Proteomic profiling of high risk medulloblastoma reveals functional biology

Abstract

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.

Keywords: cMYC; cancer; glycolysis; medulloblastoma; proteomics.

Figure 1. Effective quantification of human primary tumor cells using a super-SILAC reference standard

A. Experimental scheme of quantification analysis using mixed lysates of multiple MB tumor cell lines. Lysates of labeled cells (Lys-8, ¹³C₆ ¹⁵N₂-Lysine; Arg-10, ¹³C₆ ¹⁵N₄-Arginine) are mixed with tumor lysate (at a 1:1 ratio) and analyzed by high resolution liquid chromatography-MS/MS. B; Histograms of the ratios between the tumor protein and our super-SILAC reference, and a comparison of ratios (r = Pearson correlation coefficient) between replicates demonstrates the high quantification accuracy of our technique. Superior accuracy is achieved when quantified proteins (proportion indicated by percentage above histogram) lie within four-fold ratio between tumor and super-SILAC reference [10]

Figure 2. Differential proteome expression patterns between MYC-amplified versus non-amplified tumors

A. Unsupervised hierarchical clustering (normalized log₂ and standardized to mean signal = 0 and standard deviation = 1) of protein expression profiles from six Group 3 primary MB cells B. Differential protein expression drives segregation of tumor cells with MYC-amplification, which is not seen when similar analysis is conducted using transcriptome data (see FigS3). B, C. Top up-regulated proteins and predicted functional pathways in MYC-amplified tumors. STRING-generated protein-protein interaction pathways revealed significant (P, 0.05; t-test) connectivity in alternative splicing, ribosome biogenesis and metabolism pathways. Error bars represent standard deviation between the cultures in each subgroup (n = 6).

Figure 3. Validation of differential protein expression in independent human MB tissue samples and evidence for increased alternative splicing of PKM

A. Confirmation of proteomic alterations in independent human MB tissue samples using western blot. Significant (t-test) alterations in splicing associated factors were conserved between low passage primary tumor cell cultures and human tissue samples. B. Quantitative real-time PCR in human MB tissue and cell cultures reveals increased alternative splicing of PKM to produce the pro-glycolytic PKM2 isoform in MYC amplified tumors. C. cDNA samples from independent human tissue samples were subjected to PCR amplification using primers amplifying a 442 bp exon 8-11 region common to PKM1 and PKM2. Following incubation with PstI, the uncleaved (PKM1, 442 bp) and cleaved (PKM2, 246 and 196 bp) amplification products were separated by electrophoresis and quantified, with total signal set at 100 for each lane (Lane 1 uncut MYC-, Lane 2 uncut MYC+). There is significantly higher splicing of mRNA which is indicative of higher PKM2 isoforms.

Figure 4. MYC-amplified tumor cells have significantly altered metabolic activity compared to the rest of Group 3 MB cells (non-MYC amplified)

A. Although MYC amplified tumor cells have significantly lower reactive oxidative species (H₂O₂), they also have higher levels of total nicotinamide adenine dinucleotide phosphates (t-test; p < 0.01; error bars are standard deviation between subgroup cultures). B. ATP production under hypoxic conditions (48 hours, 0.1% O₂) is significantly reduced in most (3/4) non-MYC amplified tumor cells, but is not perturbed in MYC-amplified tumors (3/4). Error bars are standard deviation of sample replicates; t-test,* denotes P < 0.05.