected tumors by IHC staining. The results showed that ZBTB46 and the NE marker synaptophysin (SYP) increased in tumors from the mice injected with ZBTB46-expressing RasB1/SPDEF cells compared with the mice injected with RasB1/SPDEF cells (Fig. 4G and H). These data demonstrate that ZBTB46 induction is associated with NEPC diﬀer-entiation in SPDEF-expressing cells. In addition, we monitored the metastatic functions of ZBTB46 and found that the mice receiving an
intracardiac injection of ZBTB46-expressing RasB1/SPDEF cells had a lower survival rate (Fig. 4I) and increased incidences of bone metastasis (Fig. 4J and K) compared with the mice receiving an injection of RasB1/SPDEF cells. These results support our hypothesis that the abundance of ZBTB46 contributes to NE diﬀerentiation and induces bone metastasis of prostate cancer.
3.5. ZBTB46 is positively associated with PTGS1 in response to inhibition of AR signaling
ZBTB46 is highly expressed in dendritic cells (DCs), and it acts as a transcription factor selectively expressed by classical DCs and their committed progenitors . We hypothesized that the induction of ZBTB46 is associated with inflammatory responses in prostate cancer.