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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

Analysis of NEP expression and methylation in cancer databases. (a) NEP expression analysis using human clinical samples of invasive breast cancer available on the MethHC database reveals a statistically significant decrease in NEP in the cancer samples (P<0.005, n=726 cancer samples, n=84 normal samples). (b) Invasive breast cancer data from Cancer Browser was sorted for HER2-positive cancers, and NEP expression was analyzed by stage (n=67; P=0.01 for I vs II and P=0.03 for I vs III). (c) Kaplan–Meier plot using Kaplan–Meier Plotter of overall survival of invasive breast cancer samples with high (n=397) and low (n=462) NEP expression (P=0.091). (d) Analysis of human breast cancer from the MethHC database shows a significant difference between NEP methylation in normal breast tissue (n=738) and breast cancer (n=90). (e) Kaplan–Meier plot generated using Cancer Browser DNA methylation and clinical data shows survival differences between stage I IDC samples with high (n=10) and low (n=25) NEP promoter methylation (P=0.005). (f) Kaplan–Meier plot using Cancer Browser DNA methylation and clinical data of IDC samples of ER-negative samples shows significantly improved survival in patients with low NEP promoter methylation (n=11) compared with high NEP methylation (n=95) (P=0.0175).
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fig6: Analysis of NEP expression and methylation in cancer databases. (a) NEP expression analysis using human clinical samples of invasive breast cancer available on the MethHC database reveals a statistically significant decrease in NEP in the cancer samples (P<0.005, n=726 cancer samples, n=84 normal samples). (b) Invasive breast cancer data from Cancer Browser was sorted for HER2-positive cancers, and NEP expression was analyzed by stage (n=67; P=0.01 for I vs II and P=0.03 for I vs III). (c) Kaplan–Meier plot using Kaplan–Meier Plotter of overall survival of invasive breast cancer samples with high (n=397) and low (n=462) NEP expression (P=0.091). (d) Analysis of human breast cancer from the MethHC database shows a significant difference between NEP methylation in normal breast tissue (n=738) and breast cancer (n=90). (e) Kaplan–Meier plot generated using Cancer Browser DNA methylation and clinical data shows survival differences between stage I IDC samples with high (n=10) and low (n=25) NEP promoter methylation (P=0.005). (f) Kaplan–Meier plot using Cancer Browser DNA methylation and clinical data of IDC samples of ER-negative samples shows significantly improved survival in patients with low NEP promoter methylation (n=11) compared with high NEP methylation (n=95) (P=0.0175).

Mentions: Next, we analyzed invasive breast cancer using cancer genome databases MethHC41 and Cancer Browser.42 Comparing expression of the NEP promoter in breast cancer and normal tissue across a large number of invasive breast cancer samples (n=726) and normal controls (n=84) through the MethHC database revealed a statistically significant decrease in NEP mRNA expression in cancer samples (Figure 6a). To determine whether NEP expression differed within tumor samples, we grouped samples by varying characteristics (ER/PR/HER2 expression and clinical subtype) and analyzed NEP expression levels across cancer stages using RNASeq data from the Cancer Browser database.42 We observed that NEP mRNA was significantly higher in stage I HER2-positive IDC than either stage II or stage III cancers (Figure 6b), but no significant differences were seen in other subgroups. We next asked whether NEP expression associated with survival by analyzing survival between high and low NEP-expressing IDC patients using the Cancer Browser database.42 No significant difference was observed between these groups (Figure 6c; Table 1). We further sorted cohorts on the basis of ER expression, human epidermal growth factor receptor 2 (HER2), lymph node status, molecular subtype and cancer stage. Unexpectedly, we observed a statistically significant decrease in survival of high NEP-expressing patients with stage I and stage III IDC, but when the cohort was restricted to include only samples with at least 65% tumor cells, thus limiting the stromal contribution, this effect was lost (Table 1).


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)

Analysis of NEP expression and methylation in cancer databases. (a) NEP expression analysis using human clinical samples of invasive breast cancer available on the MethHC database reveals a statistically significant decrease in NEP in the cancer samples (P<0.005, n=726 cancer samples, n=84 normal samples). (b) Invasive breast cancer data from Cancer Browser was sorted for HER2-positive cancers, and NEP expression was analyzed by stage (n=67; P=0.01 for I vs II and P=0.03 for I vs III). (c) Kaplan–Meier plot using Kaplan–Meier Plotter of overall survival of invasive breast cancer samples with high (n=397) and low (n=462) NEP expression (P=0.091). (d) Analysis of human breast cancer from the MethHC database shows a significant difference between NEP methylation in normal breast tissue (n=738) and breast cancer (n=90). (e) Kaplan–Meier plot generated using Cancer Browser DNA methylation and clinical data shows survival differences between stage I IDC samples with high (n=10) and low (n=25) NEP promoter methylation (P=0.005). (f) Kaplan–Meier plot using Cancer Browser DNA methylation and clinical data of IDC samples of ER-negative samples shows significantly improved survival in patients with low NEP promoter methylation (n=11) compared with high NEP methylation (n=95) (P=0.0175).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Analysis of NEP expression and methylation in cancer databases. (a) NEP expression analysis using human clinical samples of invasive breast cancer available on the MethHC database reveals a statistically significant decrease in NEP in the cancer samples (P<0.005, n=726 cancer samples, n=84 normal samples). (b) Invasive breast cancer data from Cancer Browser was sorted for HER2-positive cancers, and NEP expression was analyzed by stage (n=67; P=0.01 for I vs II and P=0.03 for I vs III). (c) Kaplan–Meier plot using Kaplan–Meier Plotter of overall survival of invasive breast cancer samples with high (n=397) and low (n=462) NEP expression (P=0.091). (d) Analysis of human breast cancer from the MethHC database shows a significant difference between NEP methylation in normal breast tissue (n=738) and breast cancer (n=90). (e) Kaplan–Meier plot generated using Cancer Browser DNA methylation and clinical data shows survival differences between stage I IDC samples with high (n=10) and low (n=25) NEP promoter methylation (P=0.005). (f) Kaplan–Meier plot using Cancer Browser DNA methylation and clinical data of IDC samples of ER-negative samples shows significantly improved survival in patients with low NEP promoter methylation (n=11) compared with high NEP methylation (n=95) (P=0.0175).
Mentions: Next, we analyzed invasive breast cancer using cancer genome databases MethHC41 and Cancer Browser.42 Comparing expression of the NEP promoter in breast cancer and normal tissue across a large number of invasive breast cancer samples (n=726) and normal controls (n=84) through the MethHC database revealed a statistically significant decrease in NEP mRNA expression in cancer samples (Figure 6a). To determine whether NEP expression differed within tumor samples, we grouped samples by varying characteristics (ER/PR/HER2 expression and clinical subtype) and analyzed NEP expression levels across cancer stages using RNASeq data from the Cancer Browser database.42 We observed that NEP mRNA was significantly higher in stage I HER2-positive IDC than either stage II or stage III cancers (Figure 6b), but no significant differences were seen in other subgroups. We next asked whether NEP expression associated with survival by analyzing survival between high and low NEP-expressing IDC patients using the Cancer Browser database.42 No significant difference was observed between these groups (Figure 6c; Table 1). We further sorted cohorts on the basis of ER expression, human epidermal growth factor receptor 2 (HER2), lymph node status, molecular subtype and cancer stage. Unexpectedly, we observed a statistically significant decrease in survival of high NEP-expressing patients with stage I and stage III IDC, but when the cohort was restricted to include only samples with at least 65% tumor cells, thus limiting the stromal contribution, this effect was lost (Table 1).

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