Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma

Abstract

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

Keywords: FFPE tissue; Mass spectrometry; Melanoma; Proteomics; Quantitative.

Figure 1. Quantitative proteomic analysis of FFPE melanoma

Melanocytes from benign FFPE skin biopsies (25 total) and melanoma cells from primary (12 total) and metastatic melanoma (24 total) FFPE biopsies were isolated by needle dissection. Protein extracts were resolved by SDS-PAGE, visualized by Coomassie staining, excised as 24 bands per lane, and subjected to in-gel trypsin digestion. Tryptic peptides were analyzed by LC-MS/MS and relative protein levels were determined by spectral counting [9].

Figure 2. Hierarchical cluster of significantly differentiating proteins

An unsupervised cluster of both FFPE biopsies and significant proteins shows clear separation among Benign (BS), Primary Melanoma (PM), and metastatic Melanoma (M) patients. A sub-cluster of proteins shows increased expression in the primary samples compared with both benign and metastatic (in brackets). Red data points indicate increased protein expression, while green indicates decreased expression.

Figure 3. Volcano plots of significantly differentiating proteins

The negative log (base 10) of the Mann Whitney U p-values are plotted on the y-axis and the log (base 2) of the fold change are plotted on the x-axis. A) Metastatic melanoma versus benign. B) Primary melanoma versus benign. C) Metastatic melanoma versus primary melanoma. The red data points indicate proteins with a p-value < 0.01 and a fold change > 2.

Figure 4. Pathways identified for significantly differentiating proteins

The number of significantly differentiating proteins associated with each pathway is displayed in the bar graph. The pathways with the most proteins have the highest mis-regulation.

Figure 5. Detailed pathways identified for significantly differentiating proteins

The major pathways that are mis-regulated in benign nevi, primary melanoma, and metastatic melanoma and their associated proteins are shown as a pathway map modified from the multiple KEGG pathways. The pathway map highlights proteins up-regulated in benign nevi in blue, primary melanoma in green, and metastatic melanoma in red.