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

The 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.Using 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.We 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

Effect of endometrial thickness on live birth rates in fresh and frozen embryo transfers in women under 38 years of age

Many IVF clinics use endometrial thickness as a predictive factor for IVF outcomes, as research has shown a positive association between endometrial thickness and favorable IVF outcomes. A thickness of 6-8 mm is often used as a cut-off in for the decision of whether or not to transfer an embryo in both fresh and frozen cycles. However, prior studies investigating the relationship between a thin endometrium and IVF outcomes have overwhelmingly been performed in fresh cleavage stage embryo transfers. Given the recent trend toward the transfer of frozen blastocyst transfers, we aimed to determine whether endometrial thickness predicts live birth in both fresh and frozen blastocyst stage single embryo transfers

Prolyl-4-Hydroxylase 3 (PHD3) Expression Is Downregulated during Epithelial-to-Mesenchymal Transition

<div><p>Prolyl-4-hydroxylation by the intracellular prolyl-4-hydroxylase enzymes (PHD1-3) serves as a master regulator of environmental oxygen sensing. The activity of these enzymes is tightly tied to tumorigenesis, as they regulate cell metabolism and angiogenesis through their control of hypoxia-inducible factor (HIF) stability. PHD3 specifically, is gaining attention for its broad function and rapidly accumulating array of non-HIF target proteins. Data from several recent studies suggest a role for PHD3 in the regulation of cell morphology and cell migration. In this study, we aimed to investigate this role by closely examining the relationship between PHD3 expression and epithelial-to-mesenchymal transition (EMT); a transcriptional program that plays a major role in controlling cell morphology and migratory capacity. Using human pancreatic ductal adenocarcinoma (PDA) cell lines and Madin-Darby Canine Kidney (MDCK) cells, we examined the correlation between several markers of EMT and PHD3 expression. We demonstrated that loss of PHD3 expression in PDA cell lines is highly correlated with a mesenchymal-like morphology and an increase in cell migratory capacity. We also found that induction of EMT in MDCK cells resulted in the specific downregulation of PHD3, whereas the expression of the other HIF-PHD enzymes was not affected. The results of this study clearly support a model by which the basal expression and hypoxic induction of PHD3 is suppressed by the EMT transcriptional program. This may be a novel mechanism by which migratory or metastasizing cells alter signaling through specific pathways that are sensitive to regulation by O₂. The identification of downstream pathways that are affected by the suppression of PHD3 expression during EMT may provide important insight into the crosstalk between O₂ and the migratory and metastatic potential of tumor cells.</p></div

PHD3 expression in BxPC3 cells.

<p>BxPC3 cells stably transduced with retrovirus containing PHD3Wt (BxPC3-Wt), Vector (BxPC3-Vec) or anti-PHD3 shRNA (BxPC3-KD) were harvested for RNA and protein following 24 hours exposure to normoxia (21% O₂) or hypoxia (1% O₂). (A) PHD3 mRNA expression was determined by qRT-PCR in BxPC3-Vec and BxPC3-KD cells. All samples were normalized to 18S rRNA and graphed as expression relative to BxPC3-Vec Normoxia (lane 1). n = 3, error bars  = 1 S.D. p-value represents Student's 2-tailed, type 2 t-test comparison (B) Whole cell lysate from BxPC3-Wt, BxPC3-Vec and BxPC3-KD cells following 24 hours exposure to normoxia (N) or hypoxia (H) was resolved by SDS-PAGE and blotted for actin (top) and PHD3 (bottom). PHD3 band intensity was quantified relative to actin in each lane and then normalized to BxPC3-Vec (N). Relative band intensity is indicated below the figure. NS  =  non-specific band running just above PHD3 band. Data is representative of >3 biological replicates.</p

Flow cytometric analysis of E-cadherin in MDCK cells. (A) MDCK cells (parental population) were labeled with phalloidin (red) and anti-E-cadherin antibody (green) and visualized by confocal microscopy.

<p>Arrow indicates E-cadherin negative cells within the parental population. (B) Live MDCK cells (parental population) were labeled with primary anti-E-cadherin (red) or isotype-matched control antibody (blue), then with goat anti-mouse Alexa-fluor 488 (A488)-conjugated secondary antibody and analyzed by flow cytometry. (C) Flow chart for separation of mesenchymal and epithelial subpopulations of the MDCK parental cell line. (D) MDCK-L and MDCK-E3 cell lines were analyzed for surface E-cadherin expression by flow cytometry as done in (A). (E) MDCK-L and MDCK-E3 cells were subjected to normoxia (21% O₂) or hypoxia (1% O₂) for 24 hours. mRNA was harvested and PHD3 and E-cadherin expression was quantified by qRT-PCR. All data points represent the average of 3 biological replicates. Quantification of mRNA is set relative to MDCK-E3 samples at normoxia. Error bars  = +/− 1 S.D.</p

PHD3 expression correlates with a mesenchymal-like morphology in pancreatic ductal adenocarcinoma cell lines.

<p>NHF-1 (Fibroblast) MiaPaca2, Panc1, CAPAN1 and BxPC3 cells were harvested for RNA and protein following 24 hours exposure to normoxia (21% O₂) or hypoxia (1% O₂). (A) Phase-contrast images at 10× magnification were taken of MiaPaca2 (mesenchymal-like) morphology and BxPC3 cells (differentiated, epithelial morphology) under normoxic conditions. (B) PHD3 mRNA expression was determined by qRT-PCR and graphed relative to BxPC3 in normoxia. All samples were normalized to 18S rRNA and graphed as expression relative to BxPC3-Vec Normoxia (lane 1). n = 3, error bars  = 1 S.D. (C) Whole cell lysate was resolved by SDS-PAGE and blotted for β-tubulin PHD3 and PHD2. N = normoxia, H = hypoxia (1% O₂).</p

PHD3 knockdown increases the migratory capacity of cells.

<p>BxPC3 cells stably transduced with retrovirus containing PHD3Wt (BxPC3-Wt), Vector (BxPC3-Vec) or anti-PHD3 shRNA (BxPC3-KD) were seeded at confluence in 60 mm tissue culture dishes and allowed to adhere for 24 hours. (A) Scratches were made using a 5 ml stripette and photographed at 0 hr and 24 hr under normoxia (21% O₂) or hypoxia (1% O₂). Arrows highlight the directional migration of cells (B) Migration speed for each cell line was normalized to BxPC3-Vec normoxia. Data is representative of 3 independent biological replicates and 8 scratches each. Error bars  = 1 S.D. p-value represents Student's 2-tailed, type 2 t-test comparison. * samples are significantly different p<.01 than all other samples. (C) Live cells were counted using trypan blue exclusion at the indicated time points. Lines represent best fit for the data.</p

SNAIL-induced EMT in MDCKE3 Cells.

<p>(A–D). Stable pQCXIP-SNAIL (hSNAIL) or pQCXIP-vector (Vector)-expressing MDCK-E3 cells were subjected to normoxia (21% O₂) or hypoxia (1% O₂) for 24 hours. mRNA was harvested and subjected to qRT-PCR analysis for the indicated genes. All data points represent the average of 3 biological replicates. mRNA quantification is set relative to the Vector samples at normoxia. Error bars  = 1 S.D.</p

E-cadherin and PHD3 expression are inversely correlated with SNAIL expression.

<p>NHF-1 (fibroblast), MiaPaca2, Panc1, CAPAN1, and BxPC3 cells were harvested for RNA following 24 hours exposure to normoxia (21% O₂) or hypoxia (1% O₂). (A) E-cadherin and SNAIL mRNA expression was determined by qRT-PCR. All samples were normalized to 18S rRNA and graphed as expression relative to BxPC3 (lane 9). n = 3, error bars  = 1 S.D. (B) Hypoxic SNAIL mRNA expression was graphed relative to hypoxic PHD3 expression as was determined in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083021#pone-0083021-g004" target="_blank">Figure 4B</a>). ρ = Pearson's correlation coefficient. Line represents best fit for the data.</p