Clinical relevance of neutral endopeptidase (NEP/CD10) in melanoma

Abstract

Background: Overexpression of Neutral Endopeptidase (NEP) has been reported in metastatic carcinomas, implicating NEP in tumor progression and suggesting a role for NEP inhibitors in its treatment. We investigated the role of NEP expression in the clinical progression of cutaneous melanoma.

Methods: We screened 7 melanoma cell lines for NEP protein expression. NEP-specific siRNA was transfected into the lines to examine the role of gene transcription in NEP expression. Immunohistochemistry was done for 93 specimens and correlated with clinicopathologic parameters. Thirty-seven metastatic melanoma specimens were examined for NEP transcript expression using Affymetrix GeneChips. In a subset of 25 specimens for which both transcript and protein expression was available, expression ratios were used to identify genes that co-express with NEP in GeneChip analysis.

Results: NEP was overexpressed in 4/7 human melanoma cell lines, and siRNA knock-down of NEP transcripts led to downregulation of its protein expression. NEP protein overexpression was significantly more common in metastatic versus primary tumors (P = 0.002). Twelve of 37 (32%) metastatic tumors had increased NEP transcript expression, and an association was observed between NEP transcript upregulation and protein overexpression (P < 0.0001). Thirty-eight genes were found to significantly co-express with NEP (p < 0.005). Thirty-three genes positively correlated with NEP, including genes involved in the MAP kinase pathway, antigen processing and presentation, apoptosis, and WNT signaling pathway, and 5 genes negatively correlated with NEP, including genes of focal adhesion and the notch signaling pathways.

Conclusion: NEP overexpression, which seems to be largely driven by increased transcription, is rare in primary melanoma and occurs late in melanoma progression. Functional studies are needed to better understand the mechanisms of NEP regulation in melanoma.

Figure 1

NEP transcript downregulation in melanoma cell lines. Melanoma SK-MEL 19 (1a) and MeWo (1b) cell lines were transfected with 10 ug of control siRNA (left column) and NEP siRNA (right column) in one million cells. The cells were harvested 48 hours post-transfection. Following siRNA transfection, there was decreased expression of NEP in both cell lines. RAN served as a control.

Figure 2

Immunohistochemical detection of NEP expression in melanoma. Metastatic melanoma showing focal expression of NEP (red stain) (20×).

Figure 3

NEP expression in melanoma by immunohistochemistry. Metastatic melanoma showing diffuse NEP expression (red stain) (20×).

Figure 4

NEP transcript expression analysis. Results of gene transcript expression analysis of NEP in 37 metastatic melanoma samples, expressed as a ratio of NEP expression in normal lymph node tissue. Twelve of 37 samples show at least 2-fold increased gene expression.