Aberrant Promoter CpG Methylation Is a Mechanism for Impaired PHD3 Expression in a Diverse Set of Malignant Cells

Abstract

BackgroundThe prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions.Methodology/Principal FindingsUsing bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2′-deoxycytidine significantly increased the expression of PHD3.Conclusions/SignificanceWe conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.

The Prolyl Hydroxylase PHD3 Identifies Proinflammatory Macrophages and Its Expression Is Regulated by Activin A

“…Our results indicate that activin A complies with this requirement because it: 1) potentiates the activity of the EGLN3 gene regulatory region; 2) positively regulates EGLN3 expression in M1(GM-CSF) macrophages independently of low oxygen pressure; and 3) mediates the hypoxia-inducible EGLN3 gene expression in M2(M-CSF) macrophages. Therefore, unlike promoter hypermethylation or miR-20a, which inhibit EGLN3 gene expression in tumor cells (34) and cardiomyocytes (35), activin A represents a novel mechanism for positive regulation of EGLN3 expression. Along this line, soluble growth factors may turn out to exert an important control of the hypoxia-independent expression of prolyl hydroxylases in myeloid cells, as EGLN1 gene expression is upregulated by leukemia inhibitory factor in murine osteoclasts (36).…”

Modulation of hypoxia-inducible factors (HIF) from an integrative pharmacological perspective

“…Although regulation of HIF is finely controlled by PHDs, a loss of PHD3 expression by aberrant promoter CpG methylation does not correlate with an increase in HIF-1a protein levels or an increase in the transcriptional activity of HIF in cancer cell lines [21]. Currently, there is no evidence for DNA methylation-mediated regulation of other genes that regulate HIF accumulation such as von Hippel-Lindau ubiquitin ligase complex genes [21]. The gene silencing mediated by CpG island hypermethylation is associated in many cases with the deacetylation of histones within the regulatory region of genes in certain pathologies.…”

“…Recent evidence demonstrates that DNA methylation is an important process for the regulation of proteins that leads to negative regulation of HIF, such as PHD3. Although regulation of HIF is finely controlled by PHDs, a loss of PHD3 expression by aberrant promoter CpG methylation does not correlate with an increase in HIF-1a protein levels or an increase in the transcriptional activity of HIF in cancer cell lines [21]. Currently, there is no evidence for DNA methylation-mediated regulation of other genes that regulate HIF accumulation such as von Hippel-Lindau ubiquitin ligase complex genes [21].…”

Gestational hypoxia and epigenetic programming of brain development disorders

“…Abnormal methylation in the CpG island of VHL has been observed in various disorders, including bone disease [47], retinoblastoma [48], and non-small cell lung cancer [49]. One member of the HIF hydroxylase group, prolyl hydroxylase 3 (PHD3), was also regulated by DNA methylation [50,51]. It has been reported that some cancer cell lines displayed a failure of PHD3 mRNA induction when placed in a hypoxic environment [51].…”

“…One member of the HIF hydroxylase group, prolyl hydroxylase 3 (PHD3), was also regulated by DNA methylation [50,51]. It has been reported that some cancer cell lines displayed a failure of PHD3 mRNA induction when placed in a hypoxic environment [51]. Further studies investigated the major mechanism in multiple tumors, showing that the suppression of PHD3 expression was the result of a change in methylation level of the CpG island [41,50].…”