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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 promoter hypermethylation in breast cancer cells regulates expression and invasion. (a) Quantitative BSC–PCR from HMEC, MCF-7 and MDA-MB-231 cells shows significantly increased methylation in MDA-MB-231 lines compared with MCF-7 cells (P=0.038) or normal HMEC breast cells (P=0.033) over three independent experiments. Representative Ct graphs and primer melt curves are shown in Supplementary Figure 5. Right, representative gel from end point BSC–PCR; U=product amplified with unmethylated specific NEP primers; M=product amplified with methylated specific NEP primers. Gels are cropped to the appropriate molecular weight. (b) RT–PCR of cDNA from MDA-MB-231 cells treated with AZA shows increased levels of NEP mRNA after AZA treatment (P=0.012 over three independent experiments). (c) In vitro invasion assays with MDA-MB-231 cells treated with AZA or vehicle control show decreased invasion with AZA treatment (P=0.0045 over three independent experiments).
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fig4: NEP promoter hypermethylation in breast cancer cells regulates expression and invasion. (a) Quantitative BSC–PCR from HMEC, MCF-7 and MDA-MB-231 cells shows significantly increased methylation in MDA-MB-231 lines compared with MCF-7 cells (P=0.038) or normal HMEC breast cells (P=0.033) over three independent experiments. Representative Ct graphs and primer melt curves are shown in Supplementary Figure 5. Right, representative gel from end point BSC–PCR; U=product amplified with unmethylated specific NEP primers; M=product amplified with methylated specific NEP primers. Gels are cropped to the appropriate molecular weight. (b) RT–PCR of cDNA from MDA-MB-231 cells treated with AZA shows increased levels of NEP mRNA after AZA treatment (P=0.012 over three independent experiments). (c) In vitro invasion assays with MDA-MB-231 cells treated with AZA or vehicle control show decreased invasion with AZA treatment (P=0.0045 over three independent experiments).

Mentions: We next wished to investigate the mechanism of NEP downregulation in breast cancer cells. Hypermethylation of the NEP promoter has been observed in lymphoid malignancies37, 38, 39 and prostate cancer;40 we therefore asked whether methylation of the NEP promoter is increased in cancerous cells compared with primary mammary cells. Quantitative bisulfite conversion–PCR (BSC–PCR) analysis of DNA extracted from breast cancer cells with NEP methylation-specific and control beta-actin primers demonstrated that when normalized to control methylated DNA, MDA-MB-231 DNA has significantly higher methylation at the NEP promoter than either MCF-7 or HMEC DNA (Figure 4a; Supplementary Figure 5). A representative gel from end point BSC–PCR depicts the increased methylation in MDA-MB-231 DNA (Figure 4a, right). Treating the highly invasive MDA-MB-231 cells for 5 days with 5-azacytidine (AZA), a DNA methyltransferase inhibitor, resulted in increased NEP mRNA (Figure 4b) and decreased cell invasion compared with vehicle controls (Figure 4c). These results suggest that hypermethylation of the NEP promoter in breast cancer cells facilitates invasion and may represent a novel therapeutic target in breast cancer.


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 promoter hypermethylation in breast cancer cells regulates expression and invasion. (a) Quantitative BSC–PCR from HMEC, MCF-7 and MDA-MB-231 cells shows significantly increased methylation in MDA-MB-231 lines compared with MCF-7 cells (P=0.038) or normal HMEC breast cells (P=0.033) over three independent experiments. Representative Ct graphs and primer melt curves are shown in Supplementary Figure 5. Right, representative gel from end point BSC–PCR; U=product amplified with unmethylated specific NEP primers; M=product amplified with methylated specific NEP primers. Gels are cropped to the appropriate molecular weight. (b) RT–PCR of cDNA from MDA-MB-231 cells treated with AZA shows increased levels of NEP mRNA after AZA treatment (P=0.012 over three independent experiments). (c) In vitro invasion assays with MDA-MB-231 cells treated with AZA or vehicle control show decreased invasion with AZA treatment (P=0.0045 over three independent experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: NEP promoter hypermethylation in breast cancer cells regulates expression and invasion. (a) Quantitative BSC–PCR from HMEC, MCF-7 and MDA-MB-231 cells shows significantly increased methylation in MDA-MB-231 lines compared with MCF-7 cells (P=0.038) or normal HMEC breast cells (P=0.033) over three independent experiments. Representative Ct graphs and primer melt curves are shown in Supplementary Figure 5. Right, representative gel from end point BSC–PCR; U=product amplified with unmethylated specific NEP primers; M=product amplified with methylated specific NEP primers. Gels are cropped to the appropriate molecular weight. (b) RT–PCR of cDNA from MDA-MB-231 cells treated with AZA shows increased levels of NEP mRNA after AZA treatment (P=0.012 over three independent experiments). (c) In vitro invasion assays with MDA-MB-231 cells treated with AZA or vehicle control show decreased invasion with AZA treatment (P=0.0045 over three independent experiments).
Mentions: We next wished to investigate the mechanism of NEP downregulation in breast cancer cells. Hypermethylation of the NEP promoter has been observed in lymphoid malignancies37, 38, 39 and prostate cancer;40 we therefore asked whether methylation of the NEP promoter is increased in cancerous cells compared with primary mammary cells. Quantitative bisulfite conversion–PCR (BSC–PCR) analysis of DNA extracted from breast cancer cells with NEP methylation-specific and control beta-actin primers demonstrated that when normalized to control methylated DNA, MDA-MB-231 DNA has significantly higher methylation at the NEP promoter than either MCF-7 or HMEC DNA (Figure 4a; Supplementary Figure 5). A representative gel from end point BSC–PCR depicts the increased methylation in MDA-MB-231 DNA (Figure 4a, right). Treating the highly invasive MDA-MB-231 cells for 5 days with 5-azacytidine (AZA), a DNA methyltransferase inhibitor, resulted in increased NEP mRNA (Figure 4b) and decreased cell invasion compared with vehicle controls (Figure 4c). These results suggest that hypermethylation of the NEP promoter in breast cancer cells facilitates invasion and may represent a novel therapeutic target in breast cancer.

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