Melanocytes Affect Nodal Expression and Signaling in Melanoma Cells: A Lesson from Pediatric Large Congenital Melanocytic Nevi

Abstract

Expression of Nodal, a Transforming Growth Factor-beta (TGF-β) related growth factor, is associated with aggressive melanoma. Nodal expression in adult dysplastic nevi may predict the development of aggressive melanoma in some patients. A subset of pediatric patients diagnosed with giant or large congenital melanocytic nevi (LCMN) has shown increased risk for development of melanoma. Here, we investigate whether Nodal expression can help identify the rare cases of LCMN that develop melanoma and shed light on why the majority of these patients do not. Immunohistochemistry (IHC) staining results show varying degree of Nodal expression in pediatric dysplastic nevi and LCMN. Moreover, median scores from Nodal IHC expression analysis were not significantly different between these two groups. Additionally, none of the LCMN patients in this study developed melanoma, regardless of Nodal IHC levels. Co-culture experiments revealed reduced tumor growth and lower levels of Nodal and its signaling molecules P-SMAD2 and P-ERK1/2 when melanoma cells were grown in vivo or in vitro with normal melanocytes. The same was observed in melanoma cells cultured with melanocyte conditioned media containing pigmented melanocyte derived melanosomes (MDM). Since MDM contain molecules capable of inactivating radical oxygen species, to investigate potential anti-oxidant effect of MDM on Nodal expression and signaling in melanoma, melanoma cells were treated with either N-acetyl-l-cysteine (NAC), a component of the anti-oxidant glutathione or synthetic melanin, which in addition to providing pigmentation can also exert free radical scavenging activity. Melanoma cells treated with NAC or synthetic melanin showed reduced levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated melanoma cells. Thus, the potential role for Nodal in melanoma development in LCMN is less evident than in adult dysplastic nevi possibly due to melanocyte cross-talk in LCMN capable of offsetting or delaying the pro-melanoma effects of Nodal via anti-oxidant effects of MDM.

Keywords: Nodal; anti-oxidants; melanocytes; melanoma; melanosomes; pediatric nevi.

Figure 1

Immunohistochemistry results for Nodal staining. (A) Representative results show immunohistochemistry staining (brown) for Nodal in dysplastic nevi and LCMN from pediatric patients (20× original magnification); (B) Box plot representation shows that median staining scores for Nodal were similar between the two different groups analyzed.

Figure 2

Growth of C8161 cells and HeMn melanocytes in Nude mice. At the end of the observation period, final tumor volume formed from the initial 250,000 human melanoma C8161 cells was significantly reduced when grown in close proximity to 250,000 HeMn melanocytes, compared to the final tumor volume of 250,000 C8161 allowed to grow alone (* p < 0.001).

Figure 3

Co-culture (CoC) experiments. (A) Schematic diagram summarizing the co-culture experiments using HeMn melanocytes and C8161 melanoma cells. Lysates of cells growing on the bottom well (red square) were used for Western blotting; (B) Western blot results show increased expression of P-SMAD2 and P-ERK1/2 in HeMn melanocytes co-cultured with human C8161 melanoma cells compared to control HeMn cells culured alone. However, Nodal expression was not detected in HeMn cells grown alone or in co-culture with C8161 melanoma cells; and (C) Results from WB analysis show reduced levels of Nodal, P-SMAD2 and P-ERK1/2 in C8161 melanoma cells when co-cultured with HeMn melanocytes. Numbers above WB bands represent densitometric units, normalized to actin loading control, total SMAD2/3 or total ERK1/2, as appropriate, relative to control.

Figure 4

Effect of treatment of C8161 melanoma cells with HeMn melanocyte conditioned medium (HeMn-CM). (A) C8161 cells treated with HeMn-CM show morphologic changes such as dendritic shape with long cytoplasmic extensions reminiscent of melanocyte differentiation. (40× original magnification); (B) Although, Melanoma Differentiation Associated protein 7 (MDA-7) was detected in C8161 cells treated with HeMn-CM, the melanin synthesis associated enzyme tyrosinase was not; (C) C8161 cells treated with HeMn-CM showed similar proliferation rate and cell viability as non-treated control C8161 cells making potential toxic effect of HeMn-CM on C8161 unlikely; (D) Western blot analysis show that HeMn-CM treated C8161 express lower levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated control C8161 cells. Numbers below WB bands represent densitometric units, normalized to actin loading control, total SMAD2/3 or total ERK1/2, as appropriate, relative to control.

Figure 5

HeMn melanocyte derived melanosomes (MDM) affect Nodal expression and signaling components in C8161. (A) Microphotograph (40× original magnification) shows MDM containing globules (red arrows) produced by HeMn melanocytes; (B) C8161 melanoma cells treated with different concentrations of MDM collected from HeMn melanocytes showed reduction in Nodal expression compared to untreated control C8161 cells. To mimic the anti-oxidant effect of free radical scavengers contained in MDM, C8161 cells were treated with N-acetyl-l-cysteine (NAC) known to generate the anti-oxidant, glutathione; (C) Western Blot analysis show that treatment of C8161 melanoma cells with 5 mM NAC resulted in reduced levels of Nodal, P-SMAD and P-ERK1/2 compared to untreated control C8161 cells; (D) Treatment of C8161 cells with synthetic melanin, also known to have anti-oxidant properties, resulted in a dose-dependent reduction of Nodal, P-SMAD and P-ERK1/2 levels compared to untreated control C8161 cells. Numbers above WB bands represent densitometric units, normalized to actin loading control, total SMAD2/3 or total ERK1/2, as appropriate, relative to control.