Table 1.
Primers used for RT-qPCR.
Fig 1.
CYB561 is highly expressed in several cancer tissue types and exhibits an increasing trend in expression as PCa progresses to CRPC and NEPC.
(A) Publicly available microarray data were examined using the integrated data analysis and microarray repository, ONCOMINE [23]. Based on the Bittner Multi-cancer microarray data set (GSE2109), CYB561 expression was observed to be highest in prostate tumor samples and is included in the top 3% over-expressed genes in PCa compared to normal tissue equivalents. (B) Congruent analysis of RNA-seq data from the Genotype-Tissue Expression Project (GTEx) for normal prostate tissue (N) [32] and the Cancer Genome Atlas (TCGA)–prostate adenocarcinoma (PRAD; T) [33] using the online tool Gene Expression Profiling Interactive Analysis (GEPIA) [26] showed an increasing trend in CYB561 transcript levels in PRAD samples compared to normal tissue equivalents. (C-D) The TCGA–PRAD (www.cancer.gov/tcga), and castration-resistant (CRPC) and neuroendocrine prostate cancer (NEPC) [25] RNA-seq and copy number data were analyzed using the online visualization tool cBioPortal [27,34]. Increasing (C) CYB561 and (D) PAM expression correlates with disease severity and resistance to therapy, with highest amplification frequencies seen in CRPC and NEPC. (E) Baseline expression of CYB561 mRNA is higher in the PCa cell lines LNCaP, 22RV1 and PC-3 compared to the normal prostate cell lines RWPE-1 and PNT1A. (F) Baseline expression of PAM mRNA is higher in the AR-negative cell lines RWPE-1, PNT1A, and PC-3 compared to LNCaP and 22rv1 cell lines. (G) Immunoblot for CYB561 across the five cell lines show that 22rv1 and PC-3 cells have higher CYB561 expression compared to LNCaP cells with PNT1A exhibiting the highest protein expression. Bars represent mean ± SEM with statistically significant differences determined through one-way ANOVA followed by Tukey’s post-hoc test (P < 0.05; means with the same letter are not statistically different).
Fig 2.
CYB561 does not exhibit androgen-dependent gene regulation but contributes to androgen-sensitivity of LNCaP cells.
(A-B) LNCaP and (C-D) 22Rv1 cells grown in hormone-starved media (aCSS) were treated with vehicle (Veh), 10 nM DHT, or 10 nM DHT plus 10 uM Enz for 24 hr before harvest and gene expression analysis by RT-qPCR. (A) CYB561 mRNA expression was not affected by DHT and Enz treatment. (B) PAM mRNA expression decreased with DHT treatment and this effect was reversed in the presence of Enz. In 22Rv1, hormone treatment did not affect (C) CYB561 and (D) PAM expression. (E) LNCaP cells transduced with the scrambled (scr) shRNA control and shCYB561 were treated with vehicle, 10 nM DHT, or 10 nM DHT plus 10 uM Enz, and cell proliferation was tracked for seven days. LNCaP cells transduced with the shCYB561 construct and treated with DHT and DHT plus Enz have lower cell proliferation rates compared to the corresponding scr shRNA control. (F) Expression of KLK3 mRNA was induced in the scr and shCYB561 transduced cells upon DHT treatment and this effect was reduced with Enz. Notably, lower KLK3 expression was observed for the Veh and Enz treated LNCaP-shCYB561 cells compared to the matched scr shRNA controls. Data points and bars represent mean ± SEM with statistically significant differences determined through one-way ANOVA followed by Tukey’s post-hoc test for effect of hormone treatment (P < 0.05; means with the same letter are not statistically different) and Student’s t-test for the effect of CYB561 knockdown between the same hormone treatment (#P < 0.01, ##P < 0.001, ###P < 0.0001).
Fig 3.
Expression of neuroendocrine differentiation gene markers is reduced upon CYB561 knockdown in LNCaP cells.
LNCaP cells transduced with the scrambled (scr) shRNA control and shCYB561 were grown and maintained in complete media (control) or transdifferentiation media for 14 days. (A) Transdifferentiation did not affect CYB561 mRNA expression (two-way ANOVA; Treatment factor: P = 0.1079; Knockdown factor: P < 0.0001) while (B) PAM mRNA expression increased with transdifferentiation which was further enhanced with CYB561 knockdown (two-way ANOVA; Treatment factor: P < 0.0001; Knockdown factor: P = 0.0791). The neuroendocrine differentiation gene markers (C) SYP (two-way ANOVA; Treatment factor: P < 0.0001; Knockdown factor: P < 0.0001) and (D) ENO2 (two-way ANOVA; Treatment factor: P < 0.0001; Knockdown factor: P = 0.0016) mRNA expression increased with transdifferentiation and this effect was dampened with CYB561 knockdown. Bars represent mean ± SEM with statistically significant differences determined through two-way ANOVA for main effects of treatment and CYB561 knockdown and Student’s t-test for the individual effects of treatment within a shRNA type (*P < 0.001, **P < 0.0001) and CYB561 knockdown between the same treatment (#P < 0.01, ##P < 0.001, ###P < 0.0001).
Fig 4.
Transdifferentiation and CYB561 knockdown altered the Fe2+ concentrations and expression of iron responsive genes (IRGs) in LNCaP cells.
LNCaP cells transduced with the scrambled (scr) shRNA control and shCYB561 were grown and maintained in complete media (control) or transdifferentiation media for 14 days followed by Fe2+ concentration measurement. (A) CYB561 contributes to cellular iron homeostasis through its ferrireductase function to maintain vesicular redox states. This function is congruent with how other players regulate cellular iron, such as TFRC which facilitates extracellular Fe3+ uptake and ferritin (FTH) which controls Fe3+ storage within the cytosol. (B) Concentration of Fe2+ increased with transdifferentiation and this effect was dampened with CYB561 knockdown (two-way ANOVA; Treatment factor: P = 0.0001; Knockdown factor: P = 0.0007). Expression analysis of IRGs showed that (C) TFRC mRNA expression decreased with CYB561 knockdown in control and transdifferentiation media. However, TFRC expression increased in the LNCaP-shCYB561 cell line after transdifferentiation (two-way ANOVA; Treatment factor: P = 0.0220; Knockdown factor: P = 0.0013). (D) Expression of FTH1 (two-way ANOVA; Treatment factor: P < 0.0001; Knockdown factor: P = 0.0002) and (E) FPN1 (two-way ANOVA; Treatment factor: P < 0.0001; Knockdown factor: P < 0.0001) mRNA increased upon transdifferentiation of LNCaP-scr cells and this effect was enhanced with CYB561 knockdown. Bars represent mean ± SEM with statistically significant differences determined through two-way ANOVA for main effects of media treatment and CYB561 knockdown, and Student’s t-test for the individual effects of media treatment within an shRNA type (*P < 0.01, **P < 0.001, ***P < 0.0001) and CYB561 knockdown between the same media treatment (#P < 0.05, ##P < 0.01, ###P < 0.001).
Fig 5.
CYB561 contributes to the maintenance of the neuroendocrine phenotype of PC-3 cells.
Knocking down CYB561 expression had no effect on (A) PAM mRNA expression and downregulated the mRNA levels of the NED gene markers (B) SYP and (C) ENO2. (D-E) PNT1A, (F-H) LNCaP, and (H-I) 22rv1 cells were treated with conditioned media (CM) harvested from PC-3 cells transduced with scrambled (scr) shRNA control or shCYB561, and cell survival was tracked for 2 days. Treatment of (D) PNT1A, (F) LNCaP, and (H) 22rv1 cells with the positive control media (base media supplemented with 2.5% FBS) was able to sustain the proliferation of PNT1A cells in contrast to the continuous decline in the proliferation of cells treated with the negative control media (base media only). Treatment of (E) PNT1A, (G) LNCaP, and (I) 22rv1 cells with the CM from the PC-3 scr shRNA control cells was able to support cell survival when compared to the CM from PC-3 cells transduced with shCYB561. Bars and data points represent mean ± SEM with statistically significant differences determined by Student’s t-test (*P < 0.01, **P < 0.001, ***P < 0.0001).
Fig 6.
CYB561 knockdown decreased intracellular Fe2+ iron concentration and altered the expression of iron responsive genes in PC-3 cells.
Knockdown of CYB561 in PC-3 cells (A) reduced intracellular Fe2+ concentration, (B) increased TFRC, and decreased (C) FTH1 and (D) FPN1 mRNA levels. Bars represent mean ± SEM with statistically significant differences determined by Student’s t-test (*P < 0.05, **P < 0.01, ****P < 0.0001).
Fig 7.
CYB561 knockdown decreased the survival, proliferation, and migration rates of PC-3 cells.
PC-3 cells transduced with shCYB561 had lower cell proliferation rates compared to the scrambled (scr) shRNA control in (A) trypan blue exclusion assay and (B) direct cell proliferation assay. Compared to the scr shRNA control, knocking down CYB561 in PC-3 cells resulted in (C-D) fewer colonies in colony formation assays and (E-F) reduced the migration rate control as measured at 24 hr and 48 hr timepoints of a wound healing assay. Bars represent mean ± SEM with statistically significant differences determined by Student’s t-test (*P < 0.01, **P < 0.001, ***P < 0.0001).
Fig 8.
CYB561 promotes and sustains the more aggressive NEPC phenotype through its role in ascorbate recycling and iron homeostasis.
CYB561 replenishes the ascorbic acid pool which is then used by PAM to activate neuropeptides that promote transformation towards NEPC. Additionally, CYB561 increases the availability of intracellular iron which is essential for regulating cellular activities related to proliferation and survival.