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Epigenetic suppression of neprilysin regulates breast cancer invasion.

Stephen HM, Khoury RJ, Majmudar PR, Blaylock T, Hawkins K, Salama MS, Scott MD, Cosminsky B, Utreja NK, Britt J, Conway RE - Oncogenesis (2016)

Bottom Line: RT-PCR and flow cytometry analysis of MDA-MB-231 and MCF-7 breast cancer cell lines revealed decreased neprilysin expression compared with normal epithelial cells.Expression was also suppressed in invasive ductal carcinoma (IDC) compared with normal tissue.These results demonstrate that neprilysin negatively regulates the ET axis in breast cancer, and epigenetic suppression of neprilysin in invasive breast cancer cells enables invasion.

View Article: PubMed Central - PubMed

Affiliation: Lipscomb University, Department of Biology, College of Liberal Arts and Science, 1 University Park Drive, Nashville, TN, USA.

ABSTRACT
In women, invasive breast cancer is the second most common cancer and the second cause of cancer-related death. Therefore, identifying novel regulators of breast cancer invasion could lead to additional biomarkers and therapeutic targets. Neprilysin, a cell-surface enzyme that cleaves and inactivates a number of substrates including endothelin-1 (ET1), has been implicated in breast cancer, but whether neprilysin promotes or inhibits breast cancer cell progression and metastasis is unclear. Here, we asked whether neprilysin expression predicts and functionally regulates breast cancer cell invasion. RT-PCR and flow cytometry analysis of MDA-MB-231 and MCF-7 breast cancer cell lines revealed decreased neprilysin expression compared with normal epithelial cells. Expression was also suppressed in invasive ductal carcinoma (IDC) compared with normal tissue. In addition, in vtro invasion assays demonstrated that neprilysin overexpression decreased breast cancer cell invasion, whereas neprilysin suppression augmented invasion. Furthermore, inhibiting neprilysin in MCF-7 breast cancer cells increased ET1 levels significantly, whereas overexpressing neprilysin decreased extracellular-signal related kinase (ERK) activation, indicating that neprilysin negatively regulates ET1-induced activation of mitogen-activated protein kinase (MAPK) signaling. To determine whether neprilysin was epigenetically suppressed in breast cancer, we performed bisulfite conversion analysis of breast cancer cells and clinical tumor samples. We found that the neprilysin promoter was hypermethylated in breast cancer; chemical reversal of methylation in MDA-MB-231 cells reactivated neprilysin expression and inhibited cancer cell invasion. Analysis of cancer databases revealed that neprilysin methylation significantly associates with survival in stage I IDC and estrogen receptor-negative breast cancer subtypes. These results demonstrate that neprilysin negatively regulates the ET axis in breast cancer, and epigenetic suppression of neprilysin in invasive breast cancer cells enables invasion. Together, this implicates neprilysin as an important regulator of breast cancer invasion and clarifies its utility as a potential biomarker for invasive breast cancer.

No MeSH data available.


Related in: MedlinePlus

NEP negatively regulates breast cancer invasion. (a) Averages of three independent MCF-7 in vitro invasion assays in the presence or absence of 25 μg/ml thiorphan, a NEP-specific inhibitor, demonstrate increased invasion in the presence of thiorphan (P=0.025). Cell viability is not altered in the presence of thiorphan (Supplementary Figure 3). (b) Transfecting MCF-7 cells with NEP RNAi results in a significant decrease of NEP mRNA as measured by RT–PCR. Averages of semi-quantitative analysis of three independent experiments normalized to β-actin are shown (P=0.02). (c) In vitro invasion assays with MCF-7 cells transfected with control siRNA or NEP siRNA show significantly increased adhesion when NEP is knocked down (P=0.0004 from three independent experiments). (d) Transfecting an expression vector encoding NEP into MDA-MB-231 cells results in increased mRNA expression compared with cells transfected with vector only as demonstrated by RT–PCR (P=0.035). (e) MDA-MB-231 cells transfected with NEP plasmid exhibited significantly less invasion than cells transfected with the control plasmid (P=0.024 from three independent experiments).
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fig2: NEP negatively regulates breast cancer invasion. (a) Averages of three independent MCF-7 in vitro invasion assays in the presence or absence of 25 μg/ml thiorphan, a NEP-specific inhibitor, demonstrate increased invasion in the presence of thiorphan (P=0.025). Cell viability is not altered in the presence of thiorphan (Supplementary Figure 3). (b) Transfecting MCF-7 cells with NEP RNAi results in a significant decrease of NEP mRNA as measured by RT–PCR. Averages of semi-quantitative analysis of three independent experiments normalized to β-actin are shown (P=0.02). (c) In vitro invasion assays with MCF-7 cells transfected with control siRNA or NEP siRNA show significantly increased adhesion when NEP is knocked down (P=0.0004 from three independent experiments). (d) Transfecting an expression vector encoding NEP into MDA-MB-231 cells results in increased mRNA expression compared with cells transfected with vector only as demonstrated by RT–PCR (P=0.035). (e) MDA-MB-231 cells transfected with NEP plasmid exhibited significantly less invasion than cells transfected with the control plasmid (P=0.024 from three independent experiments).

Mentions: After observing decreased NEP expression in breast cancer cells and IDC, we next hypothesized that NEP negatively regulates breast cancer cell invasion. To initially test this, we measured MCF-7 cell invasion in the presence and absence of the NEP inhibitor thiorphan. Inhibiting NEP with thiorphan resulted in approximately a 1.5-fold increase in MCF-7 cell invasion (Figure 2a) but had no effect on cell viability (Supplementary Figure 3), supporting our hypothesis. Thiorphan had no effect on MDA-MB-231 invasion (data not shown), consistent with our observations that these cells express minimal NEP. Next, we transfected MCF-7 cells with siRNA targeting NEP and control siRNA. RT–PCR confirmed the knockdown of NEP mRNA in the transfected MCF-7 cells (Figure 2b), and these cells invaded significantly more than control MCF-7 cells (Figure 2c). Transfecting an expression vector encoding for NEP in MDA-MB-231 cells efficiently increased mRNA expression levels (Figure 2d) and reduced in vitro cell invasion (Figure 2e) compared with cells transfected with the vector alone. Thus, our data suggest that downregulation of NEP in breast cancer cells facilitates invasion.


Epigenetic suppression of neprilysin regulates breast cancer invasion.

Stephen HM, Khoury RJ, Majmudar PR, Blaylock T, Hawkins K, Salama MS, Scott MD, Cosminsky B, Utreja NK, Britt J, Conway RE - Oncogenesis (2016)

NEP negatively regulates breast cancer invasion. (a) Averages of three independent MCF-7 in vitro invasion assays in the presence or absence of 25 μg/ml thiorphan, a NEP-specific inhibitor, demonstrate increased invasion in the presence of thiorphan (P=0.025). Cell viability is not altered in the presence of thiorphan (Supplementary Figure 3). (b) Transfecting MCF-7 cells with NEP RNAi results in a significant decrease of NEP mRNA as measured by RT–PCR. Averages of semi-quantitative analysis of three independent experiments normalized to β-actin are shown (P=0.02). (c) In vitro invasion assays with MCF-7 cells transfected with control siRNA or NEP siRNA show significantly increased adhesion when NEP is knocked down (P=0.0004 from three independent experiments). (d) Transfecting an expression vector encoding NEP into MDA-MB-231 cells results in increased mRNA expression compared with cells transfected with vector only as demonstrated by RT–PCR (P=0.035). (e) MDA-MB-231 cells transfected with NEP plasmid exhibited significantly less invasion than cells transfected with the control plasmid (P=0.024 from three independent experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4815048&req=5

fig2: NEP negatively regulates breast cancer invasion. (a) Averages of three independent MCF-7 in vitro invasion assays in the presence or absence of 25 μg/ml thiorphan, a NEP-specific inhibitor, demonstrate increased invasion in the presence of thiorphan (P=0.025). Cell viability is not altered in the presence of thiorphan (Supplementary Figure 3). (b) Transfecting MCF-7 cells with NEP RNAi results in a significant decrease of NEP mRNA as measured by RT–PCR. Averages of semi-quantitative analysis of three independent experiments normalized to β-actin are shown (P=0.02). (c) In vitro invasion assays with MCF-7 cells transfected with control siRNA or NEP siRNA show significantly increased adhesion when NEP is knocked down (P=0.0004 from three independent experiments). (d) Transfecting an expression vector encoding NEP into MDA-MB-231 cells results in increased mRNA expression compared with cells transfected with vector only as demonstrated by RT–PCR (P=0.035). (e) MDA-MB-231 cells transfected with NEP plasmid exhibited significantly less invasion than cells transfected with the control plasmid (P=0.024 from three independent experiments).
Mentions: After observing decreased NEP expression in breast cancer cells and IDC, we next hypothesized that NEP negatively regulates breast cancer cell invasion. To initially test this, we measured MCF-7 cell invasion in the presence and absence of the NEP inhibitor thiorphan. Inhibiting NEP with thiorphan resulted in approximately a 1.5-fold increase in MCF-7 cell invasion (Figure 2a) but had no effect on cell viability (Supplementary Figure 3), supporting our hypothesis. Thiorphan had no effect on MDA-MB-231 invasion (data not shown), consistent with our observations that these cells express minimal NEP. Next, we transfected MCF-7 cells with siRNA targeting NEP and control siRNA. RT–PCR confirmed the knockdown of NEP mRNA in the transfected MCF-7 cells (Figure 2b), and these cells invaded significantly more than control MCF-7 cells (Figure 2c). Transfecting an expression vector encoding for NEP in MDA-MB-231 cells efficiently increased mRNA expression levels (Figure 2d) and reduced in vitro cell invasion (Figure 2e) compared with cells transfected with the vector alone. Thus, our data suggest that downregulation of NEP in breast cancer cells facilitates invasion.

Bottom Line: RT-PCR and flow cytometry analysis of MDA-MB-231 and MCF-7 breast cancer cell lines revealed decreased neprilysin expression compared with normal epithelial cells.Expression was also suppressed in invasive ductal carcinoma (IDC) compared with normal tissue.These results demonstrate that neprilysin negatively regulates the ET axis in breast cancer, and epigenetic suppression of neprilysin in invasive breast cancer cells enables invasion.

View Article: PubMed Central - PubMed

Affiliation: Lipscomb University, Department of Biology, College of Liberal Arts and Science, 1 University Park Drive, Nashville, TN, USA.

ABSTRACT
In women, invasive breast cancer is the second most common cancer and the second cause of cancer-related death. Therefore, identifying novel regulators of breast cancer invasion could lead to additional biomarkers and therapeutic targets. Neprilysin, a cell-surface enzyme that cleaves and inactivates a number of substrates including endothelin-1 (ET1), has been implicated in breast cancer, but whether neprilysin promotes or inhibits breast cancer cell progression and metastasis is unclear. Here, we asked whether neprilysin expression predicts and functionally regulates breast cancer cell invasion. RT-PCR and flow cytometry analysis of MDA-MB-231 and MCF-7 breast cancer cell lines revealed decreased neprilysin expression compared with normal epithelial cells. Expression was also suppressed in invasive ductal carcinoma (IDC) compared with normal tissue. In addition, in vtro invasion assays demonstrated that neprilysin overexpression decreased breast cancer cell invasion, whereas neprilysin suppression augmented invasion. Furthermore, inhibiting neprilysin in MCF-7 breast cancer cells increased ET1 levels significantly, whereas overexpressing neprilysin decreased extracellular-signal related kinase (ERK) activation, indicating that neprilysin negatively regulates ET1-induced activation of mitogen-activated protein kinase (MAPK) signaling. To determine whether neprilysin was epigenetically suppressed in breast cancer, we performed bisulfite conversion analysis of breast cancer cells and clinical tumor samples. We found that the neprilysin promoter was hypermethylated in breast cancer; chemical reversal of methylation in MDA-MB-231 cells reactivated neprilysin expression and inhibited cancer cell invasion. Analysis of cancer databases revealed that neprilysin methylation significantly associates with survival in stage I IDC and estrogen receptor-negative breast cancer subtypes. These results demonstrate that neprilysin negatively regulates the ET axis in breast cancer, and epigenetic suppression of neprilysin in invasive breast cancer cells enables invasion. Together, this implicates neprilysin as an important regulator of breast cancer invasion and clarifies its utility as a potential biomarker for invasive breast cancer.

No MeSH data available.


Related in: MedlinePlus