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Background Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). Methods Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age Results Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. Conclusion Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM. Graphical abstract

Resistance to chemotherapy-induced cell death is a major barrier to effective treatment of solid tumours such as colorectal cancer, CRC. Herein, we present a study aimed at developing a proteomics-based predictor of response to standard-of-care (SoC) chemotherapy in combination with antagonists of IAPs (inhibitors of apoptosis proteins), which have been implicated as mediators of drug resistance in CRC. We quantified the absolute expression of 19 key apoptotic proteins at baseline in a panel of 12 CRC cell lines representative of the genetic diversity seen in this disease to identify which proteins promote resistance or sensitivity to a model IAP antagonist [birinapant (Bir)] alone and in combination with SoC chemotherapy (5FU plus oxaliplatin). Quantitative western blotting demonstrated heterogeneous expression of IAP interactome proteins across the CRC cell line panel, and cell death analyses revealed a widely varied response to Bir/chemotherapy combinations. Baseline protein expression of cIAP1, caspase-8 and RIPK1 expression robustly correlated with response to Bir/chemotherapy combinations. Classifying cell lines into 'responsive', 'intermediate' and 'resistant' groups and using linear discriminant analysis (LDA) enabled the identification of a 12-protein signature that separated responders to Bir/chemotherapy combinations in the CRC cell line panel with 100% accuracy. Moreover, the LDA model was able to predict response accurately when cells were cocultured with Tumour necrosis factor-alpha to mimic a pro-inflammatory tumour microenvironment. Thus, our study provides the starting point for a proteomics-based companion diagnostic that predicts response to IAP antagonist/SoC chemotherapy combinations in CRC.

Antibodies targeting the human epidermal growth factor receptor (EGFR) are used for the treatment of RAS wild-type metastatic colorectal cancer. A significant proportion of patients remains unresponsive to this therapy. Here, we performed a reverse phase protein array-based (phospho)protein analysis of 63 KRAS, NRAS, BRAF and PIK3CA wild-type metastatic CRC tumours. Responses of tumours to anti-EGFR therapy with cetuximab were recorded in patient-derived xenograft (PDX) models. Unsupervised hierarchical clustering of pre-treatment tumour tissue identified three clusters, of which cluster C3 was exclusively composed of responders. Clusters C1 and C2 showed mixed responses. None of the three protein clusters showed a significant correlation with transcriptome-based subtypes. Analysis of protein signatures across all PDXs identified 14 markers that discriminated cetuximab-sensitive and -resistant tumours: PDK1 (S241), Caspase-8, Shc (Y317), Stat3 (Y705), p27, GSK-3β (S9), HER3, PKC-α (S657), EGFR (Y1068), Akt (S473), S6 Ribosomal Protein (S240/244), HER3 (Y1289), NF-κB-p65 (S536) and Gab-1 (Y627). Least absolute shrinkage and selection operator and binominal logistic regression analysis delivered refined protein signatures for predicting response to cetuximab. (Phospo-)protein analysis of matched pre- and post-treated models furthermore showed significant reduction of Gab-1 (Y627) and GSK-3β (S9) exclusively in responding models, suggesting novel targets for treatment. This article is protected by copyright. All rights reserved.

ObjectiveHere, we systematically investigated alterations in the bacteriome and mycobiome of CRC patients in tumours and matched adjacent mucosa resulting in the identification of microbiome-based subtypes associated with host clinico-pathological and molecular characteristics.DesignDiversity and composition of bacteriome and mycobiome of tumour and adjacent mucosa, resulting subtypes were computationally deconvoluted from RNA sequencing, using >10000 samples from in-house and publically available patient cohorts.ResultsThe bacteriome of tumours had higher dominance and lower α-diversity compared to matched adjacent local and distant mucosa. Tumours were enriched with Proteobacteria (Gammaproteobacteria class), Fusobacteria (including Fusobacterium Nucleatum species) and Basidiomycota fungi (Malasseziaceae family). Tumours were depleted of Bacteroidetes (Bacteroidia class), Firmicutes (Clostridia class) and Ascomycota (Sordariomycetes and Saccharomycotina). Tumours and adjacent mucosa samples were classified into 4 microbial subtypes, termed C1 to C4, based on the bacteriome and mycobiome composition. The bacterial Propionibacteriaceae, Enterobacteriaceae, Fusobacteriaceae, Bacteroidaceae and Ruminococcaceae and the fungal Malasseziaceae, Saccharomycetaceae and Aspergillaceae were among the key families driving the microbial subtyping. Microbial subtypes were associated with distinct tumour histology and patient phenotypes and served as an independent prognostic marker for disease-free survival. Key associations between microbial subtypes and alterations in host immune response and signalling pathways were validated in the TCGA pan-cancer cohort. The microbial subtyping demonstrated stratification value in the pan-cancer settings beyond merely representing differences in survival by cancer type.ConclusionsThis study demonstrates the translational potential of microbial subtyping in CRC patient stratification, and provides avenues to design tailored microbiota modulation therapy to further precision oncology.Statement of significanceWhat is already known on this subject?The microbiome has been implicated in the pathogenesis, progression and therapeutic response in patients diagnosed with CRC and other cancers.The vast majority of studies to date has focussed on investigating the bacteriome while the critical role played by the mycobiome in shaping cancer has begun to be explored more recently.The bacterial and fungal composition and diversity in on-tumour tissue compared to matched local and distal off-tumour mucosa is largely unexplored.Tumorigenesis may be promoted via alterations of the microbiome ecosystem that may be better recapitulated by a multi-kingdom microbial signature rather than by the abundance of individual bacterial or fungal microorganisms.What are the new findings?On-tumour tissue was enriched with Fusobacteria, Proteobacteria, Basidyomicota and depleted of Bacteroidetes, Firmicutes, Ascomycota compared with adjacent off-tumour mucosa.We stratified >600 CRC patients into four distinct microbial-based subtypes (C1-C4) according to their bacteriome and mycobiome composition. The microbial subtypes were associated with distinct prognosis, clinical phenotypes such as staging, tumour location, history, lymphovascular invasion, TP53 status and clinical outcome.Furthermore, the majority of matched adjacent mucosa samples were classified as C1 and paired tumour-matched normal samples demonstrated a robust shift towards the C1 subtype in off-tumour tissue, suggesting that the C1 subtype may recapitulate a healthier-like microbiome. This hypothesis was supported by the microbial subtyping of colon samples from healthy subjects that were categorised as C1 almost exclusively.The identified microbial subtyping demonstrated stratification value in the pan-cancer settings (n=28 additional solid cancer indications spanning >9000 samples) beyond merely representing differences in survival by cancer type, providing the strongest stratification in liver cancer.How might it impact on clinical practise in the foreseeable future?This study laids the foundation to develop a microbial signature as biomarker to clinically manage CRC and other solid cancers and potentially lead to microbiome-based companion diagnostics to design microbiota modulation therapy tailored to specific patients subgroups with distinct bacteriomes and mycobiomes.

Cancer cells’ ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells, and quantification of 18 cell lineage and apoptosis proteins. We identified an enrichment for BCL2 in immune, and BAK, SMAC, and XIAP in cancer cells. Ordinary differential equation-based modeling of apoptosis sensitivity at single-cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. Systems modeling at single-cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, but showed significant inter- and intra-tumor heterogeneity.

Resistance to chemotherapy often results from dysfunctional apoptosis, however multiple proteins with overlapping functions regulate this pathway. We sought to determine whether an extensively validated, deterministic apoptosis systems model, &lsquo, DR_MOMP&rsquo, could be used as a stratification tool for the apoptosis sensitiser and BCL-2 antagonist, ABT-199 in patient-derived xenograft (PDX) models of colorectal cancer (CRC). Through quantitative profiling of BCL-2 family proteins, we identified two PDX models which were predicted by DR_MOMP to be sufficiently sensitive to 5-fluorouracil (5-FU)-based chemotherapy (CRC0344), or less responsive to chemotherapy but sensitised by ABT-199 (CRC0076). Treatment with ABT-199 significantly improved responses of CRC0076 PDXs to 5-FU-based chemotherapy, but showed no sensitisation in CRC0344 PDXs, as predicted from systems modelling. 18F-Fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) scans were performed to investigate possible early biomarkers of response. In CRC0076, a significant post-treatment decrease in mean standard uptake value was indeed evident only in the combination treatment group. Radiomic CT feature analysis of pre-treatment images in CRC0076 and CRC0344 PDXs identified features which could phenotypically discriminate between models, but were not predictive of treatment responses. Collectively our data indicate that systems modelling may identify metastatic (m)CRC patients benefitting from ABT-199, and that 18F-FDG-PET could independently support such predictions.

AKR1B1 and AKR1B10, members of the aldo-keto reductase family of enzymes that participate in the polyol pathway of aldehyde metabolism, are aberrantly expressed in colon cancer. We previously showed that high expression of AKR1B1 (AKR1B1HIGH) was associated with enhanced motility, inflammation and poor clinical outcome in colon cancer patients. Using publicly available datasets and ex vivo gene expression analysis (n = 51, Ankara cohort), we have validated our previous in silico finding that AKR1B1HIGH was associated with worse overall survival (OS) compared with patients with low expression of AKR1B1 (AKR1B1LOW) samples. A combined signature of AKR1B1HIGH and AKR1B10LOW was significantly associated with worse recurrence-free survival (RFS) in microsatellite stable (MSS) patients and in patients with distal colon tumors as well as a higher mesenchymal signature when compared with AKR1B1LOW/AKR1B10HIGH tumors. When the patients were stratified according to consensus molecular subtypes (CMS), AKR1B1HIGH/AKR1B10LOW samples were primarily classified as CMS4 with predominantly mesenchymal characteristics while AKR1B1LOW/AKR1B10HIGH samples were primarily classified as CMS3 which is associated with metabolic deregulation. Reverse Phase Protein Array carried out using protein samples from the Ankara cohort indicated that AKR1B1HIGH/AKR1B10LOW tumors showed aberrant activation of metabolic pathways. Western blot analysis of AKR1B1HIGH/AKR1B10LOW colon cancer cell lines also suggested aberrant activation of nutrient-sensing pathways. Collectively, our data suggest that the AKR1B1HIGH/AKR1B10LOW signature may be predictive of poor prognosis, aberrant activation of metabolic pathways, and can be considered as a novel biomarker for colon cancer prognostication.

Chemotherapy-induced diarrhoea (CID) is a common dose-limiting adverse event in patients with cancer. Here, we hypothesise that chemotherapy evokes apoptosis in normal gut epithelium, contributes to CID and that patients with increased risk of CID can be identified using a systems model of BCL-2 protein interactions (DR_MOMP) that calculates the sensitivity of cells to undergo apoptosis. Normal adjacent gut epithelium tissue was collected during resection surgery from a cohort of 35 patients with stage II-III colorectal cancer (CRC) who were subsequently treated with capecitabine, XELOX or FOLFOX. Clinical follow-up, type and grade of adverse events during adjuvant chemotherapy were recorded. The level of five BCL-2 proteins required for the calculation of the DR_MOMP score was quantified together with 62 additional signalling proteins related to apoptotic pathways. Odds ratios for the occurrence of diarrhoea were determined using multinomial logistic regression (MLR). Patients treated with capecitabine who had a DR_MOMP score equal or higher than the mean had a significantly lower frequency of diarrhoea significantly compared to patients below the mean. High DR_MOMP scores indicate high apoptosis resistance. No statistical difference was observed in patients treated with XELOX or FOLFOX. Using MLR, we found that levels of apoptosis-related proteins caspase-8, p53 and XIAP statistically interacted with the DR_MOMP stress dose. Markers of MAPK signalling were prognostic for diarrhoea independently of DR_MOMP. In conclusion, apoptosis sensitivity and MAPK signalling status of the adjacent normal gut epithelium of chemotherapy-naive patients represent promising biomarkers to identify patients with CRC with increased risk of CID.

The intrinsic or mitochondrial apoptosis pathway is controlled by the interaction of antiapoptotic and pro-apoptotic members of the BCL-2 protein family. Activation of this death pathway plays a crucial role in cancer progression and chemotherapy responses. The BCL-2-related ovarian killer (BOK) possesses three BCL-2 homology domains and has been proposed to act in a similar pro-apoptotic pathway as the pro-apoptotic proteins BAX and BAK. In this study, we showed that stage II and III colorectal cancer patients possessed decreased levels of BOK protein in their tumours compared to matched normal tissue. BOK protein levels in tumours were also prognostic of clinical outcome but increased BOK protein levels surprisingly associated with earlier disease recurrence and reduced overall survival. We found no significant association of BOK protein tumour levels with ER stress markers GRP78 or GRP94 or with cleaved caspase-3. In contrast, BOK protein levels correlated with Calreticulin. These data indicate BOK as a prognostic marker in colorectal cancer and suggest that different activities of BOK may contribute to cancer progression and prognosis.

Apoptosis is essential for chemotherapy responses. We previously developed the mathematical models APOPTO-CELL and DR_MOMP to estimate the magnitude of tumor cells resilience to undergo and execute apoptosis in response to chemotherapy, and demonstrated that these models are prognostic markers of clinical responses in stage 3 colorectal cancer patients [Salvucci M et al., Clin Cancer Res 2017; Lindner AU et al., Gut 2018]. However, the heterogeneity of colorectal cancer tissue represents a hurdle that we have yet to overcome on our way to entirely understand differences in response and clinical outcome in patients. Quantification of protein levels as input for systems studies routinely reflects a local cell population average rather than protein levels in individual cells. In this study we aim to gain a better understanding of apoptosis resilience in tumors with respect to tumor cell heterogeneity and cell composition and local aspects of the tumor microenvironment. Two to three core slices of formalin-fixed and paraffin-embedded primary colorectal cancer tissue were collected from 165 stage II-IV patients from a previously used cohort [Salvucci M et al. 2017; Lindner AU et al. 2018]. The levels of 25 protein markers for cell type and immune status, apoptosis, metabolism and cell proliferation, as well as cell segmentation markers, were measured on TMA slides using a multiplexed tissue immunofluorescence imaging platform (Cell DIVE [Gerdes et al., Proc Natl Acad Sci USA 2013]). Batch correction between slides was performed using linear regression (using markers for regulatory T cell in patient tissues as housekeeping cells. Formalin-fixed gene deficient cells were used in parallel for antibody validation. The layout allowed us to create protein profiles for on average 13,079 (± 2,282 SD) cells per core per patient, and to categorize these as epithelial-like cells, regulatory T cells, T helper cells, cytotoxic T cells, other leukocytes and other cells. The data will be used to identify cells contributing most to the signal measured with traditional methods (such as reverse protein phase arrays) that were used to predict patients clinical response to therapy in our previous studies. Further, we will assess spatial differences in distribution and frequency of different cells and their contribution to clinical outcome in colorectal cancer patients. Specifically, we are investigating metrics to characterize the spatial features and layout found in each core in terms of textures, degree of lacunarity, neighboring cells composition and patterns of protein expression (diffuse vs. patchy) and co-expression among cell types. Further, we will characterize the degree of intra- and inter-patient spatial heterogeneity and will examine the association between the identified spatial motifs with macroscopical characteristics of the tumor and outcome. Citation Format: Andreas Urlich Lindner, Manuela Salvucci, Xanthi Stachtea, Steven Carberry, Philip D. Dunne, Anup Sood, Elizabeth McDonough, Sanghee Cho, Pierre Laurent-Puig, Sandra Van Schaeybroeck, Manuel Salto-Tellez, John F. Graf, Markus Rehm, Mark Lawler, Daniel B. Longley, Fiona Ginty Fiona, Jochen H. Prehn. Exploratory multiplex tissue image analysis of the impact of heterogeneity in the microenvironment of primary colorectal cancer on apoptosis markers in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-088.

In this article, we illustrate the application of difference in-gel electrophoresis for the proteomic analysis of dystrophic skeletal muscle. The mdx diaphragm was used as a tissue model of dystrophinopathy. Two-dimensional gel electrophoresis is a widely employed protein separation method in proteomic investigations. Although two-dimensional gels usually underestimate the cellular presence of very high molecular mass proteins, integral membrane proteins and low copy number proteins, this method is extremely powerful in the comprehensive analysis of contractile proteins, metabolic enzymes, structural proteins and molecular chaperones. This gives rise to two-dimensional gel electrophoretic separation as the method of choice for studying contractile tissues in health and disease. For comparative studies, fluorescence difference in-gel electrophoresis has been shown to provide an excellent biomarker discovery tool. Since aged diaphragm fibres from the mdx mouse model of Duchenne muscular dystrophy closely resemble the human pathology, we have carried out a mass spectrometry-based comparison of the naturally aged diaphragm versus the senescent dystrophic diaphragm. The proteomic comparison of wild type versus mdx diaphragm resulted in the identification of 84 altered protein species. Novel molecular insights into dystrophic changes suggest increased cellular stress, impaired calcium buffering, cytostructural alterations and disturbances of mitochondrial metabolism in dystrophin-deficient muscle tissue.*

X-linked muscular dystrophy is a highly progressive disease of childhood and characterized by primary genetic abnormalities in the dystrophin gene. Senescent mdx specimens were used for a large-scale survey of potential age-related alterations in the dystrophic phenotype, because the established mdx animal model of dystrophinopathy exhibits progressive deterioration of muscle tissue with age. Since the mdxtibialis anteriormuscle is a frequently used model system in muscular dystrophy research, we employed this particular muscle to determine global changes in the dystrophic skeletal muscle proteome. The comparison of mdx mice aged 8 weeks versus 22 months by mass-spectrometry-based proteomics revealed altered expression levels in 8 distinct protein species. Increased levels were shown for carbonic anhydrase, aldolase, and electron transferring flavoprotein, while the expressions of pyruvate kinase, myosin, tropomyosin, and the small heat shock protein Hsp27 were found to be reduced in aged muscle. Immunoblotting confirmed age-dependent changes in the density of key muscle proteins in mdx muscle. Thus, segmental necrosis in mdxtibialis anteriormuscle appears to trigger age-related protein perturbations due to dystrophin deficiency. The identification of novel indicators of progressive muscular dystrophy might be useful for the establishment of a muscle subtype-specific biomarker signature of dystrophinopathy.

Metabolic reprogramming, rewired signalling cascades, altered microbiota composition and aberrations in the tumor micro-environment have been implicated in CRC progression and response to treatment. In this study, we integrated proteomics, metabolomics, gut microbiota and predictions from network-analysis in tumor and non-cancerous tissue to build a comprehensive atlas to decipher the associations among alterations that occur in the different molecular layers. We performed our molecular fingerprinting in fresh frozen tissue samples from both tumor (centre and/or junction) and matched mucosa (5 and/or 10 cm distance) prospectively collected from a cohort of n=53 stage 0-IV CRC patients. We quantified the protein expression of a panel of 86 cancer-related targets involved in DNA damage/repair mechanisms, cell cycle regulation, growth/angiogenesis signalling, apoptosis and bioenergetics by Reverse Phase Protein Array (RPPA) in n=157 samples (n=48 from tumor and n=109 from mucosa) from n=53 patients. RPPA profiling on both tumor and matched normal tissue was available for 34 out of 53 patients. We performed High Resolution Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HR-MAS NMR) to profile metabolites in n=332 samples from n=52 patients (n=161 and n=171 from tumor and normal tissue, respectively). We characterized gut microbiota features from 16S rRNA sequencing in n=54 samples from n=18 patients. We assessed bioenergetic fitness using an ordinary-differential equations based model of core carbon metabolism that we are developing. The model can simulate the network dynamic and response to fuels (such as glucose, pyruvate and lactate) from extracellular milieu and their utilization and conversion via reactions in the cytoplasm and mitochondrial compartments. We used RPPA-based protein expression for HKII, TIGAR, PGYM, LDHA, MCT4, ATP5A as a proxy for enzymatic activities as case-specific inputs to the model. We identified distinct phenotypic differences when comparing tumors with normal tissue at each data type. Tumors showed higher protein levels for CHK1, GSK3B and LDHA; were enriched with fusobacteria; had increased metabolic levels of glycerphosphocholine, isoglutamine, phosphocholine, taurine and lactate; and were predicted by the mathematical model to have a more glycolytic phenotype. Normal mucosa had higher protein levels for GAB1 and elevated isobutyrate and lipids content. We further analysed the NMR spectra and using a genetic algorithm we identified 10 features (ppm levels) that could classify the tissue type with 88% cross-validated accuracy. We presented a unified analysis connecting dysregulations affecting the proteome, metabolome and microbiome. We provided insights into the heterogeneity of CRC which may lead to more specific molecular classifications and ultimately more targeted treatments. Citation Format: Manuela Salvucci, Liam Poynter, Reza Minerzami, Steven Carberry, Robert O'Byrne, Mattia Cremona, Bryan T. Hennessy, Kirill Veselkov, James Kinross, Jochen H. Prehn. Integrated multi-layer analysis reveals novel insights into the molecular landscape of colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4627.

Progressive x-linked muscular dystrophy represents the most commonly inherited neuromuscular disorder in humans. Although the disintegration of the dystrophin-associated glycoprotein complex triggers the initial pathogenesis of Duchenne muscular dystrophy, secondary alterations in metabolic pathways, cellular signaling and the regulation of ion homeostasis are probably crucial factors that cause end-stage fibre degeneration. The application of mass spectrometry-based proteomics for the global cataloguing of muscle biomarkers has recently been applied to the analysis of the mdx animal model of muscular dystrophy and the biochemical evaluation of experimental exon skipping therapy. The fluorescence difference in-gel electrophoretic analysis of normal versus mdx diaphragm muscle revealed changed expression levels of proteins involved in nucleotide metabolism, Ca 2+-handling, the cellular stress response and key bioenergetic processes. The swift up-regulation of small heat shock proteins, such as cvHsp, seems to form an integral part of the repair mechanisms in dystrophic fibres and may be exploitable as a new option to treat inherited muscle degeneration. Importantly, the mass spectrometry-based profiling of mdx muscle following the specific removal of exon 23 in the mutated dystrophin gene transcript showed a partial reversal of important secondary changes. Experimental exon skipping restored the expression of the dystrophin isoform Dp427, its associated glycoprotein beta-dystroglycan, neuronal nitric oxide synthase, calsequestrin, adenylate kinase and the muscle-specific stress protein cvHsp. In the future, a well defined set of signature molecules could be used to improve diagnosis, monitor disease progression, identify new therapeutic pathways, and validate the effects of novel drugs or experimental treatments such as gene therapy.

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction., Author Summary Oculopharyngeal muscular dystrophy is a genetic disease characterized by progressive degeneration of specific muscles, leading to ptosis (eyelid drooping), dysphagia (swallowing difficulties) and proximal limb weakness. The disease results from mutations in a nuclear protein called poly(A) binding protein nuclear 1 that is involved in polyadenylation of messenger RNAs (mRNAs) and poly(A) site selection. To address the molecular mechanisms involved in the disease, we have used two animal models (Drosophila and mouse) that recapitulate the features of this disorder. We show that oculopharyngeal muscular dystrophy pathogenesis depends on defects in poly(A) tail length regulation of specific mRNAs. Because poly(A) tails play an essential role in mRNA stability, these defects result in accelerated decay of these mRNAs. The affected mRNAs encode mitochondrial proteins, and mitochondrial activity is impaired in diseased muscles. These findings have important implications for the development of potential therapies for oculopharyngeal muscular dystrophy, and might be relevant to decipher the molecular mechanisms underlying other disorders that involve mitochondrial dysfunction.

In basic and applied myology, gel-based proteomics is routinely used for studying global changes in the protein constellation of contractile fibers during myogenesis, physiological adaptations, neuromuscular degeneration, and the natural aging process. Since the main proteins of the actomyosin apparatus and its auxiliary sarcomeric components often negate weak signals from minor muscle proteins during proteomic investigations, we have here evaluated whether a simple prefractionation step can be employed to eliminate certain aspects of this analytical obstacle. To remove a large portion of highly abundant contractile proteins from skeletal muscle homogenates without the usage of major manipulative steps, differential centrifugation was used to decisively reduce the sample complexity of crude muscle tissue extracts. The resulting protein fraction was separated by two-dimensional gel electrophoresis, and 2D-landmark proteins were identified by mass spectrometry. To evaluate the suitability of the contractile-protein-depleted fraction for comparative proteomics, normal versus dystrophic muscle preparations were examined. The mass spectrometric analysis of differentially expressed proteins, as determined by fluorescence difference in-gel electrophoresis, identified 10 protein species in dystrophic mdx hindlimb muscles. Interesting new biomarker candidates included Hsp70, transferrin, and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle were confirmed by immunoblotting.

Cellular aging is a fundamental biological process, and mass spectrometry-based proteomics has been widely used for the global identification of age-related changes in a variety of tissues. The proteomic profiling of senescent skeletal muscles has revealed a variety of alterations in proteins associated with the contractile apparatus, cell signaling, ion homeostasis, metabolism, and the cellular stress response. Here, we outline the two-dimensional gel electrophoretic separation and fluorescent labeling of the urea-soluble protein complement from aged diaphragm muscle. This chapter describes the various experimental steps involved in gel electrophoresis-based proteomics, including protein extraction, isoelectric focusing, slab gel electrophoresis, fluorescence labeling, image analysis, protein digestion, mass spectrometric identification of proteins and immunoblotting.

Duchenne muscular dystrophy is due to genetic abnormalities in the dystrophin gene and represents one of the most frequent genetic childhood diseases. In the X-linked muscular dystrophy (mdx) mouse model of dystrophinopathy, different subtypes of skeletal muscles are affected to a varying degree albeit the same single base substitution within exon 23 of the dystrophin gene. Thus, to determine potential muscle subtype-specific differences in secondary alterations due to a deficiency in dystrophin, in this study, we carried out a comparative histological and proteomic survey of mdx muscles. We intentionally included the skeletal muscles that are often used for studying the pathomechanism of muscular dystrophy. Histological examinations revealed a significantly higher degree of central nucleation in the soleus and extensor digitorum longus muscles compared with the flexor digitorum brevis and interosseus muscles. Muscular hypertrophy of 20-25% was likewise only observed in the soleus and extensor digitorum longus muscles from mdx mice, but not in the flexor digitorum brevis and interosseus muscles. For proteomic analysis, muscle protein extracts were separated by fluorescence two-dimensional (2D) gel electrophoresis. Proteins with a significant change in their expression were identified by mass spectrometry. Proteomic profiling established an altered abundance of 24, 17, 19 and 5 protein species in the dystrophin-deficient soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscle, respectively. The key proteomic findings were verified by immunoblot analysis. The identified proteins are involved in the contraction-relaxation cycle, metabolite transport, muscle metabolism and the cellular stress response. Thus, histological and proteomic profiling of muscle subtypes from mdx mice indicated that distinct skeletal muscles are differentially affected by the loss of the membrane cytoskeletal protein, dystrophin. Varying degrees of perturbed protein expression patterns in the muscle subtypes from mdx mice may be due to dissimilar downstream events, including differences in muscle structure or compensatory mechanisms that counteract pathophysiological processes. The interosseus muscle from mdx mice possibly represents a naturally protected phenotype.

Cellular aging is a fundamental biological process, and mass spectrometry-based proteomics has been widely used for the global identification of age-related changes in a variety of tissues. The proteomic profiling of senescent skeletal muscles has revealed a variety of alterations in proteins associated with the contractile apparatus, cell signaling, ion homeostasis, metabolism, and the cellular stress response. Here, we outline the two-dimensional gel electrophoretic separation and fluorescent labeling of the urea-soluble protein complement from aged diaphragm muscle. This chapter describes the various experimental steps involved in gel electrophoresis-based proteomics, including protein extraction, isoelectric focusing, slab gel electrophoresis, fluorescence labeling, image analysis, protein digestion, mass spectrometric identification of proteins and immunoblotting.

Colorectal cancer (CRC) is the third and second most commonly diagnosed cancer in males and females, and the second most common cause of cancer-related deaths in the developed world. Identifying the importance of epidermal growth factor (EGF) signalling pathways for the survival of CRC cells (Van Cutsem, Kohne et al. 2011) resulted in development of therapies that target EGF-receptors (EGFR). Late stage CRC patients are mainly treated with monoclonal anti-EGFR antibodies such as cetuximab. Although anti-EGFR therapies have significantly improved survival in CRC patients (Van Cutsem, Kohne et al. 2009), they are not effective in patients with activating KRAS, BRAF, NRAS and PI3KCA mutations (De Roock, Claes et al. 2010). Nevertheless, between 50-60% of patients will not benefit from the additional anti-EGFR treatment, even when these have a quadruple wild-type status (De Roock, De Vriendt et al. 2011), suggesting that novel biomarkers and targeted therapies are required. In this study, we investigate potential biomarkers for cetuximab response in a panel of 93 quadruple wild-type, liver metastatic CRC samples from patient derived xenografts (PDX) with known responses to cetuximab. Matching PDXs were used to measure changes in tumour volumes post cetuximab treatment (Bertotti, Papp et al. 2015) and were later categorised as regressing, progressing or stabilised. Protein expression levels were measured in matched PDXs, using a reverse phase protein array (RPPA) with a panel of 72 antibodies targeting key cancer related proteins as previously described). Statistical analysis of measured values showed significant correlation between high protein expression levels, such as Chk-1, PARP, HIAP-2 (cIAP-1), and PDX progression post cetuximab treatment. These proteins were significantly expressed lower in both regressing and stable PDXs. Interestingly, we found a high cross correlation between expression levels of these proteins across all the samples, giving them a great potential to act as predictive biomarkers for cetuximab response. Bioinformatics pathway enrichment of these proteins showed a significant enrichment of intrinsic apoptotic and cell cycle signalling pathways. These results have also been investigated in publically available patient data with cetuximab response and together will be used to construct a deterministic mathematical cell cycle model that will help to predict the outcome of cetuximab therapy in future. Citation Format: Naser Monsefi, Robert O’Byrne, Steven Carberry, Eugenia R. Zanella, Ana Barat, Mattia Cremona, Clare Morgan, Bryan T. Hennessy, Andrea Bertotti, Livio Trusolino, Jochen H.M. Prehn. Investigating potential molecular biomarkers for cetuximab response in metastatic colorectal cancer tissue using reverse phase protein array. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-354.

The largest human gene is represented by the X-chromosomal dystrophin gene of 2.4 million bases, which encodes for the membrane cytoskeletal protein dystrophin. The dystrophin isoform Dp427 has a subsarcolemmal location and forms a supramolecular membrane assembly with a variety of glycoproteins. In healthy muscle fibres, dystrophin acts as an actin-binding protein that links the cytoskeleton via the α/β-dystroglycan complex to the extracellular matrix protein laminin. This trans-sarcolemmal complex is believed to stabilize the muscle surface and thus prevents membrane rupturing during excitation-contraction-relaxation cycles. In the highly progressive muscle wasting disease Duchenne muscular dystrophy, the primary deficiency in dystrophin causes a drastic reduction in dystrophin-associated glycoproteins, which renders muscle fibres more susceptible to necrosis. Following the biochemical and cell biological characterization of the dystrophin-glycoprotein complex, several mass spectrometry-based proteomic studies have investigated global changes in dystrophin-deficient muscle tissues. This review briefly outlines the basic domain structure of Dp427 and the composition of the dystrophin-associated glycoprotein complex from skeletal muscle. A detailed discussion of recent proteomic analyses of the purified dystrophin-glycoprotein complex is included, as well as a summary of mass spectrometric surveys of dystrophic specimens. The study of these new areas of muscle proteomics tends to improve our understanding of the normal function of dystrophin in contractile fibres and better define the molecular mechanism of X-linked muscular dystrophy.

Duchenne muscular dystrophy is a lethal genetic disease of childhood caused by primary abnormalities in the gene coding for the membrane cytoskeletal protein dystrophin. The mdx mouse is an established animal model of various aspects of X-linked muscular dystrophy and is widely used for studying fundamental mechanisms of dystrophinopathy and testing novel therapeutic approaches to treat one of the most frequent gender-specific diseases in humans. In order to determine global changes in the muscle proteome with the progressive deterioration of mdx tissue with age, we have characterized diaphragm muscle from mdx mice at three ages (8-weeks, 12-months and 22-months) using mass spectrometry-based proteomics. Altered expression levels in diaphragm of 8-week vs. 22-month mice were shown to occur in 11 muscle-associated proteins. Aging in the mdx diaphragm seems to be associated with a drastic increase in the extracellular matrix proteins, collagen and dermatopontin, the molecular chaperone αB-crystallin, and the intermediate filament protein vimentin, suggesting increased accumulation of connective tissue, an enhanced cellular stress response and compensatory stabilization of the weakened membrane cytoskeleton. These proteomic findings establish the aged mdx diaphragm as an excellent model system for studying secondary effects of dystrophin deficiency in skeletal muscle tissue.

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Background: Colorectal cancer (CRC) is a leading cause of cancer mortality in the Western world and commonly treated with genotoxic chemotherapy. Stress in the endoplasmic reticulum (ER) was implicated to contribute to chemotherapeutic resistance. Hence, ER stress related protein may be of prognostic or therapeutic significance.Methods: The expression levels of ER stress proteins calnexin, calreticulin, GRP78 and GRP94 were determined in n = 23 Stage II and III colon cancer fresh frozen tumour and matched normal tissue samples. Data were validated in a cohort of n = 11 rectal cancer patients treated with radiochemotherapy in the neoadjuvant setting. The calnexin gene was silenced using siRNA in HCT116 cells.Results: There were no increased levels of ER stress proteins in tumour compared to matched normal tissue samples in Stage II or III CRC. However, increased calnexin protein levels were predictive of poor clinical outcome in the patient cohort. Data were validated in the rectal cancer cohort treated in the neoadjuvant setting. Calnexin gene-silencing significantly reduced cell survival and increased cancer cell susceptibility to 5FU chemotherapy.Conclusion: Increased tumour protein levels of calnexin may be of prognostic significance in CRC, and calnexin may represent a potential target for future therapies.