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.
Highlights
Neprilysin/neutral endopeptidase 24.11 (NEP), known as membrane metallo-endopeptidase, CD10 and common acute lymphoblastic leukemia antigen,[1] is a 95–100 kDa cell-surface endopeptidase that cleaves and inactivates numerous peptide substrates at hydrophobic amino acids
We found that NEP mRNA is significantly decreased in both cancer cell lines compared with the normal cells (Figure 1a)
Lower in the breast cancer cell lines, with the invasive MDA-MB-231 cells expressing the lowest levels of NEP protein (Figure 1b)
Summary
Neprilysin/neutral endopeptidase 24.11 (NEP), known as membrane metallo-endopeptidase, CD10 and common acute lymphoblastic leukemia antigen,[1] is a 95–100 kDa cell-surface endopeptidase that cleaves and inactivates numerous peptide substrates at hydrophobic amino acids. ET1 activates the mitogen-activated protein kinase (MAPK) pathway through ET receptor signaling and subsequently modulates cell survival, proliferation, invasion and angiogenesis (reviewed by Rosano et al.6) and is implicated in numerous cancers. Despite its apparent functional inhibition of ET signaling by ET1 inactivation, clinical and experimental data of NEP in various cancers are conflicting. Several reports suggest a protective role for NEP in solid cancers, including breast cancer,[7,8,9,10,11,12] studies in a number of solid cancers including colorectal, head and neck squamous cell carcinoma, lung cancer and breast cancer, implicate NEP as a possible marker for tumor progression and metastasis.[13,14,15,16,17,18,19,20,21,22,23,24,25] Despite the abundant literature detailing the contribution of the ET axis to breast cancer and NEP’s ability to regulate ET signaling,[26,27,28,29,30,31,32] the specific function of NEP and its mechanism in breast cancer invasion remains unclear
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