- Open Access
Proteomic analysis of Medulloblastoma reveals functional biology with translational potential
- Samuel Rivero-Hinojosa†1,
- Ling San Lau†1,
- Mojca Stampar†1,
- Jerome Staal1,
- Huizhen Zhang1,
- Heather Gordish-Dressman2,
- Paul A. Northcott3,
- Stefan M. Pfister3,
- Michael D. Taylor4,
- Kristy J. Brown2 and
- Brian R. Rood1, 5Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 21 April 2018
- Accepted: 17 May 2018
- Published: 7 June 2018
Genomic characterization has begun to redefine diagnostic classifications of cancers. However, it remains a challenge to infer disease phenotypes from genomic alterations alone. To help realize the promise of genomics, we have performed a quantitative proteomics investigation using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and 41 tissue samples spanning the 4 genomically based subgroups of medulloblastoma and control cerebellum. We have identified and quantitated thousands of proteins across these groups and find that we are able to recapitulate the genomic subgroups based upon subgroup restricted and differentially abundant proteins while also identifying subgroup specific protein isoforms. Integrating our proteomic measurements with genomic data, we calculate a poor correlation between mRNA and protein abundance. Using EPIC 850 k methylation array data on the same tissues, we also investigate the influence of copy number alterations and DNA methylation on the proteome in an attempt to characterize the impact of these genetic features on the proteome. Reciprocally, we are able to use the proteome to identify which genomic alterations result in altered protein abundance and thus are most likely to impact biology. Finally, we are able to assemble protein-based pathways yielding potential avenues for clinical intervention. From these, we validate the EIF4F cap-dependent translation pathway as a novel druggable pathway in medulloblastoma. Thus, quantitative proteomics complements genomic platforms to yield a more complete understanding of functional tumor biology and identify novel therapeutic targets for medulloblastoma.
- Pediatric brain tumor
In a series of landmark papers, microarray transcriptome characterization subdivided the malignant childhood brain tumor medulloblastoma into at least four distinct entities [11, 31, 32, 49, 66]. Chromosomal copy number alterations, clinical characteristics such as age and survival, and methylation array data correlated well with the transcriptomic classification, reinforcing the identity of distinct subgroups of the disease . However, the real goal of more informative, biologically based tumor taxonomy is to drive discovery of new therapeutic interventions. The WNT and SHH pathways drive their eponymous subgroups and their activity is enhanced by mutations in key elements of the pathways. But the other two subgroups, 3 and 4, do not harbor mutations at the same frequency and present a greater challenge when trying to discern their biological determinants from gene expression signatures alone . Largely, this difficulty emanates from the central assumption of transcriptomics, that a gene’s expression level predicts its contribution to cell biology. We have therefore applied quantitative proteomics in an attempt to replace this assumption with knowledge about the proteome, as proteins more directly determine the functional state of the cell.
The work of the cell is performed largely by proteins. While protein production begins with gene transcripts, a growing literature has demonstrated that transcript abundance does not predict protein quantity for a large majority of genes [21, 24, 77]. A large number of post-transcriptional regulatory mechanisms from interfering RNAs to selective RNA binding proteins affect differential translation in response to a wide range of cellular conditions . The first paper published from the CPTAC (Clinical Proteomic Tumor Analysis Consortium) initiative showed a correlation coefficient of 0.23 between transcript and protein abundance  and subsequent publications have replicated this experience [45, 78]. It is also becoming increasingly recognized that cancer cells utilize alternative pathways of translation initiation that prioritize proteins important in the cellular response to stress [41, 60, 63]. Thus, a true picture of the cancer cells’ functional state must be based not only upon genomic features but also include the protein complement if we are to decipher its biology. Further, and most importantly, in order to interfere with that biology, proteins remain the most actionable targets of pharmaceutical and immunotherapeutic intervention.
While proteomics can point the way forward toward translational progress, it can also provide a valuable lens through which to view cancer genomics. Genomic technology generates rich datasets with little inherent means to determine the relative importance of each individual finding. In contrast, clinical proteomics data is much less complete but is, by its nature, more representative of cellular biology. By employing the axiom that for a genomic event to influence phenotype it must be translated into the proteome, one can apply a filter to help discern signal from noise in the massive datasets produced by genomics platforms. In this way, these two areas of study can complement one another. Here, we present the first proteogenomic characterization of pediatric medulloblastoma and demonstrate the unique contributions to biologic understanding offered by quantitative proteomics.
Quantitative proteomics of medulloblastoma
Correlation between mRNA and protein abundance.
To test whether mRNA-protein correlation is related to the biological function of the protein, we performed KEGG Pathways enrichment analysis. We found that gene/protein pairs with higher correlation (greater than the Upper Quartile Q3) were enriched in metabolic and ribosomal pathways, whereas there was a low correlation for genes/proteins enriched in proteasome, spliceosome, oxidative phosphorylation, and RNA transport pathways (Fig. 2c).
Among other factors such as secondary mRNA structure, codon bias, ribosomal density, regulatory proteins and RNAs , the half-life of proteins and mRNA is a major factor influencing mRNA-protein correlation. From studies on mammalian cells, it is reported that proteins are 5 times more stable and 900 times more abundant than mRNA while spanning a lower dynamic range . To analyze the effect of mRNA and protein half-lives on mRNA-protein correlation, we used half-life data generated from mammalian cells by parallel metabolic pulse labeling . We found that genes with stable protein products tend to have higher mRNA-protein correlation than those with unstable proteins, however we found no differences between stable and unstable mRNA (p-values 6.4e− 06 and 0.81 respectively, two-sided Wilcoxon rank sum test) (Fig. 2d). This finding is similar to colon cancer in which the protein-mRNA correlation was found to be highest when both mRNAs and proteins were stable . These results demonstrate that mRNA transcript abundance is a poor predictor of protein complement and that both the biological function of proteins and their rate of turnover likely impact mRNA-protein correlation. Thus, extreme caution should be exercised when predicting biologic phenotype from gene expression data alone.
Effect of methylation and copy number on protein expression.
To study the effect of DNA methylation on protein expression, we calculated the differentially methylated regions (DMR) by comparing each medulloblastoma subgroup to control cerebellum samples. Differentially expressed proteins and genes were calculated in the same way. We found that the number of differentially expressed genes (mRNA) associated with a DMR was higher than the number of differentially expressed proteins associated with a DMR across all subgroups (Additional file 4: Figure S2a). We then analyzed genes with an associated DMR to correlate loss/gain of methylation with mRNA or protein expression/repression respectively. We found that for all subgroups both mRNA and protein expression showed odds ratios greater than 1, however only the mRNA correlations were significant (p-value < 0.01, Fisher exact test) (Additional file 4: Figure S2b). Together, these results suggest that there is a poor correlation between epigenetic programs, evidenced by DNA methylation, and protein quantity, indicating that additional regulation at the level of translation and/or protein stability governs protein abundance.
Significant CNA-protein correlations identify amplified sequences that translate to high protein abundance illustrating that proteomic measurements can help to prioritize genes in amplified regions for further examination . Among the 18 genes with significant CNA-protein correlation in chromosome arm 17q, we found two genes that have been previously associated with medulloblastoma biology, LASP1 and GRB2 (Additional file 7: Figure S4a) [27, 67]. Other interesting candidates included KPNB1 and ARHGDIA, both correlating between CNA and protein but not with mRNA level (Additional file 7: Figure S4a). KPNB1 is a nuclear transport receptor, which regulates glioma proliferation via the Wnt/β-Catenin pathway . ARHGDIA, encoding a Rho GDP-dissociation inhibitor, is implicated in cell migration and is upregulated in several cancers including glioma . Interestingly, the ARHGDIA protein is elevated in group 4 tumors where isochrome 17q is frequent (Additional file 7: Figure S4b). In summary, we found more correlations between CNA and mRNA compared with protein levels indicating that in many cases, CNA-driven mRNA transcript alterations do not translate to the abundance of their corresponding protein . Additionally, we have found a high frequency of positive correlations between copy number and protein expression in chromosome arm 17q genes, reinforcing this alteration as an important factor in medulloblastoma biology. When chromosomal alterations involve many genes, proteomics can be instrumental in discerning which genes are most likely to project that alteration’s impact onto cellular biology.
Proteomic subgroup classification recapitulates genomic subgroups using different data elements
While the ability to recapitulate the genomic subgroups from proteomic data supports the validity of the subgroups, it is important to note that the proteome classifies tumors based upon different data points. To test this, we attempted to cluster the tumors using the transcript abundance of each of the genes coding for the proteins used in the proteomic classification. That dataset was unable to recreate the subgroups indicating that the proteomic classification draws from a different dataset (Additional file 8: Figure S5). In general, the genes that differentiate between the subgroups have not yielded therapeutic targets, limiting the translational impact of genomic subgrouping. Therefore, the ability of proteomics to differentiate the subgroups using different data points provides an additional translational opportunity.
Identification of subgroup enriched protein isoforms from unique peptides
Another interesting set of isoforms are from the high mobility group AT-hook 1 (HMGA1) gene. We detected significantly higher expression of HMGA1 isoforms a and b in Group 3 MB (Fig. 5b). HMGA1 is a DNA binding protein commonly overexpressed in human cancers [1, 2, 8, 10, 56]. Previous studies have reported that HMGA1 is expressed in MB tumors and cell lines where it controls growth, migration and invasion [34, 36]. Here we detected both HMGA1 isoforms significantly expressed in Group 3 MB tumors (Kruskal-Wallis rank-sum test p-value = 0.00072 and 0.0178 for isoforms a and b respectively) (Fig. 5b). A significant positive correlation between HMGA1 mRNA and protein was also found (Spearman correlation coefficient = 0.478, Additional file 2: Table S2). These findings were confirmed by RNA-seq (Additional file 12: Figure S8a). Additionally, western blot analysis of the four medulloblastoma groups showed the two HMGA1 isoforms expressed at higher levels in group 3 medulloblastoma (Additional file 12: Figure S8b). Interestingly, the level of total HMGA1 expression is also correlated with poor survival in Group 3 patients (Additional file 12: Figure S8c). Finally, we have correlated HMGA1 expression with MYC expression (Spearman correlation coefficient 0.83) in medulloblastoma group 3 tumors (Additional file 12: Figure S8d), as previously described . These results highlight the potential of proteomics to identify medulloblastoma specific translated isoforms and thereby provide biological insights unavailable from genomic data alone.
Proteome based functional network analysis demonstrates the centrality of the MYC program
A potentially confounding issue with normalizing protein quantities back to cerebellum is the tendency to overemphasize proteins associated with cellular proliferation rather than individual subgroup biology. Despite that concern, we also found subgroup restricted upstream regulators including the inhibitory axis of SYNV1-p53 in SHH . This is noteworthy as SHH is the subgroup in which the majority of p53 mutations occur . We also identify the cell adhesion regulator CD44 in group 4 , the tumor suppressor BRCA1 in WNT and the anti-apoptotic MKL1 in group 3 tumors. CD44 is a cancer stem cell marker that plays a role in tumor metastasis and progression while regulating multiple signaling networks depending upon the isoforms expressed . Wild-type BRCA1 has been demonstrated to increase the nuclear form of beta-catenin, thereby enhancing the downstream activity of the WNT pathway . MKL1 is part of the RBM15-MKL1 fusion resulting in activation of a Notch pathway transcriptional activator giving rise to acute megakaryoblastic leukemia . It is also a transcriptional modulator in its own right associated with proliferation and invasion in lung and breast cancer . Thus, the top upstream regulators predicted by IPA based on the differentially abundant proteome constitute proteins with known roles in cancer.
Next, IPA assembled the most significant interaction pathways based upon the degree of representation of pathway molecules by differentially expressed proteins (Additional file 16: Figure S9, Additional file 17: Table S8). Pathways previously reported to be important in medulloblastoma were identified including the Ephrin B, mTOR, and integrin signaling pathways [4, 13, 15, 42, 74]. Elements of DNA damage repair were also represented including G2-M checkpoint control and ATM signaling. Metabolism was highlighted by the TCA cycle and oxidative phosphorylation pathways. In keeping with the focus of this report is the prominence of protein synthesis and regulation pathways including the mTOR signaling, protein ubiquitination, EIF2 and EIF4 pathways. In order to demonstrate the potential of proteomics to identify translational opportunities, we chose to validate the importance of EIF4F cap-dependent protein translation to medulloblastoma cell survival.
EIF4F pharmacologic inhibition
Genomic characterization of tumors using large sample sets has yielded an unprecedented ability to discriminate subclasses of disease based upon transcriptional programs. While mutations have proven to be the most actionable of the genomic aberrations, pediatric cancers in general have very low mutational burdens. The broad and rich datasets yielded by genomic platforms are ideal for developing a deep understanding of what divides diseases into subgroups. However this endeavor, by its very nature, handicaps efforts to translate genomic findings into therapeutic tools for two reasons. First, focusing on the characteristics that segregate tumors from one another necessarily yields ever finer distinctions. Targeting these differences may yield precise tools that nevertheless fail to address the foundational biology essential to cancer cell survival. Secondly, creating ever smaller patient groups makes it unlikely that industry will create therapeutic agents for them or that subject accrual will allow the testing of any such agent. In contrast, proteomics measures the molecules most proximate to cellular phenotype and downstream of the many regulatory processes that govern the transition from genomic instruction to gene product. As such, it offers the potential to identify those aspects of cancer biology shared between tumor subgroups and indeed even different cancer types.
Quantitative proteomics is a comparative technology. When applied to cancer, the best comparison is to the cell type of origin for that cancer. If the cell of origin is unknown or of a developmental state rendering it unavailable for study, the choice of comparison becomes more contextual. In this study, we chose to compare tumors to normal cerebellum in order to provide a standard normalization and allow for relative comparisons between subgroups. In this way, proteins that were significantly different in more than one subgroup, and thus likely contributing to the biology of those subgroups, would not be lost due to a lack of a difference between those subgroups. We felt this approach was more likely to yield biology common across medulloblastoma and thus more favorable for translation. An alternative approach would have been to perform pairwise comparisons between all subgroups, but such an analysis would have yielded little meaningful biology given the size of the sample set, breadth of protein coverage and number of possible comparisons. Future studies may better apply such an approach, particularly when technological advances enabling higher throughput make larger sample sets more feasible.
Our findings illustrate the unique advantages of proteomics that are both complementary to, and exclusive of genomic output. For example, the observation that there is a relatively poor correlation between transcript and protein abundance provides an example of how proteomics can help clarify which gene expression differences have the potential to influence cellular phenotype. Chromosomal copy number alterations can involve many genes, yet proteomics can help to refine which of those gene products is most affected by the change in gene dose. For example, we found elevated levels of the KPNB1 and ARHGDIA proteins correlating with gained chromosome 17q yet these genes are not correspondingly elevated at the level of the transcript. Thus proteomics allows for the identification and, by virtue of their translation, prioritization of gene products that are candidates to generate phenotype from CNAs. Proteomics, when coupled with RNA-seq data, can also verify the importance of context specific isoform switches by confirming their translation and abundance. For example, we identify translated isoforms of 21 proteins that are restricted to one or more subgroups, thus demonstrating a mechanism beyond genomics driving subgroup biology. Another subgroup constrained protein we have confirmed is HMGA1, a stem cell phenotype regulator that is both a MYC target gene and a regulator of MYC [59, 73]. MYC has protean transformational effects upon cells with cancer specific patterns. In this case, proteomic investigation has highlighted the association of this MYC-HMGA1 effect with group 3 medulloblastoma. Lastly, pathway analysis can be used to predict prominent biological networks from proteomic data. In contrast with genomic networks, such predictions more directly represent cellular function such as the EIF4F pathway identified here. In addition to biological discovery, proteomics offers expanded translational opportunities owing to the fact that tumor proteins are the most accessible pharmaceutical targets, constitute cancer specific antigens for immunotherapy applications, and provide potential biomarkers. Taken together, these advantages demonstrate the necessity of integrating quantitative proteomic discovery data into large scale ‘omic’ based attempts to understand cancer biology.
The tumor samples used in this manuscript are from Children’s National Health System (Rood Lab, Washington DC, United States), German Cancer Research Center (DKFZ, Pfister Lab, Heidelberg, Germany), Hospital for Sick Children (Taylor Lab, Toronto Canada). The medulloblastoma samples have been classified using the methylation profiling classifier from MolecularNeuropathology.org. Normal cerebellum samples are from the University of Maryland Brain and Tissue Bank. Tissue sample information is listed in Additional file 1: Table S1.
Cell culture and EIF4E inhibitor treatments
MB002 and MB004 were gifts from Y.J. Cho (Oregon Health & Science University, Portland, OR, United States). R002, R026 and R032 were gifts from A. Moore and W. Ingram (Queensland Children’s Tumour Bank, Brisbane, Australia). DAOY, D556 and D283 cell lines were maintained in Eagle’s Minimum Essential Medium (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 U/mL penicillin and streptomycin (ATCC). All established cell lines were verified with STR analysis (GRCF at Johns Hopkins). R026, R032 and R060 cell lines were maintained in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F12 (ATCC) without HEPES supplemented with 10% fetal bovine serum (ATCC), Glutamine (ATCC) and 100 U/mL penicillin and streptomycin (ATCC). MB002 and MB004 cells were maintained in culture media with 1:1 Dulbecco modified Eagle medium (Gibco) and neural stem cell media (Gibco) supplemented with non-essential aminoacids (Gibco), Sodium Pyruvate (Gibco), HEPES, GlutaMax (Gibco), B27 (Gibco), EGF (Gibco), FGF (Millipore), Heparin (Stem Cell),LIF (Millipore), 10% fetal bovine serum (ATCC) and 100 U/mL penicillin and streptomycin (ATCC). Primary human normal astrocytes-Cerebellar were acquired from ScienCell and maintained in Astrocyte medium (ScienCell) supplemented with Fetal Bovine Serum (ScienCell), astrocyte grow supplement (ScienCell) and penicillin/streptomycin (ScienCell). All cells lines were maintained at 37 °C with 5% CO2 in a 95% humidified atmosphere.
For EIF4E inhibitor treatment, 1000 cells were plated in a 96 well plate in triplicates with dilutions of the EIF4E inhibitors, 4EGI-1(Selleckchem) and 4E1RCat (Selleckchem), and the chemotherapy agent cisplatin (TOCRIS Bioscience). DMSO was used as control. Cells were serum starved for 24 h before treatment and seventy-two hours after treatment cell viability was analyzed using the CellTuter-Glo®2.0 Assay kit (Promega) according to the manufacture recommendations. Experiments were repeated in triplicate.
SILAC proteomic analysis
We created a pooled super-SILAC reference atlas, termed the Labeled Atlas of Medulloblastoma Proteins (LAMP), by combining equal amounts of isotopically labeled proteins from 8 primary and established cell lines chosen to represent the breadth of medulloblastoma across the four genomic subgroups (DAOY, D556, D283, R026, R032, R060, MB002, and MB004). Super-SILAC refers to the use of multiple cell lines to create the reference atlas as opposed to just one. Briefly, cell lines are passaged in growth media that has been depleted of lysine and arginine. To this media, lysine and arginine incorporating 6 carbon isotopes (13C6) was added. The proteins made by these cells incorporate the isotopic amino acids creating a predictable mass increase. Serial aliquots were tested over time until isotopically labeled proteins exceeded 95% of the total. Mass spectrometry of the LAMP itself identified 4903 proteins.
We spiked in the LAMP at a ratio of 1:1 based upon BCA protein quantitation into protein lysates from 36 medulloblastoma tumors and 5 control cerebellum samples. 100 μg of protein from each sample was run in triplicate using 1-D SDS PAGE fractionation, cut into 32 bands and in-gel trypsin digested. Concentrated peptides from each band were injected via an autosampler (6uL) and loaded onto a Symmetry C18 trap column (5 mm, 300 μm i.d. × 23 mm, Waters) for 10 min at a flow rate of 10 μL/min, 100% A. The mobile phases consisted of water with 0.1% formic acid (A) and 90% acetonitrile (B). The sample was subsequently separated by a C18 reverse-phase column (3 μm, 200A, 100 mm × 15 cm, Magic C18, Michrom Bioresources) at a flow rate of 300 nL/min using an Eksigent nano-HPLC system (Dublin, CA). A 65 min linear gradient from 5 to 60% B was employed. Eluted peptides were introduced into the mass spectrometer via Michrom Bioresources CaptiveSpray. The spray voltage was set at 1.4 kV and the heated capillary at 200 °C. The LTQ-Orbitrap-XL (ThermoFisherScientific) was operated in data-dependent mode with dynamic exclusion in which one cycle of experiments consisted of a full-MS in the Orbitrap (300–2000 m/z) survey scan in profile mode, resolution 30,000 and five subsequent MS/MS scans in the LTQ of the most intense peaks in centroid mode using collision-induced dissociation with the collision gas (helium) and normalized collision energy value set at 35%.
For protein identification and quantification we used MaxQuant version 126.96.36.199 software developed by Max Planck Institute of Biochemistry (http://www.biochem.mpg.de/5111795/maxquant). Mass spectral data were uploaded into MaxQuant software. Files from each lane were searched against the forward and reverse Uniprot human database (UniProt release 2016_05 with 20,201 entries) for partially tryptic peptides allowing one missed cleavage, and possible modification of oxidized methionine (15.99492 Da) and heavy arginine (6.0201 Da) and heavy lysine (8.0142 Da). MaxQuant uses the Andromeda search engine. Mass tolerances were set at +/− 20 ppm for first peptide search and +/− 4.5 ppm for main peptide search, with intensity threshold of 500. Data were filtered based on a 5% protein false discovery rate. All the bands from each lane were summed as one sample in the analysis.
Microarray gene expression analysis
To generate gene expression profiling data, 19 tumor and 5 control specimens were homogenized in Trizol followed by phase separation of nucleic acids with chloroform. RNA was extracted using Picopure RNA isolation kit (Arcturus Bioscience, Mountain View, CA). DNA was removed by treating columns with RNase Free DNase (Qiagen, Valencia, CA). RNA integrity and concentration were quantified using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The GeneChip WT Plus kit (Affymetrix, Santa Clara, CA) was used for cRNA synthesis from total of 250 ng of RNA. cRNA was hybridized to GeneChip Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA) and probe fluorescence intensity was detected with GeneChip® System 3000Dx v.2 (Affymetrix, Santa Clara, CA). Gene expression profiles for the rest of the tumors (16) were previously generated;7 using the GeneChip™ Human Genome U133A 2.0 Array and 9 using the Human Gene 1.1 ST Array (GEO accession number GSE37385 and GSM324067 respectively) (Additional file 1: Table S1). Expression data for each Chip type were analyzed independently in the R environment (v3.4.1) (https://www.r-project.org) with the oligo package (v1.36.1). Differentially expressed genes for each subgroup versus cerebellum were calculated using the limma R-package (v3.28.21).
Evaluating mRNA-protein correlation
mRNA and protein expression values. For mRNA, expression data values (log2) from every chip type were merged and corrected for potential chip type effect applying an Empirical Bayes method using the combat function from the sva R package (v3.20.0). For proteins, we used the ratio of heavy over light (H/L) values as described above.
Number of overlapping genes in proteomic and mRNA data sets
The proteomic data set used for mRNA-protein correlation (35 tumors, Additional file 1: Table S1) included a total of 2846 gene products quantified in at least 1 tumor; 2430 of these were present in the mRNA expression dataset. To compare mRNA and protein variation across tumors, we focused on 1240 genes with quantified protein in at least one third of the tumors. For subgroup specific analysis, we used genes with quantified protein in at least one half of the tumors.
Correlation between mRNA and protein variation
We first calculated the Spearman correlation coefficient between the mRNA expression values (log2) and the protein H/L ratio for each of the 1240 genes. Then, p-values corresponding to the coefficients were calculated with < 0.05 considered significant. The same approach was applied to each subgroup. Correlation differences among the four subgroups were evaluated based on the Kruskal-Wallis rank-sum test and differences between Group 4 and the rest was evaluated based on a two-sided Wilcoxon rank-sum test.
KEGG pathway analysis
Based on Spearman correlation coefficients, we separated the highly correlated mRNA-protein pairs (Quartile 3) from those with low correlation (Quartile 1). Next, KEGG pathway analysis using the DAVID Bioinformatics Database (DAVID Bioinformatics Resources, http://david.abcc.ncifcrf.gov/) was performed with the protein identifier of genes in each group to find enriched biological processes. Pathways with a Bonferroni corrected p-value < 0.01 in at least one group were selected.
mRNA-protein correlation versus molecular stability
To analyze the relationship between mRNA-protein correlation and stability, we used mRNA and protein half-life data from mouse fibroblast cell line . Only genes (456) in common between both our datasets and the mouse fibroblast dataset were included in the analysis. mRNA or proteins with half-life values higher than the upper quartile (Q3) were considered stable and those with half-lives in the lowest quartile (Q1) were considered unstable. Correlation differences between the 2 categories were calculated based on two-sided Wilcoxon rank-sum test.
Genome wide methylation analysis
DNA (500 ng) was obtained from 35 tumor and 5 control cerebellum tissue lysates using the Gentra Puregene DNA extraction kit (Quiagen, Valencia, CA) (Additional file 1: Table S1). DNA was prepared for methylation analysis via bisulphate conversion using EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA). Bisulphite-converted DNA was denatured and neutralized. After amplification via PCR, DNA was fragmented and hybridized onto the Infinium Methylation EPIC BeadChip (Illumina, San Diego, CA). Raw data files (.idat) generated by the Illumina iScan system were processed in the R statistical environment (v3.3.1) (https://www.r-project.org) using minfi (v 1.22.1) and ChAMP (v2.4.1) packages. Probes were removed if their p-value was above 0.01 in one or more samples. Additionally, we filtered out probes with a beadcount lower than 3 in at least 5% of samples. Finally, we removed probes on the sex chromosomes as well as those located on or close to known single nucleotide polymorphism (SNP). We retained a total of 749,678 probes for the analysis. The data was normalized using the BMIQ method. Differentially methylated regions (DMRs) were calculated for each subgroup versus cerebellum using ChAMP R-package with the DMRcate method .
Methylation array copy number analysis
Copy number segmentation was performed from the genome wide methylation arrays using the conumee R-package (v1.8.0) using normal cerebellum as diploid controls. Segmented copy number estimates were processed for input with GISTIC2  using the default parameters. The copy number by genes was used for the correlation analysis. Significant broad chromosome arm lesions were evaluated across all samples without separating them between subgroups (q-value < 0.1).
Effect of methylation and copy number on protein expression
Correlation of methylation status and gene/protein expression
Differentially methylated regions (DMRs) were calculated comparing each subgroup to cerebellum using ChAMP R-package with the DMRcate  method. Each DMR was assigned to a gene according to their coordinates in the hg19 reference genome. Differentially expressed proteins or genes (up- or down-regulated) were correlated with their associated absolute DMR values (gain or loss). Odds ratios were calculated for each subgroup using Fisher’s exact method.
Correlation of copy number alteration and gene/protein expression
The matched CNA (Copy Number alteration), protein and mRNA abundance measurements from 32 tumors (Additional file 1: Table S1) were used to study the impact of CNA on gene and protein expression. We focused on 1148 genes with quantified proteins in at least 33% of the tumors that were also included in the CNA and mRNA datasets. For each of those genes, we calculated the Spearman correlation coefficient between CNA and mRNA/protein measurements. Significant calls were made based on a p-value cutoff of 0.05.
NMF clustering was performed from the proteomic ratios H/L using the NMF R-package (v0.20.0) . For NMF clustering we used proteins quantified in at least 40% (1158 proteins) of the samples in the dataset. The missing ratio L/H values (NAs) were replaced by the lowest value found in the dataset (0.05) to approximate the lower limit of detection. We used the NMF algorithm brunet  with 5000 runs for each factorization rank k between 2 and 10 using the default seed method. The same NMF settings were applied to the transcript abundance of each of the genes coding for the proteins used in the proteomic classification.
Differentially expressed proteins
Proteins from each medulloblastoma subgroup were compared to control cerebellum in a pairwise fashion using the R environment (v3.4.1) with statistical confidence measured using the Wilcoxon rank-sum test at a p-value cut-off of 0.05. Proteins were also required to be present in at least half of the total replicates of both subgroups in a comparison. To these differentially abundant proteins were added the subgroup restricted proteins, those that were present in at least half of the samples of one subgroup and absent in the remaining samples.
Pathway analysis of differentially expressed proteins was performed using Ingenuity Pathway Analysis (IPA) v. 01–04. Lists of differentially expressed proteins and their fold changes for each genomic subgroup, compared to control cerebellum, were imported into IPA. For this analysis, we removed from consideration the 2 tumor samples that clustered separately given their distinctiveness. Proteins that were present in one group and absent in another were arbitrarily assigned a fold change value equal to the average fold change of quantifiable proteins +/− two standard deviations. Core expression analysis was created for each of the lists considering direct and indirect relationships using Ingenuity Knowledge Base genes to calculate p-values. Top pathways for each subgroup were then selected based on highest ranked p-values (Additional file 14: Table S6).
Protein isoform quantification
To create the customized MB specific isoform database, we used the R package customProDB (v1.16) . RNA-seq data from 167 publicly available MB tumors (Datasets: EGAD00001001899, EGAD00001002683, EGAD00001001210, EGAD00001001620, and EGAD00001000328) were mapped to human genome GRCh38/hg38 using STAR(v.2.5.1) . Protein coding transcripts (UCSC RefSeq annotation) were quantified using the calculateRPKM function of the customProDB package. Only protein coding transcripts expressed in at least 5% of the samples and belonging to the top 80% by expression were included in the database. For protein isoform identification and quantification, we used MaxQuant software (v.188.8.131.52). Files from each lane were searched against the MB specific isoforms database for partially tryptic peptides allowing one missed cleavage, and possible modification of oxidized methionine (15.99492 Da), heavy arginine (6.0201 Da) and heavy lysine (8.0142 Da). Mass tolerances were set at +/− 20 ppm for first peptide search and +/− 4.5 ppm for main peptide search, with an intensity threshold of 500. Data were filtered based on a 5% protein false discovery rate. All the bands from each lane were summed as one sample in the analysis. For differential isoform expression, we focused on genes that contain isoforms in more than one protein group. The statistical confidence of the differentially expressed isoforms was measured using the Kruskal-Wallis rank-sum test. Significant calls were made based on a p-value cutoff of 0.05 (Additional file 17: Table S8).
RNA-seq data from 167 publicly available MB tumors (Datasets: EGAD00001001899, EGAD00001002683, EGAD00001001210, EGAD00001001620, and EGAD00001000328) were mapped to human genome GRCh38/hg38 using STAR(v.2.5.1) . Genes and isoform expression was quantified using the cufflinks software (v2.2.1)  and hg38 UCSC transcript annotation.
Overall survival functions were estimated using the Kaplan-Meier method and p-values were calculated using the log-rank test. The statistical analysis was performed in the R statistical environment using the R package survival (v2.41–3) and survminer (v0.4.0). Gene expression and overall survival data from 113 Group 3 medulloblastoma tumors were downloaded from a previously published dataset  (GEO accession data: GSE85218). Group 3 medulloblastoma tumors were separated into samples with HMGA1 expression greater and less than the median.
Protein extracts were prepared by lysing tumor tissue cells in RIPA Lysis buffer (Millipore) containing 50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1× protease inhibitor cocktail (Roche Applied Science). Tumor lysates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with HMGA1 antibodies (D6A4, Cell Signaling). Protein detection was performed using ECL Western blotting detection reagents (SuperSignal West Dura, Thermo Scientific).
Quantitative proteomics offers an important dimension beyond genomics, proximate to the molecular disease processes underlying malignancy. Here, we present the largest tissue based Super-SILAC quantitative proteomics study to date demonstrating how proteomics complements genomic platforms to yield a more complete understand of functional tumor biology and identify novel therapeutic targets for medulloblastoma. We conclude that there is a poor correlation between epigenetic features/transcript level and protein abundance. However, proteomics can help distill the most salient genomic features based upon the axiom that a genetic event must be translated to the proteome in order to exert a functional effect upon the cell. Proteomics can also help identify which genes are affected by dosage changes resulting from chromosomal copy number alterations. In concert with RNA-seq, proteomics can identify disease-specific translated splice isoforms as well as novel protein isoforms. While proteomic data can be used to recapitulate the genomic subgroups, the gene products it uses to do so are different than those identified with genomic platforms, thus offering a new cohort of targets for attempted intervention. Indeed, signaling networks built from protein data are more reflective of cell biology and therefore a more robust source of therapeutic targets. Thus proteomics can be used retrospectively to help interpret and filter the wealth of genomic findings as well as prospectively to map out functional cellular biology to be exploited for therapeutic development.
This work has been supported by the Lilabean Foundation, the Prevent Cancer Foundation, and Jeff Gordon Children’s Foundation. This project was also supported by Award Number 1U54HD090257-01 from the NIH, District of Columbia Intellectual and Developmental Disabilities Research Center Award (DC-IDDRC) program. We wish to thank Y.J. Cho (OHSU Portland, OR A. Moore and W. Ingram, (Queensland Children’s Tumour Bank, Brisbane, AU) for kindly providing us with the MB002, MB004, R002, R026 and R032 cell lines. Human cerebellar tissue was obtained from University of Maryland Brain and Tissue Bank which is a Brain and Tissue Repository of the NIH NeuroBioBank. The authors also thank Andrey Korshunov for the provision of medulloblastoma tissue samples.
Availability of data and materials
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner repository with the dataset identifier PXD008750. The expression and methylation microarray data has been deposited in Gene Expression Omnibus (GEO) under the accession number GSE109403. The previously published expression microarray data used in this manuscript is available under the accession numbers: GSE37385 and GSM324067. The previously published RNA-seq data used in this work is available in the European Genome-phenome Archive (EGA) under the accession numbers: EGAD00001001899, EGAD00001002683, EGAD00001001210, EGAD00001001620, and EGAD00001000328.
LSL, MS, JS, KB and HZ performed the experiments. SR-H, MS and HG-D analyzed the data. PAN, SP and MDT contributed tissue samples and expert manuscript review. SR-H and BRR designed the study. SR-H, MS and BRR wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Ethics approval has been obtained for the use of de-identified human tissue in this study.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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