Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma-An In Vitro and a Clinical Study

Abstract

Alpha-enolase (ENO1) is a glycolytic metalloenzyme, and its overexpression occurs in numerous cancers, contributing to cancer cell survival, proliferation, and maintenance of the Warburg effect. Patients with an overexpression of ENO1 have a poor prognosis. The aim of the present study was to investigate the prognostic significance of ENO1 in surgical resections from 112 melanoma patients and to assess its expression and enzymatic activity in normoxia and hypoxia in several melanoma cell lines. Overexpression of ENO1 in tumor cells from patients was correlated with unfavorable prognosticators such as Breslow thickness, Clark level, mitotic activity, and the presence of ulceration. The expression of ENO1 also positively correlated with a greater thickness of the neoplastic infiltrate and a worse long-term prognosis for patients with cutaneous melanoma. We report significantly higher expression of ENO1 in melanoma cell lines in comparison to normal melanocytes. To conclude, our in vitro and clinical models showed that overexpression of ENO1 promotes invasiveness of melanoma cells and correlates with aggressive clinical behavior. These observations open the way to further search of a potential prognostic and therapeutic target in cutaneous melanoma.

Keywords: ENO1; cutaneous melanoma; immunohistochemistry; melanoma cell lines.

Figure 1

The expression level of ENO1 in the cell lysates from primary melanocytes and melanoma cell lines. Representative Western blots showing ENO1 and Akt 1/2/3 expression (for normalization) in protein lysates obtained from the primary human melanocytes (HEM) and indicated melanoma cell lines (a). Densitometric ENO1/Akt ratios are shown as mean values (n = 3 except for HEM, n = 2) ± standard error of the mean (SEM) (b). The significance level was set at p = 0.001–0.0001 (***).

Figure 2

Enolase expression in melanoma cell lines determined by indirect immunofluorescence and confocal microscopy. (a) Single optical sections showing cells stained for ENO1 (green) and DAPI (blue). In the second column ENO1 signal was enhanced by brightness adjustment for the sake of better visualization. Raw images (shown in the last column) were subjected to fluorescence signal intensity analysis. Bar—15 μm. (b) Fluorescence signal intensity of the ENO1 presented as a mean ± standard deviation. The significance level was set at p = 0.05–0.01 (*). Subsequent number of cells were analyzed: A375—40 cells, WM1341D—71 cells, WM9—25 cells, Hs294T—45 cells.

Figure 3

The enolase activity in protein lysates prepared from A375, Hs294T, WM1341D, and WM9 melanoma cells cultured under normoxia or hypoxia. Bars represent mean values (n = 4) ± standard error of the mean (SEM). The significance level was set at p = 0.05–0.01 (*), p = 0.01–0.001 (**).

Figure 4

Representative results of immunohistochemical analysis of ENO1 expression in cutaneous melanoma patients. Low cytoplasmic ENO1 immunoreactivity in melanoma cells ((a), 200×; (b), 400×). High expression of ENO1 in tumoral cells ((c), 200×; (d), 400×).

Figure 5

ENO1 expression and AJCC (American Joint Committee on Cancer) staging. The lowest ENO1 expression in tumoral cells was observed in patients with stage I cutaneous melanoma. In stages II-IV, it was observed a significant increasing of ENO1 immunoreactivity in neoplastic cells.

Figure 6

Kaplan–Meier analysis of the prognostic significance of ENO1 expression in cutaneous melanoma patients. Overexpression of ENO1 correlated with shorter cancer-specific overall survival (a) and shorter disease-free survival (b). p levels of the log-rank test.

Figure 7

Multivariable regression model for disease-free survival and cancer-specific overall survival in cutaneous melanoma patients (DFS (disease-free survival), CSOS (cancer-specific overall survival).