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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 hypermethylation and decreased expression in IDC samples. (a) DNA isolated from human IDC tumor samples was subjected to BSC and PCR amplification using methylated specific (‘M') and unmethylated specific (‘U') primers from the NEP promoter. Samples are grouped by lymph node metastasis status; the third N0/N1 sample was run on the same gel as the other samples, but was rearranged for organizational purposes. The normal samples were run on a separate gel but processed and analyzed with the cancer samples. The samples on the right show BSC analysis of matched Cancer (‘Ca') and Uninvolved (‘Un') tissue. Gels are cropped to the appropriate molecular weight. (b) Methylation index analysis from quantitative BSC–PCR reveals a non-significant trend of increased methylation in cancer samples compared with normal/uninvolved tissue (n=6 for each; P=0.09). (c) Methylation index from quantitative BSC–PCR analysis reveals that metastatic tumor samples (scored as N2/N3 by lymph node analysis; n=3) have significantly higher NEP promoter methylation than normal breast tissue (n=3; P=0.04), uninvolved breast tissue (n=3; P=0.039), and cancer tissue with little or no metastasis (scored as N0/N1; n=3, P=0.039).
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fig5: NEP hypermethylation and decreased expression in IDC samples. (a) DNA isolated from human IDC tumor samples was subjected to BSC and PCR amplification using methylated specific (‘M') and unmethylated specific (‘U') primers from the NEP promoter. Samples are grouped by lymph node metastasis status; the third N0/N1 sample was run on the same gel as the other samples, but was rearranged for organizational purposes. The normal samples were run on a separate gel but processed and analyzed with the cancer samples. The samples on the right show BSC analysis of matched Cancer (‘Ca') and Uninvolved (‘Un') tissue. Gels are cropped to the appropriate molecular weight. (b) Methylation index analysis from quantitative BSC–PCR reveals a non-significant trend of increased methylation in cancer samples compared with normal/uninvolved tissue (n=6 for each; P=0.09). (c) Methylation index from quantitative BSC–PCR analysis reveals that metastatic tumor samples (scored as N2/N3 by lymph node analysis; n=3) have significantly higher NEP promoter methylation than normal breast tissue (n=3; P=0.04), uninvolved breast tissue (n=3; P=0.039), and cancer tissue with little or no metastasis (scored as N0/N1; n=3, P=0.039).

Mentions: To determine whether our observations in cultured breast cancer cell lines are representative of clinical findings, we analyzed primary IDC samples (n=6), along with normal and uninvolved breast tissue (n=6), from the Cooperative Human Tissue Network (CHTN). We performed BSC–PCR analysis of DNA extracted from these samples and observed increased amplification with the methylation-specific primers in the metastatic breast cancers (N2/3) compared with the poorly metastatic IDC (N0/1) and normal controls (Figure 5a). Quantitative BSC analysis revealed that there was a non-significant trend of increased NEP methylation in cancer samples compared with controls (Figure 5b). Interestingly, we observed a significant increase in methylation in highly metastatic IDC samples when tumors were grouped by lymph node metastasis. Tumors given a lymph node metastasis score (‘N') of 2–3 had an eightfold increase in methylation index compared with normal tissue, uninvolved tissue or cancer tissue with an N score of 0–1 (Figure 5c). Thus, methylation of the NEP promoter is associated with increased IDC metastasis in the samples we tested.


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 hypermethylation and decreased expression in IDC samples. (a) DNA isolated from human IDC tumor samples was subjected to BSC and PCR amplification using methylated specific (‘M') and unmethylated specific (‘U') primers from the NEP promoter. Samples are grouped by lymph node metastasis status; the third N0/N1 sample was run on the same gel as the other samples, but was rearranged for organizational purposes. The normal samples were run on a separate gel but processed and analyzed with the cancer samples. The samples on the right show BSC analysis of matched Cancer (‘Ca') and Uninvolved (‘Un') tissue. Gels are cropped to the appropriate molecular weight. (b) Methylation index analysis from quantitative BSC–PCR reveals a non-significant trend of increased methylation in cancer samples compared with normal/uninvolved tissue (n=6 for each; P=0.09). (c) Methylation index from quantitative BSC–PCR analysis reveals that metastatic tumor samples (scored as N2/N3 by lymph node analysis; n=3) have significantly higher NEP promoter methylation than normal breast tissue (n=3; P=0.04), uninvolved breast tissue (n=3; P=0.039), and cancer tissue with little or no metastasis (scored as N0/N1; n=3, P=0.039).
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fig5: NEP hypermethylation and decreased expression in IDC samples. (a) DNA isolated from human IDC tumor samples was subjected to BSC and PCR amplification using methylated specific (‘M') and unmethylated specific (‘U') primers from the NEP promoter. Samples are grouped by lymph node metastasis status; the third N0/N1 sample was run on the same gel as the other samples, but was rearranged for organizational purposes. The normal samples were run on a separate gel but processed and analyzed with the cancer samples. The samples on the right show BSC analysis of matched Cancer (‘Ca') and Uninvolved (‘Un') tissue. Gels are cropped to the appropriate molecular weight. (b) Methylation index analysis from quantitative BSC–PCR reveals a non-significant trend of increased methylation in cancer samples compared with normal/uninvolved tissue (n=6 for each; P=0.09). (c) Methylation index from quantitative BSC–PCR analysis reveals that metastatic tumor samples (scored as N2/N3 by lymph node analysis; n=3) have significantly higher NEP promoter methylation than normal breast tissue (n=3; P=0.04), uninvolved breast tissue (n=3; P=0.039), and cancer tissue with little or no metastasis (scored as N0/N1; n=3, P=0.039).
Mentions: To determine whether our observations in cultured breast cancer cell lines are representative of clinical findings, we analyzed primary IDC samples (n=6), along with normal and uninvolved breast tissue (n=6), from the Cooperative Human Tissue Network (CHTN). We performed BSC–PCR analysis of DNA extracted from these samples and observed increased amplification with the methylation-specific primers in the metastatic breast cancers (N2/3) compared with the poorly metastatic IDC (N0/1) and normal controls (Figure 5a). Quantitative BSC analysis revealed that there was a non-significant trend of increased NEP methylation in cancer samples compared with controls (Figure 5b). Interestingly, we observed a significant increase in methylation in highly metastatic IDC samples when tumors were grouped by lymph node metastasis. Tumors given a lymph node metastasis score (‘N') of 2–3 had an eightfold increase in methylation index compared with normal tissue, uninvolved tissue or cancer tissue with an N score of 0–1 (Figure 5c). Thus, methylation of the NEP promoter is associated with increased IDC metastasis in the samples we tested.

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