New panel of biomarkers to discriminate between amelanotic and melanotic metastatic melanoma

Abstract

Melanoma is a form of skin cancer that can rapidly invade distant organs. A distinctive feature of melanomas is their pigmentation status, as melanin is present in most skin melanomas, whilst many metastatic tumors could become amelanotic. Besides the obvious malfunction of the key genes of the melanin pathway, the amelanotic tumors could bear a characteristic molecular signature accounting for their aggressivity. Using mass spectrometry-based proteomics we report here a distinctive panel of biomarkers for amelanotic aggressive melanoma that differ from the less invasive pigmented cells. The developed method allows the label-free quantification of proteins identified by LC-MS/MS analysis. We found a set of proteins comprising AHNAK, MYOF, ANXA1, CAPN2, ASPH, EPHA2, THBS1, TGM2, ACTN4 along with proteins involved in cell adhesion/migration (integrins, PLEC, FSCN1, FN1) that are highly expressed in amelanotic melanoma. Accompanying the down regulation of pigmentation specific proteins such as tyrosinase and TYRP1, these biomarkers are highly specific for a type of highly invasive melanoma. Interestingly, the LC-MS/MS proteomics analysis in hypoxia revealed that the abundance of this specific set of proteins found in normoxia was rather unaltered in these conditions. These biomarkers could therefore predict a metastatic behaviour for the amelanotic cells in the early stages of the tumor development and thus serve in melanoma prognostic. Applying this algorithm to related databases including melanoma samples published by independent laboratories/public databases we confirm the specificity of the newly found signatures. Overall, we begin to unravel the molecular alterations in the amelanotic melanoma and how basic proteomics offers insights into how to assess the clinical, pathological and misdiagnosis differences between the main subtypes of melanoma.

Keywords: amelanotic melanoma; mass spectrometry; melanoma biomarkers; melanoma diagnostic; melanoma prognostic; proteomics.

Figure 1

Expression of melanin related proteins in pigmented and amelanotic melanoma cells and assessment of cells migration. (A) Visual comparison between amelanotic cells (A375, SKMEL28) and pigmented cells (MNT1, SKMEL23, Me290). (B) Heatmap representation of log2 transformed LFQ intensity values for proteins annotated for pigmentation (GO:0043473). (C) Western blot analysis of TYR, DCT, TYRP1, PMEL, microphthalmia-associated transcription factor (MITF) protein expressions in A375, MNT1, SKMEL28, SKMEL23 and Me290 and quantitative representation of relative protein expressions (one-way ANOVA analysis; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; n=3). Data are presented as mean ± SEM (D) Migration assay for A375, MNT1, SKMEL28, SKMEL23 and Me290 melanoma cell lines over a period of 2 days. (E) Measurements of area coverage indicating the significant differences between the five cell lines (one-way ANOVA analysis; ****p < 0.0001; ***p < 0.001). The measurements were conducted on at least 3 replicates.

Figure 2

LC-MS/MS proteome comparison of amelanotic versus pigmented melanoma cells. (A) Principal Component Analysis (PCA) showing clustering of samples according to their features. (B) Volcano plot of protein expression differences (log2FC) vs -log10(q value) from two sample t test of melanoma samples vs control (q-value<0.05 and absolute log2FC≥1) ( Supplementary Table 1, S1 ). (C) Venn diagram depicting shared upregulated proteins from the pairwise comparisons between A375 vs MNT1 and SKMEL28 vs MNT1. (D) Heatmap of log2 transformed LFQ intensity values showing the statistically significant shared up-regulated proteins from the comparisons between amelanotic cell lines (A375 and SKMEL28) and highly pigmented MNT1 cells (pairwise comparisons, two sample t test, q-value<0.05 and log2FC≥1) ( Supplementary Table 1, S1 ). (E) Immunofluorescent staining of AHNAK in A375, SKMEL28, SKMEL23, Me290 and MNT1 cell lines. (F) Volcano plots highlighting differentially expressed proteins from the pairwise comparisons between amelanotic cells and highly pigmented MNT1 cells (q-value<0.05 and absolute log2FC≥1) ( Supplementary Table 1, S1 ). (G) Western blotting assays and intensity band quantification for ITGA3, FN1, CTSD and CTNNB1 protein levels (one-way ANOVA analysis; ***p <0.001; **p<0.01; *p<0.05, n=3). Calnexin (CNX) was used as internal control. Data are presented as mean ± SEM. (H) Box plot of log2 transformed LFQ intensity values of CTNNB1, CTSD, FN1 and ITGA3 ( Supplementary Table 1, S1 ). (I) Box plot of log2 transformed LFQ intensity values for EPHA2, FSCN1, LAMB1, PAGE5, TGM2 and THBS1 ( Supplementary Table 1, S1 ).

Figure 3

Proteins involved in cellular adhesion process are downregulated in highly pigmented melanoma cells. (A) Box plot of log2 transformed LFQ intensity of selected integrins showing the differences between cell lines and a prevalence of integrins to have an increased expression in A375 cell line. (B) Pearson’s protein-protein correlation matrix of integrins and proteins annotated for pigmentation (GO:0043473). (C) Heatmap of log2 transformed LFQ intensity values of proteins related to epithelial to mesenchymal transition (GO:0001837) identified in our proteomic data. This set of proteins differentiate low migrating cells (Me290 and MNT1) from the more aggressive counterparts, A375, SKMEL28 and SKMEL23. (D) Dot plots showing the results of GSEA analysis associated with “biological process”, “molecular function” and “cellular component” terms (p.adjust<0.05), coming from the pairwise comparison between amelanotic (A375 and SKMEL28) and highly pigmented cells (MNT1) ( Supplementary Table 1 , GSEA A375 vs MNT1 and GSEA SKMEL28 vs MNT1).

Figure 4

Hypoxia influence on several processes upon melanoma cells cultivation under low oxygen condition (1% O₂, 5%CO₂ and 94% N₂). (A) Western blot assay of hypoxia-inducible factor 1-alpha (HIF1α), hypoxia-inducible factor 2-alpha (HIF2α), phosphorylated extracellular signal-regulated kinase (p-ERK) and extracellular signal-regulated kinases (ERK) protein expressions in HEK293T, A375, MNT1 and SKMEL28 cells. HEK293T, A375, MNT1 and SKMEL28 cells were kept in normoxia and hypoxia (1% O2) for 24 hours, at 37°C. Cells were harvested in RIPA buffer and specific proteins were identified. Expression levels of HIF1α and HIF2α proteins were normalized to calnexin (n=3). (B) Western blotting of autophagy related proteins (LC3 and p62). Quantitative analysis of LC3-II to LC3-I protein ratio, (n=3). (C) The same experiment as in (A) was performed for cathepsin D protein expression. Densitometric quantification of cathepsin D protein bands was assessed using calnexin as internal control. Data are represented as mean ± SEM (two-way ANOVA with Sidak multiple comparisons test; **p < 0.01, *p < 0.05). (D) Box plot of log2 transformed LFQ intensity values of cathepsin D in normoxia and hypoxia, as emerge from mass spectrometry data results.

Figure 5

LC-MS/MS proteome analysis of melanoma cells in hypoxia. (A) Volcano plots representing the protein expression changes under hypoxia in the comparisons between amelanotic and highly pigmented cells (pairwise comparisons, q value<0.05, absolute log2FC ≥1) ( Supplementary Table 1, S1 ). (B) Venn diagrams depicting unique and shared sets of up-regulated proteins from the comparisons between amelanotic and highly pigmented cells in normoxia and hypoxia (C) Box plot of log2 transformed LFQ intensity level of AHNAK and PLEC. (D) Venn diagram depicting unique and shared sets of up-regulated proteins in melanoma cell lines after cell exposure to hypoxia (log2FC≥1). (E) Cnet plot depicting the linkages between core enriched proteins for processes that were significantly enriched, coming from the comparison between melanotic cells cultured under hypoxia vs normoxia ( Supplementary Table 1 , GSEA MNT1 Hypoxia vs Normoxia). (F) Western blot analysis of TYR and DCT under normoxia and hypoxia. Calnexin was used as loading control. Western blot densitometry band quantification for TYR and DCT is presented as the mean ± SEM (two-way ANOVA with Sidak multiple comparisons test; ***p < 0.001, n=3).

Figure 6

Heatmap representations showing the relationship between melanogenetic proteins and selected proteins identified in this study. The analysis was conducted on three different public datasets. (A) Heatmap of z-score of normalized protein expression values for selected proteins identified in 33 melanoma cell lines (52). (B) Heatmap of z-score of normalized protein area (log2) for selected proteins. Three biological replicates isolated from patients with melanoma are represented (53). (C) Heatmap of RNA-seq expression z-scores (RSEM z score) for selected protein-coding genes of 442 melanoma patients (54).