Article pubs.acs.org/jpr
Large-Scale Proteomic Characterization of Melanoma Expressed Proteins Reveals Nestin and Vimentin as Biomarkers That Can Potentially Distinguish Melanoma Subtypes Veneta Qendro,† Deborah H. Lundgren,† Karim Rezaul,† Forrest Mahony,† Nicholas Ferrell,† Andrew Bi,† Ardian Latifi,† Daniyal Chowdhury,† Steven Gygi,‡ Wilhelm Haas,‡ Lori Wilson,§ Michael Murphy,∥ and David K. Han*,† †
Department of Cell Biology, Center for Vascular Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030, United States ‡ Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, United States § Howard University Medical Center, 520 W Street NW, Washington, D.C. 20059, United States ∥ Department of Dermatology and Laboratory of Dermatopathology, University of Connecticut Health Center, 21 South Road, Farmington, Connecticut 06030, United States S Supporting Information *
ABSTRACT: Melanoma is an aggressive type of skin cancer, which accounts for only 4% of skin cancer cases but causes around 75% of skin cancer deaths. Currently, there is a limited set of protein biomarkers that can distinguish melanoma subtypes and provide an accurate prognosis of melanoma. Thus, we have selected and profiled the proteomes of five different melanoma cell lines from different stages of progression in comparison with a normal melanocytes using tandem mass spectrometry. We also profiled the proteome of a solid metastatic melanoma tumor. This resulted in the identification of 4758 unique proteins, among which ∼200− 300 differentially expressed proteins from each set were found by quantitative proteomics. Correlating protein expression with aggressiveness of each melanoma cell line and literature mining resulted in the final selection of six proteins: vimentin, nestin, fibronectin, annexin A1, dipeptidyl peptidase IV, and histone H2A1B. Validation of nestin and vimentin using 40 melanoma samples revealed pattern of protein expression can help predict melanoma aggressiveness in different subgroups of melanoma. These results, together with the combined list of 4758 expressed proteins, provide a valuable resource for selecting melanoma biomarkers in the future for the clinical and research community. KEYWORDS: melanoma, proteomics, biomarkers, nestin, vimentin, spectral counting, quantitative proteomics
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prognosis.2 Measured from the deepest cell of the tumor to the epidermal granular layer, an increase in tumor thickness directly correlates with a decrease in the 5 year survival for patients with melanoma.3 Various other factors such as sex, age, family history, tumor location, mitoses, and ulceration are also considered in prognosis of melanoma.3 Clark level of staging has also been widely used to qualitatively define confinement of tumor within the epidermis (Level I) or the extent of tumor invasion into the dermis (Levels II−IV) and subcutaneous tissue (Level V).3
INTRODUCTION The American Cancer Society predicts that 76 690 Americans will be affected by melanoma in 2013, and 9480 will die of the disease. Melanoma incidence has increased over the past several decades to become one of the most common types of cancer behind only prostate, breast, lung, and colon cancers (cancer. org, 2013). Its growing prevalence has increased the necessity for research into improved diagnostic, preventative, and treatment methods. A diverse and proliferative cancer, melanoma continues to present diagnostic challenges. Visual inspection of suspicious cutaneous pigmented lesions remains a principal method of melanoma detection.1 The ABCDE acronym (asymmetry, border irregularity, color variegation, large diameter, and evolution) applied to any suspected lesion determines the need for further investigation.1 By light microscopy, Breslow’s tumor thickness represents the most important criterion used to determine melanoma © XXXX American Chemical Society
Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: July 22, 2014
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dx.doi.org/10.1021/pr5006789 | J. Proteome Res. XXXX, XXX, XXX−XXX
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Figure 1. (A) Schematic representation of cell lines belonging to different stages of melanoma progression as categorized by the American Joint Committee on Cancer. (B) Schematic flow diagram of proteomic analysis performed on indicated cell lines and melanoma tissue.
Proteomics has emerged as a possible tool for the advancement of diagnostic and prognostic biomarkers in melanoma.4 Proteomics in melanoma research involves the search for differentially regulated proteins that indicate the onset of, or the progression toward, metastatic disease.5 As cells differentiate and proliferate, their proteomes may reflect changes associated with the aggressiveness of melanoma. Discovery of a single protein, or group of proteins, indicative of these effects would significantly improve the diagnostic ability of physicians and aid in cancer prognosis and treatment. The potential for protein biomarkers could also extend to the differentiation of aggressive from less or nonaggressive strains of melanomas. Progress in this area would greatly aid personalized treatment plans and improve prognoses for patients with this disease. To identify potential biomarker(s) of melanoma, we performed large-scale proteomic profiling for five melanoma cell lines, a tissue sample of metastatic melanoma, and a benign melanocyte cell strain as the control group. The analysis resulted in a combined list of 4758 unique proteins expressed among the samples analyzed. A spectral count analysis of each of the melanoma cell lines compared with normal melanocytes revealed approximately 200−300 differentially expressed proteins from each set. Following an in-depth examination of these differentially expressed proteins, including a literature search to identify the entries most relevant to melanoma, we narrowed our list of biomarker candidates down to six key
proteins for further experimentation (nestin, vimentin, annexin A-1, fibronectin, dipeptidyl peptidase IV, histone H2A 1-B). On the basis of the availability of paraffin-reactive antibodies, we tested 48 tissue arrays using nestin and vimentin antibodies (Figure 1B). Major findings are that nestin expression correlates with the most aggressive melanoma, while vimentin expression varies among different melanoma subtypes. These results, together with the identified list of over 4758 unique proteins, serve as a rich resource for further exploration of melanoma-expressed proteins for selecting useful biomarkers.
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METHODS AND MATERIALS
Patient Tumor Samples
Formalin-fixed paraffin-embedded (FFPE) tissue sample of deidentified metastatic melanoma, 0.8 cm × 0.8 cm in diameter, characterized by a nodular homogeneous proliferation of tumor cells was obtained from the Dermatopathology Laboratory, University of Connecticut Health Center and was used for proteomic identification and nestin staining (Figure 2). Cell Lines
A total of five melanoma cell lines and a melanocyte cell strain as a control were generated at the Wistar Institute by Meenhard Herlyn and colleagues6 and obtained from the American Type Culture Collection and used for mass spectrometry analysis (Bethesda, MD). All melanoma cell lines as well as the melanocyte cell strain were grown in cell culture dishes (100 B
dx.doi.org/10.1021/pr5006789 | J. Proteome Res. XXXX, XXX, XXX−XXX
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Figure 2. Immunohistochemistry of metastatic melanoma stained with antinestin antibody depicting the heterogeneity of protein expression even within the same tumor.
mm × 20 mm) containing Medium 254 supplemented with human growth supplement (HMGS) at 37 °C in a humidified atmosphere containing 5% CO2. Medium was changed every 3 days, and cells were lysed at ∼80% confluence. The melanoma cells were purchased from the American Type Culture Collection (ATCC) at passage zero. Because it is well known that melanoma cells can lose their phenotype during prolonged culture, we maintained a low passage number for protein extraction and analyzed all melanoma cells and melanocytes at passage four or five. To identify useful biomarkers whose expression levels are related to the aggressiveness of melanoma, we organized the melanoma cell lines using two criteria: Vertical Growth Phase/ Radial Growth Phase and the manual of the American Joint Committee on Cancer (AJCC) (Figure 1; Supplemental Table 1A in the Supporting Information). On the basis of the Vertical Growth Phase/Radial Growth Phase criteria, melanoma cell lines were organized from benign to malignant in the following order: WM35, WM1552C, WM39, WM793B, and 1205Lu. According to AJCC criteria, melanoma cell lines were organized from benign to malignant in the following order: WM35, WM793B, WM1552C, and 1205Lu; WM39 could not be staged based on this criterion.
glycerol, 5 mM EDTA, and a mixture of protease inhibitors (Roche)] for 1 h on ice and centrifuged at 16 000g for 15 min, and supernatant was saved. The insoluble pellets were resolubilized in 200 μL of lysis buffer II (buffer I containing 0.5% SDS), briefly sonicated, and incubated on ice for 30 min. After centrifugation at 16 000g for 15 min, supernatant was saved and pooled together. Protein concentration was determined using a Micro-BCA protein concentration determination kit (Pierce, Rockford, IL). Protein samples were boiled with SDS sample buffer at 95 °C for 10 min and stored at −20 °C until sample separation. Sample Separation and In-Gel Trypsin Digestion
An equal amount of total cell lysates (50 μg) from each cell line was separated on a 10% NuPage gel (Invitrogen). Gels were lightly stained with Coomassie blue R-250 (50% methanol, 10% acetic acid, 0.1% R-250) for 10 min and destained overnight in destaining solution (5% methanol, 7% acetic acid). After imaging, the area from the top to the bottom of each lane of the Coomassie-stained gel was cut into 15 slices. Each gel slice was chopped into small pieces (∼1 mm cubes) and transferred to 500 μL microcentrifuge tubes. The gel pieces were completely destained by repeated washes with NH4HCO3 (50 mM) and NH4HCO3/CH3CN (50 mM/25%). The destained gel pieces were dehydrated with 100% CH3CN and dried briefly in a vacuum concentrator (CentriVap). Gel pieces were rehydrated with 50 μL of trypsin solution (20 ng/μL in 100 mM NH4HCO3) on ice for 45 min. In-gel digestion was performed
Sample Preparation
For harvesting, cells were washed twice with 10 mL of PBS and were solubilized in 1.0 mL of lysis buffer I [50 mM Tris-HCl (8.0), 250 mM NaCl, 1% n-dodecyl-β-D-maltoside, 10% C
dx.doi.org/10.1021/pr5006789 | J. Proteome Res. XXXX, XXX, XXX−XXX
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at 37 °C for 18−20 h. The resulting peptides were extracted according to the established protocol of Shevchenko et al.7 Extracted peptides were dried in Centrivap, redissolved in solvent A [5% acetonitrile, 0.4% acetic acid, and 0.005% heptafluorobutyric acid (HFBA)], and stored at −20 °C until a mass spectrometric analysis was performed.
In addition, to include proteins that are highly expressed in metastatic melanoma, the 10 most highly expressed proteins in the 1205Lu metastatic cell line based only on the averaged spectral counts were also analyzed for (1) a consistent trend in expression across all other melanoma cell lines and (2) high level expression in the metastatic melanoma tissue sample. This triage analysis of the 10 most highly expressed proteins from the 1205Lu cell line resulted in the selection of 6 additional proteins, for a total of 20 proteins of interest. Next, we performed pathway analysis and a literature search for the relevance of these 20 proteins in melanoma and carcinogenesis and their possible utility as biomarkers, resulting in a final selection of six proteins provided in Table 2.
Mass Spectrometry
Total cell lysates from the five different melanoma cell lines and cell-rich areas from the tissue sample of metastatic melanoma were manually microdissected and trypsin-digested for protein extraction.8 50 μg of protein isolated from each cell line and 50 μg of protein recovered from the FFPE tissue sample were further analyzed using mass spectrometry. For each lysate, approximately 90 LC−MS/MS runs were carried out, each run lasting 95 min, for a combined total of 270 LC/MS runs. At least ≥2 high-scoring peptides were required for protein identification and maintenance of a low protein false discovery rate [false discovery rate (FDR) ≤ 1.3%] (Table 2).9
Bioinformatics Analysis
The Uniprot accession IDs of the differentially regulated proteins in each melanoma cell line were submitted to DAVID bioinformatics tool (http://david.abcc.ncifcrf.gov) to perform functional annotation clustering and assign over-represented GO terms (cellular components) to each group of proteins. GO terms meeting Bonferroni-corrected p values
