Fig 1: PKLR is downregulated by ZBTB10 in PCa cells.A Immunoblots showing ZBTB10, PKLR, ENO2, and CHGA protein levels in PC3 and LASCPC01 cells stably transfected with an empty vector (EV) or ZBTB10 cDNA vector. B, C RT-qPCR showing PKLR, NE marker, and ZBTB10 mRNA levels in PC3 (B) and LASCPC01 (C) cells stably transfected with an EV or ZBTB10 cDNA vector. * vs. the EV; by a one-way ANOVA. Quantification of mRNA is presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. D Schematic of the predicted ZBTB10 response element (ZRE) and various introduced binding site mutants in regulatory sequence reporter constructs of human PKLR (GRCh38:1); and sequence diagram of wild-type ZRE (ZRE-WT) and ZRE mutants (ZRE1~ZRE4). E ChIP assay showing binding of ZBTB10 and acetyl-H3 to predicted ZREs of the PKLR gene regulatory sequence following treatment of C4-2 cells with charcoal-stripped serum (CSS)-containing medium (left) or 10 μM MDV3100 (MDV, right) for 48 h. Sheared chromatin from nuclear extracts was precipitated with antibodies to ZBTB10 and acetyl-H3, and predictive primers (D, black arrows) were used to quantify the precipitated DNA by a qPCR. Enrichment of each protein to each site is given as a percentage of the total input and then normalized to IgG. * vs. -CSS (left) or -MDV (right); by a one-way ANOVA. F ChIP assay showing binding of ZBTB10 and acetyl-H3 to predicted ZREs of the PKLR gene regulatory sequence in PC3 cells following stable transfection with an EV or ZBTB10 cDNA vector (left) or in LNCaP cells following a non-target control (NC) or ZBTB10 shRNA vector (right) transfection. * vs. the EV (left) or NC (right); by a one-way ANOVA. G Relative median fluorescent intensity (MFI) of the GFP reporter gene containing ZRE-WT from the PKLR regulatory sequence in C4-2 and LNCaP cells following CSS-containing medium treatment for 24 h, and further treatment with 10 nM dihydrotestosterone (DHT) for 24 h. * vs. -CSS; # vs. +CSS; by a two-way ANOVA. H Relative MFI of the GFP reporter gene containing WT-ZRE from the PKLR regulatory sequence in PC3 (left) or C4-2 (right) cells following stable transfection with the EV or ZBTB10 cDNA vector (left) or in C4-2 cells following NC or ZBTB10 shRNA vector transfection (right). * vs. the EV (left) or NC (right); by a one-way ANOVA. I Relative MFI of the GFP reporter gene containing ZRE-WT or ZREMs from the PKLR regulatory sequence in PC3 cells following stable transfection with the EV or ZBTB10 cDNA vector. * vs. ZRE-WT; # vs. the EV; by a two-way ANOVA. Quantification of the ChIP assay and relative MFI values are presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: Inhibition of PKLR may be a promising therapeutic strategy of NE-like PCa.A Various prostatic cell lines were treated with 0, 1, 5, 10, 25, and 50 μM vilanterol (left) or saquinavir (right) for 24 h, and cell viability was determined by an MTT colorimetric assay. n = 8 per group. * vs. the vehicle (0 μM); by a one-way ANOVA. B Proliferation analysis of C4-2 cells expressing an empty vector (EV) or PKLR cDNA vector, and treated with 0, 1, 5, 10, 25, and 50 μM vilanterol (left) or saquinavir (right) for 24 h. n = 8 per group. * vs. the vehicle (0 μM); by a one-way ANOVA. C Proliferation analysis of C4-2 and C4-2-MDVR cells treated with 0, 1, 5, 10, 25, and 50 μM vilanterol (left) or saquinavir (right) for 24 h. n = 8 per group. * vs. the vehicle (0 μM); by a one-way ANOVA. D Sphere-formation assays of C4-2/PKLR, C4-2-MDVR, PC3, and LASCPC01 cells exposed to the vehicle (DMSO), vilanterol (10 μM), or saquinavir (10 μM) for 1 week. n = 8 per group. * vs. DMSO; by a one-way ANOVA. Quantification of cell viability and sphere formation presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. E Relative CHGA (left) and SOX2 (right) mRNA levels in C4-2/PKLR, C4-2-MDVR, PC3, and LASCPC01 cells with DMSO, 10 μM vilanterol, or 10 μM saquinavir treatment for 24 h. * vs. DMSO; by a one-way ANOVA. Quantification of mRNA is presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. F Scheme for the treatment of LASCPC01 and C4-2-MDVR xenografts with vilanterol and saquinavir. Male nude mice bearing established LASCPC01 or C4-2-MDVR tumors were treated with DMSO, 25 mg/kg body weight (BW) vilanterol, or 25 mg/kg BW saquinavir starting 1 month after the tumor injection, twice a week for 4 weeks. G–I Tumor growth analysis of mice subcutaneously inoculated with LASCPC01 cells. Tumor sizes were monitored once a week (G), images (H), and tumor weights (I) were obtained at the end of the experiment (n = 5 mice per group). * vs. DMSO. *p < 0.05, **p < 0.01; by a one-way ANOVA. J IHC staining and representative intensities of PKLR, ENO2, MKI67, and PCNA in subcutaneous tumors from (H). * vs. DMSO. Significance was determined by a two-tailed Student’s t-test. *p < 0.05, **p < 0.01.
Fig 3: PKLR rescues ZBTB10-suppressed malignancy of PCa.A, B Proliferation in C4-2 cells stably transfected with an empty vector (EV) or PKLR cDNA vector (A) or in PC3 cells with a non-target control (NC) or PKLR shRNA vector transfection (B). n = 8 per group. * vs. the EV (A) or NC (B); by a one-way ANOVA. C, D Sphere formation of C4-2 (C) and PC3 (D) cells with PKLR overexpression (C) or PKLR-knockdown (KD) (D), respectively. n = 8 per group. * vs. the EV (C) or NC (D); by a one-way ANOVA. E Protein levels of PKLR in C4-2 or PC3 cells with PKLR overexpression or PKLR-KD. F, G Proliferation of LNCaP and C4-2 cells stably transfected with the EV, ZBTB10, or ZBTB10+PKLR cDNA vector. n = 8 per group. * vs. the EV; # vs. ZBTB10; by a two-way ANOVA. H Sphere formation of LNCaP and C4-2 cells stably transfected with the EV, ZBTB10, or ZBTB10+PKLR cDNA vector. n = 8 per group. * vs. the EV; # vs. ZBTB10; by a two-way ANOVA. I, J Relative migration (I) and invasion (J) of LNCaP and C4-2 cells stably transfected with the EV, ZBTB10, or ZBTB10 + PKLR cDNA vector. n = 5 per group. * vs. the EV; # vs. ZBTB10; by a two-way ANOVA. Quantification of proliferation, sphere formation, migration, and invasion assays presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. K, L Relative PKLR, ZBTB10, proliferation marker, and EMT marker mRNA levels in LNCaP (K) and C4-2 (L) cells expressing the EV, PKLR, or ZBTB10 + PKLR cDNA vector. * vs. the EV; # vs. ZBTB10; by a two-way ANOVA. Quantification of mRNA is presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: ZBTB10 is negatively correlated with PKLR expression.A, B IHC staining of PKLR and ZBTB10 on consecutive sections of a PCa TMA (CA4) in two selected cases. Scale bars, 100 μm. C Correlation analysis of the intensities of PKLR and ZBTB10 of the PCa TMA (n = 49). PKLR expression was negatively associated with ZBTB10-expressing PCa samples. R, correlation coefficient; p, two-tailed p value. Significance was determined by correlation XY analyses in GraphPad Prism. D, E Tumor grade association analysis using a Chi-squared test of the CA9 PCa TMA. Intensities of PKLR (D) and ZBTB10 (E) staining were semiquantitatively scored using the H-index as follows: negative, weakly positive, moderately positive, and strongly positive. p values were calculated by a Chi-squared test performed using SPSS statistical 18.0 software. p < 0.001. F Proposed model for ADT-induced PKLR drives hormone-refractory PCa. Hormone-sensitive PCa activates AR signaling by increasing ZBTB10 leading to increased binding to the PKLR regulatory sequence and mediation of its transcriptional suppression. ADT induced inactivation of the AR-ZBTB10 pathway, leading to an abundance of PKLR through ZBTB10 loss of function. Overexpression of PKLR may upregulate glucose metabolism and NED progression of PCa cells. Targeting PKLR by potential PKLR inhibitors may reduce the PKLR-driven glycolysis and NED of ADT-resistant PCa.
Fig 5: Increased PKLR and decreased ZBTB10 are associated with NED of PCa after ADT.A Mean expression of ZBTB10 and PKLR in LNCaP cells from the GDS3358 database during 11 months of ADT. * vs. the control. **p < 0.01, ***p < 0.001; by a one-way ANOVA. B PKLR and ZBTB10 expression in patient samples of the GSE48403 dataset showed an increase in PKLR and a decrease in ZBTB10 expression after ADT. *p < 0.05, **p < 0.01; by a one-way ANOVA. C Protein levels of ZBTB10 in various PCa cells. D PKLR and ZBTB10 mRNA levels in C4-2 cells during 5 months of 20 μM MDV3100/enzalutamide treatment compared to parental C4-2 cells, by RT-qPCR analysis. * vs. parental C4-2; by a one-way ANOVA. E RT-qPCR showing PKLR, ZBTB10, NE marker, stem cell marker, and androgen-responsive gene mRNA levels in C4-2 cells following treatment with charcoal-stripped serum (CSS)-containing medium for 48 h, and further treatment with 10 nM dihydrotestosterone (DHT) for 24 h. * vs. -CSS; # vs. +CSS; by a two-way ANOVA. Quantification of mRNA is presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. F Immunoblots showing PKLR, ENO2, CHGA, ZBTB10, NKX3-1, and KLK3 protein levels in LNCaP and C4-2 cells following treatment with CSS-containing medium for 5 and 10 days and 10 nM DHT for 24 h. G, H Representative images of IHC staining and intensity analysis (by an H-score analysis) of PKLR (G) and ZBTB10 (H) in PCa tissue sections from the same patients before and after ADT. n = 17 samples from Taipei Medical University-Wan Fang Hospital. Scale bars, 100 µm. Statistical analysis by two-tailed Student’s t-test. ****p < 0.0001.
Supplier Page from Abcam for Anti-PKLR antibody