Nectin like-5 overexpression correlates with the malignant phenotype in cutaneous melanoma

Abstract

NECL-5 is involved in regulating cell-cell junctions, in cooperation with cadherins, integrins and platelet-derived growth factor receptor, that are essential for intercellular communication. Its role in malignant transformation was previously described. It has been reported that transformation of melanocytes is associated with altered expression of adhesion molecules suggesting the potential involment of NECL-5 in melanoma development and prognosis. To shed light on this issue, the expression and the role of NECL-5 in melanoma tissues was investigated by bioinformatic and molecular approaches. NECL-5 was up-regulated both at the mRNA and the protein levels in WM35, M14 and A375 cell lines compared with normal melanocytes. A subsequent analysis in primary and metastatic melanoma specimens confirmed "in vitro" findings. NECL-5 overexpression was observed in 53 of 59 (89.8%) and 12 of 12 (100%), primary melanoma and melanoma metastasis, respectively; while, low expression of NECL-5 was detected in 12 of 20 (60%) benign nevi. A significant correlation of NECL-5 overexpression was observed with most of known negative melanoma prognostic factors, including lymph-node involvement (P = 0.009) and thickness (P = 0.004). Intriguingly, by analyzing the large series of melanoma samples in the Xu dataset, we identified the transcription factor YY1 among genes positively correlated with NECL-5 (r = 0.5). The concordant computational and experimental data of the present study indicate that the extent of NECL-5 expression correlates with melanoma progression.

Figure 1. Expression analyses of NECL-5 in WM35, M14 and A375 cell lines

1A) Western blot in WM35, M14 and A375 cell lines, compared with NHEM. The upper bands represent NECL-5 protein, and the lower bands are β-Actin used as an internal control. The intensities of these bands were quantified by the Kodak 1D image analysis software. 1B) mRNA expression of NECL-5 gene by RT-PCR using G6PD for normalization. 1C) Flow cytometry analysis of NECL-5 expression. Histograms compare staining with specific antibody (black line) with the appropriate isotype control antibody (gray line).

Figure 2. (A) siRNA of NECL-5 gene in both A375 and M14 cells was used to understand the effect on cell migration

(B) siRNA of NECL-5 gene resulted in a significant decreased in transwell migration compared to control cells.

Figure 3. Immunostaining using a specific anti-NECL-5 antibody in a representative fraction of human melanocytic lesions of benign nevi, primary and metastatic melanoma

Immunohistochemical analysis of NECL-5 in normal skin, benign nevi and melanoma tissues. a: Normal skin tissue shows no immunostaining for NECL-5 (bar: 35 μm; inset, 15 μm). b: Skin dermis of benign nevus tissue is weakly labelled for NECL-5 (bar: 32 μm). c: In primary melanoma lesion, immunolabelled melanocytes are immersed in a matrix also immunostained for NECL-5 (bar: 50 μm). d: Dermis and melanocytes of metastatic melanoma skin tissue are strongly labelled for NECL-5 molecule (bar: 50 μm). e: thickness ≤ 1 mm; f: thickness > 1 mm; NECL-5 expression is higher in melanoma sections with thickness > 1 mm in comparison with those with thickness ≤ 1 mm (bar: 50 μm).

Figure 4. Correlation of YY1 with NECL-5 in melanoma

Heat map of genes positively correlated with NECL-5 (r = 0.5) by Pearson correlation analysis in the Xu melanoma dataset (A). Immunohistochemistry evaluation of NECL-5 and YY1 in a representative case of melanoma respectively in Panel (a) and (b) (B).