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  • INCB018424 br Fig ZBTB induces NE marker expressions of prostate

    2022-05-19


    Fig. 1. ZBTB46 induces NE marker expressions of prostate cancer. (A) Immunoblotting for AR, ZBTB46, and ENO2 in various prostate cancer cell lines. (B–D) ZBTB46, CHGA, and ENO2 mRNA levels after MDV3100 treatment at 10 μM in FBS-containing medium for 2 weeks of various prostate cancer cell lines. (E) Immunoblotting of extracts from 22Rv1 and C4-2B INCB018424 following MDV3100 treatment for 2 weeks. (F) ZBTB46 and NE markers (CHGA, CHGB, and ENO2) in 22Rv1 and C4-2B cells following transient transfection with ZBTB46 or an empty vector (EV) by qRT-PCR. (G) ZBTB46, CHGA, and ENO2 proteins in 22Rv1 and C4-2B cells following stable transfection with EV and ZBTB46 expression vectors. (H) ZBTB46 and NE markers in RasB1 and PC3 cells stably expressing ZBTB46 shRNA or control vector (shLuc) by qRT-PCR. (I) Immunoblots showing ZBTB46, CHGA, and ENO2 in RasB1, PC3, and NE-1-8 cells following control (NC) or ZBTB46 siRNA (si46) transfection. (J) ZBTB46 and NE markers (CHGA, CHGB, and ENO2) in C4-2B cells transiently transfected with NC or ZBTB46 siRNA and then treated with MDV3100 in FBS-containing medium for 24 h by qRT-PCR. The quantification results of mRNA are 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.
    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 INCB018424 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 antibodies 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.