Castration-resistant prostate cancer (CRPC) is associated with resistance to androgen deprivation therapy, and an increase in the population of neuroendocrine (NE) differentiated cells. It is hypothesized that NE differentiated cells secrete neuropeptides that support androgen-independent tumor growth and induce aggressiveness of adjacent proliferating tumor cells through a paracrine mechanism. The cytochrome b561 (CYB561) gene, which codes for a secretory vesicle transmembrane protein, is constitutively expressed in NE cells and highly expressed in CRPC. CYB561 is involved in the α-amidation-dependent activation of neuropeptides and contributes to regulating iron metabolism which is often dysregulated in cancer. These findings led us to hypothesize that CYB561 may be a key player in the NE differentiation process that drives the progression and maintenance of the highly aggressive NE phenotype in CRPC. In our study, we found that CYB561 expression is upregulated in metastatic and NE prostate cancer (NEPC) tumors and cell lines compared to normal prostate epithelia and that its expression is independent of androgen regulation. Knockdown of CYB561 in androgen-deprived LNCaP cells dampened NE differentiation potential and transdifferentiation-induced increase in iron levels. In NEPC PC-3 cells, depletion of CYB561 reduced the secretion of growth-promoting factors, lowered intracellular ferrous iron concentration, and mitigated the highly aggressive nature of these cells in complementary assays for cancer hallmarks. These findings demonstrate the role of CYB561 in facilitating transdifferentiation and maintenance of NE phenotype in CRPC through its involvement in neuropeptide biosynthesis and iron metabolism pathways.

Protein expression data was evaluated using a western blot. In silico analysis of gene expression data was performed by examining the Bittner Multi-cancer clinical microarray data set (GSE2109) from the International Genomics Consortium Project for Oncology (www.intgen.org) for neuropeptide receptor mRNA expression across different clinical tumor types. Gene expression analysis was performed by reverse transcription-quantitative PCR (RT-qPCR). Data for gene expression analysis (normalized to 18s rRNA, GAPDH, or β-Actin (ACTB) transcript levels) were log10 transformed before statistical analysis. The basal and androgen-regulated gene expression data were analyzed using one-way ANOVA followed by Tukey's post hoc test. Results from the transdifferentiation experiment were analyzed using two-way ANOVA to determine the main effects of CYB561 knockdown and transdifferentiation, after which Student's unpaired t-test was done to determine the effect of transdifferentiation within an shRNA group and the effect of CYB561 knockdown in cells maintained in the same growth condition. Data from the LNCaP hormone treatment and starvation gene expression analysis were analyzed using the Student's unpaired t-test. All statistical analyses were done using GraphPad Prism version 8.0 (GraphPad Software, La Jolla California USA, www.graphpad.com), and P < 0.05 was accepted as statistically significant.

Funding provided by: Department of Science and TechnologyCrossref Funder Registry ID: https://ror.org/05tgxx705Award Number: Funding provided by: University of the Philippines DilimanCrossref Funder Registry ID: https://ror.org/03tbh6y23Award Number: Funding provided by: Department of Science and TechnologyCrossref Funder Registry ID: https://ror.org/05tgxx705Award Number: