Tumor specimens from patients
We collected 64 formalin-fixed and paraffin-embedded (FFPE) samples of primary cutaneous MM from the Surgical Pathology Unit of the S. Chiara Hospital in Trento, Italy. The study was approved by the Research Ethics Committee for Clinical Experimentation of the Trentino Public Healthcare Agency, Italy, and each patient signed formal written informed consent for sampling and research. Samples were diagnosed by expert pathologists (SB and MB), according to the classification system of the American Joint Committee on Cancer [22]. Clinical features of the primary MMs and patients’ follow-up data are summarized in Additional file 1: Table S1. The sample ID indicated in any of the tables cannot be linked back to any of the patients.
Cell lines
MM cell lines SK-MEL-28 and G-361 were a gift of Alberto Inga (CIBIO, University of Trento, Italy) and were originally obtained from the ICLC Interlab Cell Line Collection (Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy). SK-MEL-28 cells were grown in DMEM supplemented with 10 % fetal bovine serum (FBS), 2 mM L-Glutamine, 1 % non-essential amino acids, penicillin, and streptomycin. G-361 cells were cultured in EMEM, supplemented with 10 % FBS, 2 mM L-Glutamine, 1 % non-essential amino acids, penicillin, and streptomycin. SK-MEL-28-TrkA and G-361-TrkA or SK-MEL-28-E and G-361-E, were obtained by lentiviral infection with TrkA-containing plasmid or empty vector, respectively, and were maintained in the same culture medium as the original stock.
Genome profiling of clinical samples
Genomic copy number aCGH profiles of 31 MM samples, available as series GSE45354, at Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/), were analyzed as previously described [15]. In brief, the array CGH was performed using the Agilent 8x60K human CGH oligo microarray chip (Agilent Technologies, Santa Clara, CA; 021924 SurePrint G3 Human CGH 8x60K Microarray, cat. G4450A), mapped to the human genome (USCS genome browser Human, Feb. 2009, GRCh37/hg19). The scanned microarray TIFF images were acquired with the Agilent DNA Microarray Scanner G2505C, by the manufacturer’s software (Agilent ScanControl 8.1.3), and analyzed using the Agilent Feature Extraction Software version 10.7.7.1. The analysis of raw aCGH data was then conducted via the R environment for statistical computing (http://www.r-project.org/) using packages provided by the Bioconductor library (http://www.bioconductor.org/). Hotspots of minimal common regions of amplification were defined as the minimal regions of overlap shared by at least three samples with a maximum length of 2.5 Mb [20].
DNA extraction from clinical samples and genomic real-time quantitative PCR
Genomic DNA (gDNA) was isolated from all FFPE archival samples using an optimized DNA isolation protocol based on a Qiagen extraction kit (#51306; Qiagen), as previously detailed [15]. Quantitative PCR (qPCR) validation of genomic copy number was performed by using the laminin alpha 1 (LAMA1) gene, located in 18p11.31, as reference gene, since this locus showed absence of CNAs in 97 % of cases from our aCGH dataset. As diploid calibrator, a pooled FFPE gDNA of 10 healthy patients with inflammation of the vermiform appendix was used. Two benign nevi were used as an additional diploid control. The reaction was performed by using the commercially available FAM-labeled TaqMan Copy Number Assay (Life Technology) for LAMA1 exon 3 (Hs00282410_cn), CDKN2A exon 5 (Hs03714372_cn), and NTRK1 intron 3 (Hs05769842_cn). A 10 μl reaction was prepared with 5 μl of KAPA PROBE-FAST qPCR Master Mix (2X) ABI Prims (Kapabiosystems), 0.5 μl of TaqMan assay (20X), and 10 ng of template gDNA. Thermal cycling conditions consisted of an initial cycle at 95 °C for 10 min, followed by 40 cycles each of 15 s 95 °C and 1 min 60 °C. Comparative cycle threshold (Ct) values for each target gene were calculated by Bio-Rad CFX Manager 2.1 software (Bio-Rad Laboratories Inc.) using regression mode and relative copy number ratio was measured by the E ΔCt method over the reference gene LAMA1, where E is the PCR efficiency calculated by standard curves generated from dilution series of calibrator gDNA, as previously described [15]. Experiments were repeated in two independent replicates, where PCR for each assay was performed in three internal replicates. Diploid copy number was set as a fold change of 1; gain of one extra genomic copy was defined when fold change over diploid calibrator was between 1.25 and 1.75; amplification was defined as an increase in fold change above 1.75; hemizygous deletion was determined as a fold change between 0.75 and 0.5; homozygous deletion was defined as fold-change decrease below 0.5 [15, 23].
Quantitation of DNA copy number and mRNA expression for cell lines
Total gDNA from MM cells was extracted with DNeasy Blood & Tissue kit (Qiagen). Genomic copy number of TrkA and CDKN2A was quantified by comparison with gDNA of normal melanocytes (#C-024-5C; HEMaLP, Life Technology), using the same primer set and protocols as previously described for tissue samples. Relative copy number ratio was measured by applying regression mode, as calculated by the Bio-Rad CFX Manager 2.1 software, and the ΔΔCt method Ct for normalization of Ct values to LAMA1 as internal reference gene [24]. The experiment was repeated twice.
Total RNA from MM cells was extracted by using RNeasy Plus mini Kits (Qiagen) and reverse-transcribed using iScriptTM cDNA Synthesis Kit (Bio-Rad). The obtained cDNA was subjected to real-time qPCR by TaqManGene Expression Assay (Life Technologies). Commercially available FAM-labeled TaqMan assays were used for LAMA1 (Hs00300550_m1) and NTRK1 (Hs01021011_m1). A 10 μl reaction was prepared with 5 μl of KAPA PROBEFAST qPCR Master Mix (2X) ABI Prims (#KK4702; Kapabiosystems, Woburn, MA), 0.5 μl of TaqMan assay (20X), 100 ng of template cDNA, and run on Bio-Rad CFX384 Real-Time PCR Detection System (Bio-Rad). PCR cycles were: 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. Values of Ct were calculated by Bio-Rad CFX Manager 2.1 software, using regression mode, and ΔΔCt method was used for expression quantification using the Ct of LAMA1 for normalization [24]. Results were obtained as a mean of three experiments.
Vectors and lentiviral transduction
The human TrkA gene (splice variant NTRK1-001, RefSeq NM_001012331.1) was subcloned from the original pCMV5-TrkA (Addgene Plasmid 15002; ref. [25]) into SalI-XbaI sites of the doxycycline-inducible Tet-On lentiviral vector pLenti-CMV/TO-eGFP-Puro (Addgene Plasmid 17481; ref. [26]), by replacing the eGFP sequence, and the construct was verified by Sanger sequencing. MM cells SK-MEL-28 and G-361 were transduced with the tetracycline-repressor expression vector pLenti-CMV-TetR-Blast (Addgene Plasmid 17492; ref. [26]) before transduction with pLenti-CMV/TO-TrkA-Puro or the pLenti-CMV/TO-Puro empty vector. Lentiviral particles were produced by co-transfecting the transfer plasmids with packaging vector pCMV delta R8.2 (Addgene plasmid 12263; Didier Trono) and envelop plasmid pMD2.G (Addgene plasmid 12259; Didier Trono) into HEK-293-T cells (ICLC Interlab Cell Line Collection), in a penicillin/streptomycin-free Opti-MEM® culture medium (Life Technology), with 0.5 mg/ml Polyethylenimine (Sigma-Aldrich), based on Trono lab protocols (http://tronolab.epfl.ch). Viral titer in the supernatant was established at 0.5 transducing units (TU) per reaction, as measured by SYBR Green I-based PCR-enhanced reverse transcriptase (SG-PERT) assay [27]. Parallel infection efficiency of pLenti-CMV/TO-eGFP-Puro control plasmid was above 60 % at 96 h post infection, as quantified by the GFP signal. Transduced cells were selected for 6 days with puromycin 3 μg/ml (Sigma-Aldrich), starting at 48 h post-infection.
Cell treatments
Before performing the experiments, transduced cells were allowed to adhere to the plate by growing for 16 h in complete melanoma cell medium. Afterwards, to induce TrkA expression cells were pre-treated with 500 ng/ml doxycycline (Sigma-Aldrich) for 48 h, either in medium 2 % FBS or FBS-free medium, and doxycycline was maintained during the entire course of the experiments. To test the activation of NGF-TrkA downstream pathways, cells were treated with 100 ng/ml β-NGF (#PHG0126; Life Technology) for 15 min in FBS-free medium. A dose–response curve was measured by incubating the cells for 15 min in FBS-free medium with 6.25, 12.5, 25, 50, and 100 ng/ml β-NGF. To activate NGF-TrkA signaling before phenotypic assays, cells were treated with 100 ng/ml β-NGF for 24 h or 48 h. To specifically block the MAPK pathway, cells were incubated with 5 μM U0126 (Promega) in the presence or absence of NGF. To inhibit the AKT pathway, cells were incubated with 25 μM LY294002 (Promega) in the presence or absence of NGF. CEP-701 (Sigma-Aldrich) was used at 10 μM, as a broad inhibitor of kinase signaling. Control experiments were conducted in the absence of doxycycline in 2 % FBS medium or FBS-free medium plus vehicle (DMSO). During treatment experiments, vehicle was either water (for NGF controls) or DMSO (for kinase inhibitor controls).
Western blot analysis
Cells (approximately 0.5 x 106) were harvested on ice in lysis buffer (50 mMTris-HCL pH 8, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS) supplemented with 1 μg/ml Pepstatina A (Sigma-Aldrich), protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktails 1/2 (Sigma-Aldrich). After determination of total protein content by the Bradford reagent (Sigma-Aldrich), 30 μg of protein extracts were resolved by SDS-PAGE gels and then blotted onto 0.2 μm nitrocellulose membrane (Bio-Rad). Unspecific protein binding was blocked by incubation for 1 h in 5 % Blotto non-fat dry milk (Santa Cruz Biotechnologies Inc.) in 0.1 % TBS-tween and membranes were incubated overnight at 4 °C with primary antibodies: rabbit anti-TrkA, 1:1000 (#06-574; Upstate); rabbit anti-phospho (Try490)-TrkA, 1:1000 (#9141S; Cell Signaling Technology Inc.); rabbit anti-ERK1/2, 1:2000 (sc-153; Santa Cruz); rabbit anti-phospho-ERK1/2, 1:1000 (#4370S; Cell Signaling); rabbit anti-AKT(pan), 1:1000 (#4691S; Cell Signaling); rabbit anti-phospho-AKT1, 1:1000 (Ab66138; abcam); mouse anti-p21cip1 (sc-397; Santa Cruz, 1:2000); mouse anti-eIF4E, 1:1000 (SC9976; Santa Cruz); mouse anti-p53, 1:5000 (sc-377567; Santa Cruz); mouse anti-Cyclin D1, 1: 1000 (ab101430; Abcam); mouse anti-β-tubulin (sc-53140; Santa Cruz, 1:5000); mouse anti-α-actinin (sc-17829; Santa Cruz, 1:6000); mouse anti-GAPDH (sc-32233; Santa Cruz; 1:5000). After washing, membranes were incubated for 1 h at room temperature, with goat anti-rabbit (sc-2004; Santa Cruz) or goat anti-mouse (sc-2005; Santa Cruz) secondary HRP-conjugated antibodies, diluted 1:10000 in blocking solution. Membranes were then washed and developed by using the ECL detection assay (Amersham Biosciences). After detection of phospho-TrkA, phospho-AKT, and phospho-ERK signals, the membranes were stripped with Re-Blot Plus Mild Solution (Merck Millipore) and re-blotted for total protein staining. Protein expression was quantified from digital images by Image Lab software (Bio-Rad), setting the global subtraction method for background. TrkA proteins typically correspond to two WB bands: the mature cell surface 140-kDa form and the immature 110-kDa form, which is subsequently modified by glycosylation in the ER before translocation to the membrane [28].
Cell-cycle analysis
Cells were seeded (0.4 × 105 cells/well) in a 6-well plate and allowed to adhere for 16 h in complete medium. After treatment, cells were centrifuged and processed with the Cycletest™ Plus DNA Reagent Kit (BD Biosciences) and incubated in Propidium Iodide (PI) labeling solution, following the manufacturer’s indications. Cell cycle analysis, by measuring DNA content, was performed by flow cytometry using a FACS Canto II instrument (BD Biosciences). FACSDiva™ Software V8.0 (BD Biosciences) was used to quantify the distribution of cells in each cell cycle phase: sub-G1 (dead cells), G1, S and G2/M. Results were displayed as the average of three separate experiments.
Real-time proliferation analysis
Cell proliferation was monitored by the xCELLigence RTCA DP Analyzer (Roche) for at least 48 h after treatment, following manufacturer’s indications. This apparatus makes it possible to follow the cellular response to treatment in real-time using electrical impedance as the readout. The continuous monitoring of cell viability by the xCELLigence system allows us to distinguish between cell death and reduced proliferation [29]. Cells (5 × 103 cells/well) were seeded into E-plates 16 (Acea Biosciences Inc.) and impedance was continuously recorded in 15 min intervals until the end of the experiment. Cell index (CI) values, derived from the measured impedances, were acquired by the RTCA Software V1.2 (Roche) and exported to Microsoft Excel for normalization of data of each single well to the first measurement after starting the treatment. Statistical analysis and graphical representation of data were performed by the Prism GraphPad Software V5.0 (GraphPad Software Inc., La Jolla, CA, USA). Data displayed in the graphs is the average value of three biological replicates, each consisting of two technical replicates.
Cell number quantification, proliferation assay and detection of apoptosis
To assess proliferation after treatment by measuring the amount of newly synthetized DNA, cells were plated in a 96-well plate (5 × 103 cells/well) and the Click-iT® EdU cell proliferation assay (Life Technologies) was used following the manufacturer’s instructions. Cells were incubated with 10 μM of the nucleoside analog EdU for 2 h and immediately fixed in 4 % formaldehyde and permeabilized. To detect apoptosis, cells were stained for 1 h at room temperature with anti-active-caspase-3 antibody, 1:600 (ab13847; Abcam) followed by goat anti-rabbit secondary antibody staining, Alexa Fluor® 488, 1:1000 (#A-11070; Life Technologies), for 1 h at room temperature. The total DNA was stained with Hoechst 33342 (Life Technologies) and used for quantifying the absolute number of cells present in the plate. Quantification of fluorescent cells that incorporated Hoechst 33342, EdU or were stained for caspase-3 was carried out by using the Operetta® High Content Imaging System equipped with the Harmony software (PerkinElmer Inc.). Fractions of EdU labeled cells were calculated based on Hoechst signal. Three independent experiments, with two internal replicates, were performed for each condition.
Statistical analysis
All statistical analysis were performed by Prism GraphPad Software V5.0 (GraphPad Software Inc.) except for the association of copy number amplifications, detected by aCGH, with tumor thickness, which was calculated by the Mann–Whitney test in the R software environment for statistical computing. Detailed methods for the identification of CNAs from the aCGH data are provided in ref. [15]. The Mann–Whitney test was used to evaluate the association between MM thickness and copy number levels of TrkA derived from aCGH and genomic qPCR analysis. Pearson’s correlation coefficient was use to assess correlation between the aCGH copy number log2 ratio and the log2 of the qPCR fold changes of TrkA. Spearman’s correlation test was used to evaluate the correlation between TrkA copy number and mRNA expression data extracted from publically available resources: Cancer Cell Line Encyclopedia (CCLE, http://www.broadinstitute.org/ccle/home) and The Cancer Genome Atlas data (TCGA, http://www.cbioportal.org/index.do; ref. [30, 31]).
The Kaplan-Meier method and log-rank test were used to assess the difference in overall survival and metastatic outcome between TrkA-amplified patients and TrkA-diploid patients. One-way ANOVA test, followed by Tukey’s post-test to compare two groups, was performed to explore differences of proliferation rates in the xCELLigence proliferation assay. Student’s t test (two-tailed, unpaired) was used to compare means for all other statistical analyses. Results for cellular experiments are given as the mean of three independent experiments; p values were considered significant when lower than 0.05.