Human-like hyperplastic prostate with low ZIP1 induced solely by Zn deficiency in rats

Abstract

Prostate cancer is a leading cause of cancer death in men over 50 years of age, and there is a characteristic marked decrease in Zn content in the malignant prostate cells. The cause and consequences of this loss have thus far been unknown. We found that in middle-aged rats a Zn-deficient diet reduces prostatic Zn levels (P = 0.025), increases cellular proliferation, and induces an inflammatory phenotype with COX-2 overexpression. This hyperplastic/inflammatory prostate has a human prostate cancer-like microRNA profile, with up-regulation of the Zn-homeostasis-regulating miR-183-96-182 cluster (fold change = 1.41-2.38; P = 0.029-0.0003) and down-regulation of the Zn importer ZIP1 (target of miR-182), leading to a reduction of prostatic Zn. This inverse relationship between miR-182 and ZIP1 also occurs in human prostate cancer tissue, which is known for Zn loss. The discovery that the Zn-depleted middle-aged rat prostate has a metabolic phenotype resembling that of human prostate cancer, with a 10-fold down-regulation of citric acid (P = 0.0003), links citrate reduction directly to prostatic Zn loss, providing the underlying mechanism linking dietary Zn deficiency with miR-183-96-182 overexpression, ZIP1 down-regulation, prostatic Zn loss, and the resultant citrate down-regulation, changes mimicking features of human prostate cancer. Thus, dietary Zn deficiency during rat middle age produces changes that mimic those of human prostate carcinoma and may increase the risk for prostate cancer, supporting the need for assessment of Zn supplementation in its prevention.

Keywords: dietary Zn intake; prostate cancer metabolic phenotype; prostate cancer risk; untargeted metabolomics profiling; untargeted miRNA profiling.

Fig. 1.

Establishment of a Zn-deficient middle-aged Sprague–Dawley rat model with prostatic Zn loss. (A) Study design: 1-mo-old male rats received a Zn-deficient or Zn-sufficient diet for 1.5, 4, or 10 mo to form six Zn-modulated age groups (n = 10–20 rats per group): Zn-deficient young-adult, adult, and middle-aged and Zn-sufficient young-adult, adult, and middle-aged. (B and C) Testis Zn content (B) and prostate Zn content (C) (measured in micrograms per gram dry weight) of young-adult, adult, and middle-aged rats on a Zn-deficient or a Zn-sufficient diet (n = 7–12 rats per group). (D) The PCNA-labeling index in middle-aged prostate is expressed as the percent of intensely stained PCNA-positive nuclei (S-phase) per ∼500 prostate epithelial nuclei evaluated in a microscope field at 200× magnification (n = 9 rats per group). (E) NF-κβ p65 DNA-binding activity of nuclear extracts from middle-aged rat prostates was measured by the TransAM NF-κβ p65 assay kit (n = 9 rats per group). (F) qPCR analysis of four selected inflammation genes, S100a8, S100a9, Cxcl5, and Ptgs2, in middle-aged rat prostates (n = 6–10 rats per group, measurements were performed in triplicate with Psmb6 as a normalizer). Data are expressed as mean ± SD. All statistics are two-sided; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Zn-deficient middle-aged rat prostate shows increased cellular proliferation and inflammation. H&E analysis of histology and IHC analysis of PCNA, NF-κβ p65, and COX-2 protein expression were performed in FFPE prostate tissues. (A) Zn-deficient middle-aged Wistar-Unilever rat prostate vs. Zn-sufficient counterparts (n = 10 rats per dietary group). (Top Row) H&E-staining shows representative prostates from Zn-deficient middle-aged rats no. 21 and 28 displaying proliferative epithelia (arrowheads) and prostate from Zn-sufficient middle-aged rat 1 showing a thin epithelium (arrowhead) with infolding. (Scale bars: 100 μm in main panels and 50 μm in Insets.) (Second Row) In PCNA IHC, Zn-deficient middle-aged prostates show abundant PCNA-positive nuclei (red; 3-amino-9-ethylcarbazole substrate-chromogen; arrowheads); Zn-sufficient counterparts display few PCNA-positive nuclei. (Scale bars: 50 μm in main panels and 25 μm in Insets.) Additionally, Zn-deficient middle-aged prostates showed strong cytoplasmic NF-κβ p65 (Third Row) and intense COX-2 (Bottom Row) expression with typical perinuclear cytoplasmic staining; Zn-sufficient middle-aged prostates showed no NF-κβ p65 staining and occasional COX-2–positive staining (brown, DAB). (Scale bars: 25 μm.) (B) Compared with Zn-deficient young-adult rat prostate, Zn-deficient middle-aged prostate was proliferative (H&E staining, Top Row) (Scale bars: 100 μm in main panels and 50 μm in Insets) with frequent PCNA-stained nuclei (Second Row) (Scale bars: 50 μm in main panels and 50 μm in Insets), moderately strong cytoplasmic NF-κβ p65 expression (Third Row) and intense COX-2 expression (Bottom Row) (Scale bars: 100 μm).

Fig. 3.

Prostate miRNA expression profiling by the nanoString nCounter rat miRNA assay in Sprague–Dawley rats (A–C) and Wistar-Unilever rats (D and E) growing from young adult into middle age on a Zn-deficient diet or a Zn-sufficient diet. (A) Bar plot showing fold-change of 14 miRNAs up-regulated in Zn-deficient middle-aged vs. Zn-deficient young-adult prostates that are similarly up-regulated in human PCa compared with 11 such miRNAs up-regulated in Zn-sufficient middle-aged vs. Zn-sufficient young-adult prostates (cutoff: fold change ≥1.4, P < 0.05, n = 6 rats per cohort). Asterisks denote up-regulation of the oncogenic miR-183-96-182 cluster. (B) Validation of five selected miRNAs (identified by the nanoString platform) in Zn-deficient middle-aged vs. Zn-deficient young-adult rat prostates by the TaqMan miRNA assay (with snoRNA as normalizer, measurements performed in triplicate, n = 8 rats per cohort). (C) Validation of five selected miRNAs (identified by nanoString platform) in Zn-sufficient middle-aged vs. young-adult rat prostates by the TaqMan miRNA assay (snoRNA as normalizer, measurements performed in triplicate, n = 8 rats per cohort). (D) Prostate Zn content (measured in micrograms per gram wet weight; mean ± SD, n = 9–11 rats per cohort). (E) Expression of the miR-183-96-182 cluster and miR-21 in prostates of Zn-deficient middle-aged Wistar-Unilever rats (qPCR, snoRNA as normalizer, assays performed in triplicate, n = 9–12 rats per group). Data are expressed as fold-change in Zn-deficient vs. Zn-sufficient middle-aged rat prostates. All statistics are two-sided.

Fig. 4.

Zip and ZnT expression profiles of Zn-deficient middle-aged rat prostates resemble profiles of human prostate cancer and the relationship between miR-182 up-regulation and ZIP1 mRNA/protein down-regulation. (A and B) qPCR analyses of Zn importers (Zip 1–14; Zip12 is not detectable) (A) and Zn exporters (ZnT 1–10) (B) in prostates from Zn-deficient vs. Zn-sufficient middle-aged Wistar-Unilever rats (Oaz1 as normalizer, measurements performed in triplicate, n = 9–11 rats per cohort; two-sided t test). Asterisks denote Zip and ZnT expression similarly up- or down-regulated in human prostate cancer. (C) ISH cellular localization of miR-182 by mmu-miR-182 detection probe (double digoxigenin-labeled at the 5′ and 3′ ends) and IHC analysis of ZIP1 protein expression in FFPE prostate tissues of Zn-deficient vs. Zn-sufficient middle-aged rat prostates. Intense/frequent miR-182 ISH signal (blue) was detected in Zn-deficient middle-aged prostate (two representative samples are shown) vs. very weak and diffuse miR-182 ISH signals (blue) in Zn-sufficient middle-aged prostate (blue, 4-nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyl phosphate counterstained by nuclear fast red). ZIP1 protein expression was diffuse and weak (brown staining, DAB) in representative ZN-deficient middle-aged rat prostate epithelia vs. strong ZIP1 expression (brown staining) in epithelial cells of a Zn-sufficient middle-aged rat prostate. n = 10 rats per group. (Scale bars: 50 μm in main panels and 25 μm in Insets.)

Fig. 5.

Human prostate adenocarcinoma tissue shows an inverse relationship between miR-182 up-regulation and hZIP1 down-regulation. (A) A nanoString nCounter human miRNA assay showing miR-182 up-regulation in human prostate adenocarcinoma vs. adjacent nonneoplastic prostate (n = 4 paired samples; P = 0.08). (B) qPCR validation of the miR-182 result by nanoString (TaqMan miRNA assay, hsa-miR-182-5p, RNU44 as normalizer, measurements performed in triplicate, n = 6 paired samples; P = 0.02). (C) qPCR analysis showing hZIP1 down-regulation in human prostate adenocarcinomas vs. adjacent nonneoplastic prostate (OAZ1 as normalizer, measurements performed in triplicate; n = 6 paired samples; P = 0.02). Data are expressed as mean ± SD. (D) ISH cellular localization of miR-182 using the hsa-miR-182 detection probe (double digoxigenin-labeled at the 5′ and 3′ ends) and IHC analysis of hZIP1 protein expression (n = 6 paired human prostate adenocarcinomas and adjacent prostate tissue). Representative patient cases 1 and 2 show moderate/frequent miR-182 ISH signals (blue, 4-nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyl phosphate; nuclear fast red counterstain) in prostate adenocarcinoma but no miR-182 ISH signals in adjacent nonneoplastic prostate tissue. hZIP1 immunostaining was diffuse and weak (brown, DAB) in prostate adenocarcinoma but was strong in nonneoplastic prostate epithelial cells.

Fig. 6.

Untargeted metabolomics profiling by GC-TOF-MS reveals a human PCa-associated metabolic phenotype in Zn-deficient middle-aged Wistar-Unilever rat prostates with marked down-regulation of citrate (n = 9 rats per group). (A) ChemRICH set enrichment statistics plot showing that TCA metabolites were down-regulated in Zn-deficient middle-aged vs. Zn-sufficient middle-aged rat prostates, with pentose metabolites and saturated fatty acids up-regulated. The node color scale shows the proportion of increased (red) or decreased (blue) compounds in Zn-deficient vs. Zn-sufficient middle-aged prostate. Purple nodes have both increased and decreased metabolites. (B) Box-and-whisker plots (data log10 transformed) for citric acid and uracil (two metabolites similarly dysregulated in human PCa) in the Zn-deficient and Zn-sufficient middle-aged rat prostates. (C) Metabolomics network of biochemical differences between Zn-deficient and Zn-sufficient middle-aged rat prostates. A biochemical and chemical similarity network was calculated for all measured metabolites with KEGG and PubChem CIDs. Molecules not directly participating in biochemical transformations but sharing many structural properties were connected at a threshold of Tanimoto similarity coefficient ≥0.7. Each node denotes an identified metabolite (blue, down-regulated; red, up-regulated; yellow, insignificant change; P < 0.05; Mann–Whitney U test). Metabolites are connected based on biochemical relationships (red lines) or structural similarity (light-blue lines). Metabolite size reflects median fold-change.