br Proliferation assay br The
2.4. Proliferation assay
The cells were stably transfected with a gene encoding an SPDEF or ZBTB46 expression vector or a ZBTB46 or SPDEF short hairpin (sh)RNA vector and seeded at a density of 2000 cells/well in 96-well plates. The cells were treated with MDV3100 at 10 μM and NS-398 at 1.77 or 75 μM for 24 h in 10% FBS-containing medium, as described in Supplementary Methods.
2.5. Colony-formation assay
A colony-formation assay was performed using a starting number of 1000 cells/well seeded in 0.3% agarose (Aﬀymetrix) on top of a 0.6% agarose layer in 12-well plates. Single-cell suspensions of ZBTB46 complementary (c)DNA-transfected 22Rv1 and C4-2B cells, SPDEF/ ZBTB46 cDNA-transfected RasB1 cells, or ZBTB46 shRNA vector-transfected PC3 and RasB1 cells were performed as described in Supplementary Methods.
2.6. Tumorigenicity and metastasis assays in mice
The animal work was performed in accordance with a protocol approved by the Taipei Medical University Animal Care and Use Committee (approval No. LAC-2015-0185, Taiwan) as described in Supplemental Materials and Methods. Five-week-old male nude mice were injected with ZBTB46 shRNA vector-transfected RasB1 cells and treated with 20 mg/kg NS-398 or DMSO as the control for 1 month by an intraperitoneal injection twice a day. For the analysis of metastasis, 5-week-old male nude mice (NLAC, Taiwan) were subjected to in-tracardiac injections of 105 cells per mouse of RasB1/EV, RasB1/SPDEF, and RasB1/SPDEF/ZBTB46 cells harboring a luciferase expression vector.
2.7. Immunohistochemical (IHC) staining
We used tissue microarray (TMA) sections, including 16 normal prostatic epithelial samples, 100 primary prostate adenocarcinomas, and eight SCNCs from the Duke University School of Medicine (NC, USA). Twenty-one clinical tissue samples from prostate cancer patients before and after they were treated with ADT were collected from Taipei
Medical University-Wan Fang Hospital (Taiwan). Tissue samples were obtained and used according to the protocols approved by the Duke University School of Medicine-Institutional Review Board (protocol ID: Pro00070193) and the Taipei Medical University-Joint Institutional Review Board (approval No. N201712051). IHC was performed using ZBTB46 (HPA013997, Sigma), SPDEF (TA324209, OriGene), and PTGS1 (ab695, Abcam) 1449-05-4 at respective dilutions of 1:250, 1:200, and 1:200, as described in Supplementary Methods.
2.8. Immunofluorescence (IF) staining
TMA sections from the Duke University School of Medicine were rinsed in 2% BSA in PBS for 30 min and then incubated overnight at 4 °C with PTGS1 (ab695, Abcam) and CHGA (ab15160, Abcam) anti-bodies in 2% BSA/PBS at respective dilutions of 1:200 and 1:150, as described in Supplementary Methods.
2.9. Statistical analysis
All data are presented as the mean ± standard error of the mean (SEM). Statistical calculations were performed with GraphPad Prism analytical tools (GraphPad Software). Diﬀerences between individual groups were determined by Student's t-test or a one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for compar-isons among three or more groups. The method for determining the cutoﬀs was pre-decided by half of the number of patients. p < 0.05 was considered statistically significant.
3.1. Antagonized AR signaling-induced ZBTB46 exhibited NEPC diﬀerentiation
We investigated the role of ZBTB46 in the NE diﬀerentiation of prostate cancer cells. We examined the amount of ZBTB46 in a panel of human prostate cancer cells and found that both AR-negative cells (PC3 and RasB1) and the NE-like cell line (NE-1-8) expressed more ZBTB46 and NE markers compared with the AR-positive cells (LNCaP-AR, LNCaP, 22Rv1, and C4-2B) (Fig. 1A and Supplementary Fig. S1A). To examine the abundance of ZBTB46 associated with NEPC diﬀerentia-tion following ADT, we found that long-term treatment with MDV3100 induced ZBTB46 and NE markers, such as chromogranin A (CHGA) and enolase 2 (ENO2) in the AR-positive cells (22Rv1, C4-2, C4-2B, and LNCaP) (Fig. 1B–E). Our results support the stimulation of NE trans-formation of prostate cancer with continued ADT treatment [8–11,47]. Moreover, our results show that the CRPC C4-2 and 22Rv1 cells re-sponded better to the MDV3100 than the androgen-dependent LNCaP cells (Fig. 1B and D), consistent with the studies showing that an-drogen-independent cases have better NE diﬀerentiation than the an-drogen-dependent cases [48–50]. Overexpression of ZBTB46 induced an abundance of NE markers in the 22Rv1 and C4-2B cells (Fig. 1F and G). In contrast, ZBTB46-knockdown decreased the level of NE markers in the RasB1 and PC3 cells (Fig. 1H and I, left), and similar results were found in the NE-1-8 cells (Fig. 1I, right). We further performed ZBTB46-knockdown in the C4-2B cells and treated the cells with MDV3100. The results showed that ZBTB46-knockdown reduced NE markers regardless of MDV3100 treatment (Fig. 1J), confirming that ZBTB46 regulates NEPC diﬀerentiation. Moreover, we found that in addition to the NE markers (CHGA, chromogranin B (CHGB), and ENO2) being reduced, the reprogramming transcription factor, SOX2, and a key factor in the EMT (SNAI1), also decreased in the ZBTB46-knockdown NE-1-8 cells and mouse TRAMP-C1 cells (Supplementary Fig. S1B). These results suggest that ZBTB46 induction is associated with NEPC diﬀerentiation.