Supplementary material: CYB561 supports the neuroendocrine phenotype in castration-resistant prostate cancer
Data files
Apr 02, 2024 version files 289.90 KB
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Figure_1E_F.xlsx
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Figure_2A_B_C_D.xlsx
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Figure_2E.xlsx
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Figure_2F.xlsx
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Figure_3A_B_C_D.xlsx
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Figure_4B.xlsx
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Figure_4C_D.xlsx
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Figure_4E.xlsx
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Figure_5A_B_C.xlsx
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Figure_5D_E.xlsx
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Figure_5F_G.xlsx
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Figure_5H_I.xlsx
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Figure_6A.xlsx
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Figure_6B_C_D.xlsx
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Figure_7A.xlsx
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Figure_7B.xlsx
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Figure_7C.xlsx
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Figure_7E.xlsx
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README.md
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Abstract
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.
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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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 < 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.
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.