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. 2016 Dec 20;7(51):84893-84906.
doi: 10.18632/oncotarget.12753.

Elevated expression of ZNF217 promotes prostate cancer growth by restraining ferroportin-conducted iron egress

Xingkang Jiang  1 , Changwen Zhang  1 , Shiyong Qi  1 , Shanqi Guo  2 , Yue Chen  1 , E Du  1 , Hongtuan Zhang  1 , Xiaoming Wang  1 , Ranlu Liu  1 , Baomin Qiao  1 , Kuo Yang  1 , Zhihong Zhang  1 , Yong Xu  1
Affiliations

Elevated expression of ZNF217 promotes prostate cancer growth by restraining ferroportin-conducted iron egress

Xingkang Jiang et al. Oncotarget. .

Abstract

Although we and other studies indicated ZNF217 expression was increased in prostate cancer (PCa), the factors mediating its misregulated expression and their oncogenic activity remain largely unexplored. Recent evidence demonstrated that ferroportin (FPN) reduction lead to decreased iron export and increased intercellular iron that consequently aggravates the oncogenic effects of iron. In the present study, ZNF217 was identified as a transcriptional repressor that inhibits FPN expression. Increased of ZNF217 expression led to decreased FPN concentration, coupled with resultant intracellular iron retention, increased iron-related cellular activities and enhanced tumor cell growth. In contrast, decreased of ZNF217 expression restrained tumor cell growth by promoting FPN-driven iron egress. Mechanistic investigation manifested that ZNF217 facilitated the H3K27me3 levels of FPN promoter by interacting with EZH2. Besides, we also found that MAZ increased the transcription level of ZNF217, and subsequently inhibited the FPN expression and their iron-related activities. Strikingly, the expression of MAZ, EZH2 and ZNF217 were concurrently upregulated in PCa, leading to decreased expression of FPN, which induce disordered iron metabolism. Collectively, this study underscored that elevated expression of ZNF217 promotes prostate cancer growth by restraining FPN-conducted iron egress.

Keywords: EZH2; MAZ; ZNF217; ferroportin; prostate cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Elevated ZNF217 expression aggravates iron-related tumor cell growth
A. The mRNA and protein level of ZNF217 in four different PCa cell lines (i.e. PC3, DU145, LNCaP and C4-2) and prostatic epithelial cell line RWPE-1 by qPCR and Western blot analysis, respectively. B, F. The mRNA and protein level of ZNF217 were assessed upon ZNF217 knockdown in PC3 cells or overexpression in LNCaP cells. C, G. The cell growth were examined upon ZNF217 knockdown in PC3 cells or overexpression in LNCaP cells. D, H. The cell cycle was detected upon ZNF217 knockdown in PC3 cells or overexpression in LNCaP cells. E, J. The final tumor weight of tumors derived from ZNF217 knockdown PC3 cells or ZNF217 transfected LNCaP cells. F, K. The tissue iron levels in tumor samples upon ZNF217 knockdown in PC3 cells or overexpression in LNCaP cells. Data depict the mean ± SD and representative of six independent experiments. Asterisk (*) indicates P < 0.05
Figure 2
Figure 2. Increased ZNF217 expression promotes tumor cell iron metabolism
A, D. Intracellular iron content was examined after ZNF217 knockdown in PC3 cells or overexpression in LNCaP cells. B, E. DNA replication was assessed upon ZNF217 downregulation in PC3 cells or forced ZNF217 expression in LNCaP cells. C, F. Relative ATP levels was determined after ZNF217 knockdown in PC3 cells or ZNF217 overexpression in LNCaP cells. Data depict the mean ± SD and representative of six independent experiments. Asterisk (*) indicates P < 0.05
Figure 3
Figure 3. ZNF217 inhibits the expression of FPN to modulating iron metabolism
A, C. The mRNA level of ZNF217 was detected upon ZNF217 knockdown in PC3 cells or overexpression in LNCaP cells. B, D. The protein levels of ZNF217, FPN, ferritin and TfR were assessed upon ZNF217 downregulation in PC3 cells or forced ZNF217 expression in LNCaP cells. E. The putative ZNF217 binding site in FPN promoter regions. F. ChIP assay showed that ZNF217 expression was increased in the promoter of FPN in PC3 and LNCaP cells upon ZNF217 overexpression. G. Luciferase assay showed that ZNF217 binds to the promoter of FPN. Data depict the mean ± SD and representative of six independent experiments. Asterisk (*) indicates P < 0.05
Figure 4
Figure 4. ZNF217 facilities the H3K27me3 levels of FPN promoter by cooperating with EZH2
A, B. The binding ability ZNF217, EZH2, H3K27me3, H4K9me3 and H3K4me3 levels on the promoter of FPN were assessed ZNF217 downregulation in PC3 cells or forced ZNF217 expression in LNCaP cells. C, D. The binding ability ZNF217, EZH2, H3K27me3, H4K9me3 and H3K4me3 levels on the promoter of FPN were assessed EZH2 downregulation in PC3 cells or forced EZH2 expression in LNCaP cells. E, F. The mRNA and protein levels of FPN were examined upon EZH2 knockdown in PC3 cells or overexpression in LNCaP cells. G. Co-IP assay showed a directly interaction between ZNF217, EZH2 and H3K27me3. H. The mRNA and protein levels of FPN were examined upon EZH2 knockdown in ZNF217-transfected LNCaP cells. I-J. The cell growth and intracellular iron level were assessed upon EZH2 knockdown in ZNF217-transfected LNCaP cells. Data depict the mean ± SD and representative of six independent experiments. Asterisk (*) indicates P < 0.05
Figure 5
Figure 5. MAZ transcriptional activates ZNF217 expression
A. Six putative MAZ binding sites (MT1-MT6) are underlined in the promoter of ZNF217 promoter region. +1 indicates the first nucleotide upstream of ghe transcription start site (TSS). B. Transcriptional activity of ZNF217 was determined by luciferase assay under MAZ overexpression. PC3 cells was transfected with pGL3-ZNF217 promoter (WT, MT1 mutation, MT2 mutation, MT3 mutation, MT4 mutation, MT5 mutation, MT6 mutation) when cells were transfected with MAZ plasmids. C. ChIP assay was performed to detect the six binding sites in ZNF217 promoter region using anti-MAZ antibody or normal IgG. D, H. The binding ability of MAZ on the promoter of ZNF217 was assessed upon MAZ knockdown in PC3 cells or overexpression in LNCaP cells. E, I. The mRNA expression of MAZ, ZNF217 and FPN were examined MAZ knockdown in PC3 cells or overexpression in LNCaP cells. F, G, J, K. The cell growth and intracellular iron content were determined in MAZ knockdown PC3 cells or MAZ-transfected LNCaP cells. Data depict the mean ± SD and representative of six independent experiments. Asterisk (*) indicates P < 0.05
Figure 6
Figure 6. ZNF217 expression is upregulated in PCa
A, B, C. Oncomine database showed that ZNF217 was significantly upregulated in PCa samples, compared with those of normal tissues. D-F. The mRNA expression of ZNF217, MAZ and FPN were assessed in our PCa samples and their adjacent tissues. F, G. Spearman correlation analysis revealed that ZNF217 expression was positively correlated with MAZ and EZH3 expression, and H. negatively correlated with FPN expression in PCa tissues. Data depict the mean ± SD and representative of six independent experiments. Asterisk (*) indicates P < 0.05
Figure 7
Figure 7. A scheme diagram deciphering the mechanism underlying ZNF217 exerts oncogenic role in prostate cancer by restraining ferroportin-conducted iron egress
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