DOI: 10.5061/dryad.9s4mw6mgq
title: Supplementary material: CYB561 supports the neuroendocrine phenotype in castration-resistant prostate cancer

description: 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.
description: Protein expression data was evaluated using a western
 blot. <em>In silico</em> 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 <em>18s
 rRNA</em>, <em>GAPDH,</em> or
 β-<em>Actin </em>(<em>ACTB</em>) transcript
 levels) were log<sub>10</sub> 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 <em>CYB561 </em>knockdown and
 transdifferentiation, after which Student’s
 unpaired <em>t</em>-test was done to determine the effect of
 transdifferentiation within an shRNA group and the effect
 of <em>CYB561</em> 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 <em>t</em>-test. All statistical analyses were done
 using GraphPad Prism version 8.0 (GraphPad Software, La Jolla California
 USA, www.graphpad.com), and <em>P</em> &lt; 0.05 was
 accepted as statistically
 significant.
description: # Supplementary material: CYB561 supports the neuroendocrine phenotype in
 castration-resistant prostate cancer ## Data All data were analyzed using
 GraphPad Prism version 8.0. The statistical analyses included in each file
 are taken from the summary of results shown by GraphPad. ### Figure1E,
 F.xlxs Data are presented as tables with table headings denoting the mRNA
 measured and column titles denoting the cell lines where the mRNA
 expression was measured. In some cases, the term "NORM" precedes
 the gene name indicating that the data shown are already expression values
 normalized to the housekeeping gene. **Statistical analyses**: Ordinary
 one-way ANOVA and Tukey's multiple comparisons were used. The summary
 of the results is included in the data file. A P &lt; 0.05 was set to
 indicate significance. ### Figure2A, B, C, D.xlxs Data are presented as
 tables with table headings denoting the cell line used and mRNA measured
 and column titles denoting the hormone treatment done. **Statistical
 analyses:** Ordinary one-way ANOVA and Tukey's multiple comparisons
 were used. The summary of the results is included in the data file. A P
 &lt; 0.05 was set to indicate significance. ### Figure2E.xlxs Data are
 presented as tables with table headings denoting the cell line used
 (scrambled or sh) and the time of cell proliferation measurement in hours.
 Column titles denote the hormone treatment done to the cells. In some
 cases, the term "LOG" is used in table headings indicating that
 the data shown are log-transformed values. **Statistical analyses:**
 Ordinary one-way ANOVA and Tukey's multiple comparisons were used.
 The summary of the results is included in the data file. A P &lt; 0.05 was
 set to indicate significance. ### Figure2F.xlxs Data are presented as
 tables with table headings denoting the mRNA measured and column titles
 denoting the cell lines where the mRNA expression was measured and the
 hormone treatment was done. In some cases, the term "LOG" is
 used in table headings indicating that the data shown are log-transformed
 values. **Statistical analyses:** Ordinary one-way ANOVA and Tukey's
 multiple comparisons were used. The summary of the results is included in
 the data file. A P &lt; 0.05 was set to indicate significance. ###
 Figure3A, B, C, D.xlxs Data are presented as tables with table headings
 denoting the mRNA measured, column titles denoting the state of the cell
 (control or transdifferentiated), and row titles indicating the cell lines
 used. In some cases, the term "t-test" or "transform"
 is used in table headings indicating that the data shown are
 log-transformed values used for statistical analysis. **Statistical
 analyses:** Ordinary one-way ANOVA and Tukey's multiple comparisons
 were used. The summary of the results is included in the data file. A P
 &lt; 0.05 was set to indicate significance. ### Figure4B.xlxs Data are
 presented as tables with column titles denoting the state of the cell
 (control or transdifferentiated) and row titles indicating the cell lines
 used. **Statistical analyses:** Ordinary two-way ANOVA and unpaired
 t-tests were used. The summary of the results is included in the data
 file. A P &lt; 0.05 was set to indicate significance. ### Figure4C, D.xlxs
 Data are presented as tables with table headings denoting the mRNA
 measured, column titles denoting the state of the cell (control or
 transdifferentiated), and row titles indicating the cell lines used. In
 some cases, the term "t-test" or "transform" is used
 in table headings indicating that the data shown are log-transformed
 values used for statistical analysis. **Statistical analyses:** Ordinary
 two-way ANOVA and unpaired t-tests were used. The summary of the results
 is included in the data file. A P &lt; 0.05 was set to indicate
 significance. ### Figure4E.xlxs Data are presented as tables with table
 headings denoting the mRNA measured, column titles denoting the state of
 the cell (control or transdifferentiated), and row titles indicating the
 cell lines used. In some cases, the term "t-test" or
 "transform" is used in table headings indicating that the data
 shown are log-transformed values used for statistical analysis.
 **Statistical analyses**: Ordinary two-way ANOVA and unpaired t-tests were
 used. The summary of the results is included in the data file. A P &lt;
 0.05 was set to indicate significance. ### Figure5A, B, C.xlxs Data are
 presented as tables with table headings denoting the mRNA measured and
 column titles denoting the cell lines where the mRNA expression was
 measured and the hormone treatment was done. In some cases, the term
 "LOG" or "NORM" is used in table headings indicating
 that the data shown are log-transformed values or the data shown are
 already expression values normalized to the housekeeping gene.
 **Statistical analyses**: Unpaired t-tests were used. The summary of the
 results is included in the data file. A P &lt; 0.05 was set to indicate
 significance. ### Figure5D, E.xlxs Data are presented as tables with table
 headings denoting the cell line used (parental, scrambled, or sh). Column
 titles denote the media used to grow the cells. Row titles indicate the
 time of cell proliferation measurement in hours. In some cases, the term
 "LOG" is used in table headings indicating that the data shown
 are log-transformed values. **Statistical analyses**: Multiple t-tests
 were used. The summary of the results is included in the data file. A P
 &lt; 0.05 was set to indicate significance. ### Figure5F, G.xlxs Data are
 presented as tables with table headings denoting the cell line used
 (parental, scrambled, or sh). Column titles denote the media used to grow
 the cells. Row titles indicate the time of cell proliferation measurement
 in hours. In some cases, the term "LOG" is used in table
 headings indicating that the data shown are log-transformed values.
 **Statistical analyses**: Multiple t-tests were used. The summary of the
 results is included in the data file. A P &lt; 0.05 was set to indicate
 significance. ### Figure5H, I.xlxs Data are presented as tables with table
 headings denoting the cell line used (parental, scrambled, or sh). Column
 titles denote the media used to grow the cells. Row titles indicate the
 time of cell proliferation measurement in hours. In some cases, the term
 "LOG" is used in table headings indicating that the data shown
 are log-transformed values. **Statistical analyses**: Multiple t-tests
 were used. The summary of the results is included in the data file. A P
 &lt; 0.05 was set to indicate significance. ### Figure6A.xlxs Data are
 presented as tables with column titles denoting the state of the cell
 lines used. **Statistical analyses**: Unpaired t-tests were used. The
 summary of the results is included in the data file. A P &lt; 0.05 was set
 to indicate significance. ### Figure6B, C, D.xlxs Data are presented as
 tables with table headings denoting the mRNA measured and column titles
 denoting the cell lines where the mRNA expression was measured. In some
 cases, the term "LOG" is used in table headings indicating that
 the data shown are log-transformed values. **Statistical analyses**:
 Unpaired t-tests were used. The summary of the results are included in the
 data file. A P &lt; 0.05 was set to indicate significance. ###
 Figure7A.xlxs Data are presented as tables with column titles denoting the
 cell lines counted. **Statistical analyses**: Unpaired t-tests were used.
 The summary of the results is included in the data file. A P &lt; 0.05 was
 set to indicate significance. ### Figure7B.xlxs Data are presented as
 tables with column titles denoting the cell line used (scrambled or sh).
 Row titles indicate the time of cell proliferation measurement in hours or
 days. In some cases, the term "LOG" is used in table headings
 indicating that the data shown are log-transformed values. **Statistical
 analyses**: Multiple t-tests were used. The summary of the results is
 included in the data file. A P &lt; 0.05 was set to indicate significance.
 ### Figure7C.xlxs Data are presented as tables with column titles denoting
 the state of the cell lines used. **Statistical analyses**: Unpaired
 t-tests were used. The summary of the results is included in the data
 file. A P &lt; 0.05 was set to indicate significance. ### Figure7E.xlxs
 Data are presented as tables with column titles denoting the cell line
 used (scrambled or sh). Row titles indicate the time of measurement in
 hours. The term "WHA" is used in table headings which is short
 for "wound healing assay." **Statistical analyses**: Multiple
 t-tests were used. The summary of the results is included in the data
 file. A P &lt; 0.05 was set to indicate significance. ###
 Raw\_western\_blot\_images.pdf Data are presented as raw images of blots.
 Lane titles indicate cell lines.