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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. Electronic supplementary material The online version of this article (10.1186/s40478-018-0548-7) contains supplementary material, which is available to authorized users.

Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.

The genomic characterization of medulloblastoma at the level of the chromosome, epigenome and transcriptome has radically redefined diagnostic classifications and reoriented disease definitions toward a biological underpinning. However, it still remains a challenge to leverage this wealth of data toward a true understanding of disease biology and subsequently to capitalize on the promise of biologically based therapies. One reason for this difficulty is the loose correlation between the genetic and the functional aspects of the cancer cell resulting from multilayered regulatory mechanisms governing protein production, the predominant functional moiety of the cell. To better actualize the promise of genomics, it is necessary to add an understanding of the proteomic dimension to cancer cell biology. We have undertaken a super-SILAC based quantitative proteomics survey of 42 tissue samples spanning the 4 genomic 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 one additional subgroup. Adding array gene expression data to this analysis, we calculate a relatively poor correlation between mRNA and protein abundance with the best concordance found among stable proteins. EPIC 850k methylation array data performed on the same samples extend this analysis to the epigenome yielding chromosomal copy number variations and correlations to gene promoter methylation. From the quantitative proteomic data, we are able to discern networks of functional pathways with differential activation between the medulloblastoma subgroups and normal control cerebellum yielding potential avenues for clinical intervention. This work demonstrates that quantitative proteomics can further elucidate the biology inherent in a genomically based classification regime.

Diffuse axonal injury (DAI) is an evolving axonopathy commonly characterized clinically as widespread damage to the white matter tracts. In recent electrophysiological studies, researchers have proposed that myelinated and unmyelinated axons differ in their vulnerability and functional recovery following DAI. In this study we present for the first time an in vitro stretch-injury approach that utilizes a novel myelinating co-culture system to determine the differential response between myelinated and non-myelinated axon bundles to injury. In implementing this technique we demonstrate that myelinated axon bundles are less vulnerable to stretch injury compared to caliber-matched non-myelinated bundles. Interestingly, moderate axonal strain did not induce demyelination, but instead caused an increase in the proportion of degenerated myelin basic protein over time. Additionally, there were no significant differences in the expression of axonal swellings, which is indicative of disrupted axonal transport. In summary, we present an ideal in vitro model that permits further mechanistic investigations into the role of myelin and oligodendrocyte-neuron interactions in response to DAI.

Acute axonal shear and stretch in the brain induces an evolving form of axonopathy and is a major cause of ongoing motor, cognitive and emotional dysfunction. We have utilized an in vitro model of mild axon bundle stretch injury, in cultured primary cortical neurons, to determine potential early critical cellular alterations leading to secondary axonal degeneration. We determined that transient axonal stretch injury induced an initial acute increase in intracellular calcium, principally derived from intracellular stores, which was followed by a delayed increase in calcium over 48 h post-injury (PI). This progressive and persistent increase in intracellular calcium was also associated with increased frequency of spontaneous calcium fluxes as well as cytoskeletal abnormalities. Additionally, at 48 h post-injury, stretch-injured axon bundles demonstrated filopodia-like sprout formation that preceded secondary axotomy and degeneration. Pharmacological inhibition of the calcium-activated phosphatase, calcineurin, resulted in reduced secondary axotomy (p < 0.05) and increased filopodial sprout length. In summary, these results demonstrate that stretch injury of axons induced an initial substantial release of calcium from intracellular stores with elevated intracellular calcium persisting over 2 days. These long-lasting calcium alterations may provide new insight into the earliest neuronal abnormalities that follow traumatic brain injury as well as the key cellular changes that lead to the development of diffuse axonal injury and secondary degeneration.

The ubiquitin proteasome system (UPS) plays a vital role in the regulation of protein degradation. Ubiquitination of proteins has been implicated in the pathological cascade associated with neuronal degeneration in both neurodegenerative disease and following acquired central nervous system (CNS) injury. In the present study, we have investigated the role of the UPS following mild to moderate in vitro axonal stretch injury to mature primary cortical neurons, a model of the evolving axonal pathology characteristic of diffuse axonal injury following brain trauma. Transient axonal stretch injury in this model does not involve primary axotomy. However, delayed accumulation of ubiquitin in neuritic swellings at 48 h post-injury (PI) was present in axonal bundles, followed by approximately 60% of axonal bundles progressing to secondary axotomy at 72 h PI. This delayed accumulation of ubiquitin was temporally and spatially associated with cytoskeletal damage. Pharmacological inhibition of the UPS with both MG132 and lactacystin prior to axonal injury resulted in a significant (p < 0.05) increase in the number of axonal bundles progressing to secondary axotomy at 48 and 72 h PI. These results demonstrate that, following mild to moderate transient axonal stretch injury, UPS activity may assist structural reorganization within axons, potentially impeding secondary axotomy. Protein ubiquitination in the axon may therefore have a protective role relative to the diffuse axonal changes that follow traumatic brain injury.

The genomic characterization of medulloblastoma at the level of the chromosome, epigenome and transcriptome has radically redefined diagnostic classifications and reoriented disease definitions toward a biological underpinning. However, it still remains a challenge to leverage this wealth of data toward a true understanding of disease biology and subsequently to capitalize on the promise of biologically based therapies. One reason for this difficulty is the loose correlation between the genetic and the functional aspects of the cancer cell resulting from multilayered regulatory mechanisms governing protein production, the predominant functional moiety of the cell. To better actualize the promise of genomics, it is necessary to add an understanding of the proteomic dimension to cancer cell biology. We have undertaken a super-SILAC based quantitative proteomics survey of 42 tissue samples spanning the 4 genomic 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 one additional subgroup. Employing a custom built RNA-seq based searchable proteome database, we are also able to use peptide sequence data to discover medulloblastoma specific protein isoforms. Adding array gene expression data to this analysis, we calculate a relatively poor correlation between mRNA and protein abundance with the best concordance found among stable proteins. From the quantitative proteomic data, we are able to discern networks of functional pathways with differential activation between the medulloblastoma subgroups and normal control cerebellum yielding potential avenues for clinical intervention. This work demonstrates that quantitative proteomics can further elucidate the biology inherent in a genomically based classification regime.

Brain tumors are the leading cause of cancer-related deaths in children, and medulloblastoma is the most prevalent malignant childhood/pediatric brain tumor. Providing effective treatment for these cancers, with minimal damage to the still-developing brain, remains one of the greatest challenges faced by clinicians. Understanding the diverse events driving tumor formation, maintenance, progression, and recurrence is necessary for identifying novel targeted therapeutics and improving survival of patients with this disease. Genomic copy number alteration data, together with clinical studies, identifies c-MYC amplification as an important risk factor associated with the most aggressive forms of medulloblastoma with marked metastatic potential. Yet despite this, very little is known regarding the impact of such genomic abnormalities upon the functional biology of the tumor cell. We discuss here how recent advances in quantitative proteomic techniques are now providing new insights into the functional biology of these aggressive tumors, as illustrated by the use of proteomics to bridge the gap between the genotype and phenotype in the case of c-MYC-amplified/associated medulloblastoma. These integrated proteogenomic approaches now provide a new platform for understanding cancer biology by providing a functional context to frame genomic abnormalities.

Despite landmark advances in the genomics of medulloblastoma, significant ambiguity exists in the translation these findings into therapeutic advances. We propose that quantitative proteomics can be used to better discern driver aberrations and more directly view the functional, and therefore targetable, biology of this disease. METHODS: We created a Stable isotope Labeling of Amino acids in cell Culture (SILAC) reference atlas of proteins from 4 medulloblastoma cell lines and spiked it into cell lysates of low passage medulloblastoma cultures. A Hybrid LTQ-Orbitrap mass spectrometer was used to quantitate proteins relative to the atlas. RESULTS AND DISCUSSION: We used the SILAC reference atlas to quantitate the proteome of myc-amplified and non-myc-amplified group 3 low passage medulloblastoma cells. Accurate quantitation was achieved for an average of 1215 proteins per primary cell line. Technical replicates resulted in r2 values between 0.91 and 0.98. The differentially abundant proteins myc-amplified tumors reveal protein-protein connectivity networks associated with alternative splicing, ribosome biogenesis and metabolism. At the junction of splicing and metabolism, we confirmed the upregulation of specific splicing factors (HNRNPs) in the myc-amplified group. We demonstrate the HNRNP control of PKM isoform generation and the subsequent effect upon tumor cell metabolism and resistance to hypoxia. Furthermore, we confirm these proteomic findings using independent medulloblastoma tissue samples and show they are conserved in low passage primary tumor cultures. CONCLUSIONS: Our results demonstrate the advantages of quantitative proteomic profiling to discover oncogenic driver molecules. This is particularly significant given the emerging evidence of the poor correlation between mRNA abundance and protein abundance. Ultimately, insight into the protein level biology provided by these types of studies will yield more readily accessible therapeutic targets and allow clearer inferences of disease biology.

We report a new model of transient axonal stretch injury involving pressurized fluid deflection of bundles of axons, resulting in a transient 1-6% increase in original axon length to investigate the slow progression of axonal alterations that are characteristic of diffuse axonal injury (DAI). We found no discernable difference in axon bundle morphology or cytoskeletal neurofilament protein arrangement between unstretched and stretched axonal bundles at 24 h post-injury. However, by 48 h post-injury, there was a stereotypical response of stretched axons involving characteristic neurofilament alterations that bear similarities to in vivo neuronal responses associated with DAI that have been reported previously. For instance, neurofilament protein immunoreactivity (SMI-312) was increased in axons contained within 51% of all injured axon bundles at 48 h compared to surrounding unstretched axon bundles, suggestive of neurofilament compaction. Furthermore, axonal bundle derangement occurred in 25% of injured axon bundles, with individual fibres segregating from each other and becoming undulating and wavy. By 72 h post-stretch, 70% of injured axon bundles underwent secondary axotomy, becoming completely severed at the site of initial stretch injury. While these results suggest a temporal series of stereotypical responses of axons to injury, we were able to distinguish very clear differences between mildly (100-103% increase in original axonal length) injured and strongly injured (106%+) axons. For instance, mildly injured axons developed increased neurofilament immunoreactivtity (SMI-312) within 48 h, and the marked development of ring-like neurofilament immunoreactive structures within axonal bundles, which were rarely axotomized. Conversely, at more severe strain levels increased neurofilament immunoreactivity was less apparent, while axons often became distorted and disorganised within axonal bundles and eventually became completely disconnected. Almost no ring-like neurofilament structures were observed in these severely injured axonal bundles. This suggests that axons do not respond in a stereotypical manner to a transient stretch insult, and indeed that variable degrees of stretch injury activate different responses within axons, with dramatically different outcomes. Hence, it is possible that the cytoskeletal characteristics that we have used in this study may be useful parameters for discriminating between mildly and severely injured axons following TBI.