Hepcidin regulation in prostate and its disruption in prostate cancer

Abstract

Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression.

Figure 1. Hepcidin is upregulated in prostate cancer cells and regulates ferroportin and intracellular iron in prostate cells

(A) Hepcidin in conditioned medium from normal prostate epithelial cells (PEC) and prostate cancer cell lines. (B) Western blot of pro-hepcidin and ferroportin in normal prostate cells and prostate cancer cell lines. Shown is a single exposure of one blot; dashed line indicates where an irrelevant lane was cropped out; (C) PEC cells were treated with hepcidin in growth medium for 4hr and levels of ferroportin assessed by western blot (D) Immunofluorescence of ferroportin in untreated PEC and PEC treated for 24 hours with 800 nM hepcidin; ferroportin (red) and nuclei (blue). (E) labile iron pool in PEC treated with iron (ferric ammonium citrate) or hepcidin. (F) Western blot of ferroportin in PC3 cells treated with anti-hepcidin antibody or IgG control for 24 hours. Means and standard deviations of triplicate determinations; data are representative of one of three independent experiments.

Figure 2. Hepcidin is upregulated and ferroportin is down-regulated in prostate cancer patient samples

(A) Transcript levels of ferroportin and hepcidin in normal prostate (N; green, n=29), primary tumor (P; blue, n=131) and metastatic tumor (M; red, n=19). Data from (19). (B) Immunohistochemistry of hepcidin in prostate tissue. Top: Prostate cancer [Gleason score (3+5) = 8] .Middle: Benign prostatic glands and stroma. Bottom: Control IgG staining. CaP, prostate cancer; NP, normal prostate glands. (C) Labile iron pool in normal PEC and DU145 prostate cancer cells. (D) MTS assay of DU145 cells treated with either IgG control or anti-hepcidin antibody. (E) Clonogenic assay of DU145 using 3 μg/ml anti-hepcidin antibody from Amgen (#1) or Abcam (#2). Means and standard deviations of triplicate determinations; data are representative of one of three independent experiments. P values reported in Fig. 2D represent differences between anti-hepcidin-treated (1 μg/ml and 3 μg/ml) and IgG control; difference between untreated and IgG control was not significant (p>0.05).

Figure 3. IL6 and BMP4/7 mediate induction of hepcidin in prostate cancer cells

(A) Phosphorylated and total STAT3 in DU145 cells treated with IL6. (B) Extracellular hepcidin following treatment of DU145 cells with 100 or 200 ng/ml IL6. (C) Extracellular hepcidin in DU145 cells treated with anti-IL6 antibody or IgG control antibody. (D) Extracellular hepcidin in DU145 cells treated with 100 ng/ml or 200 ng/ml BMP4, BMP6, or BMP7. (E) Extracellular hepcidin in DU145 cells treated with 3μg/ml anti-BMP4, anti-BMP6, anti-BMP7, anti-IL6 antibody, or IgG control antibody. Means and standard deviations of triplicate determinations; data are representative of one of three independent experiments.

Figure 4. SOSTDC1 antagonizes BMP and Wnt-mediated induction of Hepcidin

(A) Phosphorylation of Smad-1-5-8 in DU145 cells treated with BMP4 or BMP7 in the presence or absence of SOSTDC1 for 24 hours. (B) Extracellular hepcidin in DU145 cells treated with 50 or 100 ng/ml rSOSTDC1 for 24 hours. (C) Hepcidin transcript levels in DU145 cells treated with 20 mM LiCl, 20 mM LiCl plus 100 ng/ml SOSTDC1, 50 ng/ml Wnt3a, 50 ng/ml Wnt3a plus 100 ng/ml SOSTDC1 or 50 uM endo-IWR for 24 hrs. (D) Hepcidin promoter-driven luciferase activity in DU145 cells treated as in C. Inset is a cartoon of the location of BMP and STAT3 (44) sites and a candidate TCF/LEF site in the human hepcidin promoter. (E) Hepcidin secretion in DU145 cells treated with 1 μg/ml anti-IL6 antibody, 1 ug/ml control IgG, 100 ng/ml recombinant SOSTDC1, or the combination of anti-IL6 and SOSTDC1 for 24 hours. Means and standard deviations of triplicate determinations; data are representative of one of three independent experiments.

Figure 5. Expression of SOSTDC1 is controlled by promoter methylation in prostate cancer cells

(A) Transcript levels of SOSTDC1 in DU145 and normal PEC cells. (B) Extracellular SOSTDC1 in DU145 and PEC cells. (C) Methylation of the SOSTDC1 promoter at the designated positions in DU145 cells treated with DMSO or 5-Aza-2’-deoxycytidine. (D) SOSTDC1 transcripts in DU145 cells treated with 2 μM 5-Aza-2’-deoxycytidine or DMSO in 3 independent experiments. Means and standard deviations of triplicate determinations; data in A and B are representative of one of three independent experiments.

Figure 6. SOSTDC1 promoter is hypermethylated in prostate tumors

(A) Methylation of SOSTDC1 in benign and malignant human prostate tissue (data from (14)). (B) SOSTDC1 expression in prostate tissue, analyzed by immunohistochemistry (IHC). Benign prostatic glands at left are more intensely staining than the high grade [Gleason score (5+4) = 9] prostatic carcinoma to the right. (C) Probability of biochemical recurrence in prostate cancer patients with SOSTDC1 methylation in tumor tissue that is above or below the mean.

Figure 7

Working model of transcriptional control of hepcidin in the prostate.