Search

Cancer is a highly heterogeneous disease, even within the same patient. Biopsies taken from different regions of a tumor may stand in stark molecular contrast to each other. Therefore, the ability to generate meaningful data from multiple platforms using the same biopsy is crucial for translating multi-omics characterizations into the clinic. However, it is generally a cumbersome and lengthy procedure to generate DNA, RNA and protein material from the same biopsy. The Qiagen AllPrep kit is an accessible, straightforward, and widely used kit in clinics worldwide to process biopsies and generate genomic and transcriptomic data from tumors and we asked if high quality proteomics data could be obtained from the remaining material. Here we develop a procedure for generating deep and quantitatively accurate proteomic information in high throughput from AllPrep flowthrough. With a number of refinements, we obtain in excess of 9,000 quantified proteins, a substantial coverage of the total proteome. We also successfully characterize the signaling state of the tumors using phosphoproteomics. Combining a standard kit with in-depth proteomics will be an attractive approach for clinics seeking to implement multi-omics-based precision oncology.Graphical abstract

mRNA isoforms

Pharmacodynamic changes (pathways)

Response markers

Extended Materials and Methods with in depth details about experimental procedures and protocols

Pharmacodynamic markers

Supplementary tables 1 and 2; Clinical information about the patients that the PDX models were established from and pathways that are regulated in the sensitive tumors after buparlisib treatment, respectively.

Response markers (pathways)

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro. A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4–dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy.Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732–46. ©2018 AACR.

Supplementary Figures 1-10 supporting the study Mundt et al., "Proteomic profiling of PI3K inhibitor resistance"

Aberrant activation of the RAS family of guanosine triphosphatases (GTPases) is prevalent in lung adenocarcinoma, with somatic mutation of KRAS occurring in ~30% of tumors. We previously identified somatic mutations and amplifications of the gene encoding RAS family GTPase RIT1 in lung adenocarcinomas. To explore the biological pathways regulated by RIT1 and how they relate to the oncogenic KRAS network, we performed quantitative proteomic, phosphoproteomic, and transcriptomic profiling of isogenic lung epithelial cells in which we ectopically expressed wild-type or cancer-associated variants of RIT1 and KRAS. We found that both mutant KRAS and mutant RIT1 promoted canonical RAS signaling, and that overexpression of wild-type RIT1 partially phenocopied oncogenic RIT1 and KRAS, including induction of epithelial-to-mesenchymal transition. Our findings suggest that RIT1 protein abundance is a factor in its pathogenic function. Therefore, chromosomal amplification of wild-type RIT1 in lung and other cancers may be tumorigenic.

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy., SigPath is a targeted, quantitative mass spectrometry assay that measures 284 phosphosites spanning 200 phosphoproteins with high throughput and quantitative precision across a broad swath of signaling biology of known interest.

Mantle cell lymphoma (MCL), a malignancy of B-lymphocytes, has a poor prognosis. It is thus necessary to improve the understanding of the pathobiology of MCL and identify factors contributing to its aggressiveness. Our studies, based on Affymetrix data from 17 MCL biopsies, real-time quantitative polymerase chain reaction data from 18 sorted primary MCL cells and 108 MCL biopsies compared to non-malignant tissue, reveals that GNAZ expression predicts poor clinical outcome of MCL patients (Cox regression, P = 0·014) and lymphocytosis (Mann-Whitney, P = 0·011). We show that GNAZ translates to Gαz protein - a signalling molecule within the G-protein coupled receptor network. Our findings suggest that GNAZ/Gαz contribute to the MCL pathobiology.

Purpose: The purpose of this study was to identify mechanisms of resistance to BRAF targeted therapy using proteomics together with genomics and transcriptomics in patients with BRAFV600E mutated solid tumors. Experimental procedures: A total of nine patients with BRAFV600E mutated advanced solid tumors (5 with colorectal cancer, 2 with neuroendocrine carcinoma, 1 with cholangiocarcinoma and 1 with breast cancer) treated with BRAF targeted therapy (BRAF inhibitor in combination with either MEK inhibitor and/or EGRF antibody) as part of the Copenhagen Prospective Personalized Oncology study, were included in this study. Tumor biopsies at baseline and at disease progression were analyzed with whole exome/genome sequencing (WES/WGS), transcriptomics (RNA sequencing) and proteomics. Genomic variants were analyzed together with changes in protein expression. Three filtering steps were used to identify potential resistance mechanisms from the proteomics measurements. Proteins were filtered for 1) proteins with a high shift in abundance between baseline and progression 2) proteins with known associations to the patient’s primary cancer based on text-mining and 3) proteins in pathways where BRAF is also involved. Results: Alterations conferring resistance were identified in 2 out of 9 patients when comparing data from WES/WGS and RNA sequencing at baseline and at disease progression (one patient with PTBP2-BRAF fusion and one with NRAS mutation). Genomic or transcriptomic alterations conferring resistance were not detected in the remaining 7 patients. Notably, there was not a single protein shared between all 9 patients after our filtering, but when comparing overlap on pathway level, six pathways related to RAF and MAPK signaling were affected in all 9 patients. Particularly, paradoxical activation of RAF signaling seems to be an interesting candidate to explain resistance, where formation of RAF dimer structures happens as a response to treatment with BRAF inhibitors, selectively inhibiting BRAF monomer structures (i.e. BRAFV600E). Conclusion: With a multiomic approach using proteomics together with genomics and transcriptomics, potential mechanisms of resistance were detected in all patients at disease progression, where resistance to BRAF targeted therapies had occurred. Six mechanisms of resistance were shared independently of diagnosis and BRAF inhibitor combination regimes. The potential of integrating proteomics with genomics and transcriptomics is promising and may potentially guide therapy for future patients with treatment resistant BRAFV600E mutated solid tumors. Citation Format: Martina Eriksen, Annelaura Bach Nielsen, Filip Mundt, Josephine Kerzel Duel, Matthias Mann, Ulrik Lassen, Christina Westmose Yde, Camilla Qvortrup, Martin Højgaard, Iben Spanggaard, Kristoffer Staal Rohrberg. Multiomics detect potential mechanisms of resistance to BRAF targeted therapy in patients with BRAFV600E mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3254.

Defects in pancreatic islets and the progression of multi-tissue insulin resistance in combination with environmental factors are the main causes of type 2 diabetes (T2D). Mass spectrometry-based proteomics of five key-metabolic tissues on a cohort of 42 multi-organ donors provided deep coverage of the proteomes of pancreatic islets, visceral adipose tissue (VAT), liver, skeletal muscle and serum. Enrichment analysis of gene ontology (GO) terms built a tissue-specific map of the chronological order of altered biological processes across healthy controls (CTRL), pre-diabetes (PD) and T2D subjects. This unique dataset allowed us to explore alterations of entire biological pathways and individual proteins in multiple tissues. We confirmed the significant decrease of the citric acid cycle and the respiratory electron transport in VAT and muscle of T2D and we provided a thorough visual representation of the complete set of downregulated proteins. Importantly, we found widespread novel alterations in relevant biological pathways including the increase in hemostasis in pancreatic islets of PD, the increase in the complement cascade in liver and pancreatic islets of PD and the elevation in cholesterol biosynthesis in liver of T2D. Overall, our findings suggest inflammatory, immune and vascular impairments in pancreatic islets as potentially causal factors of insufficient insulin production and increased glucagon levels in the early stages of T2D. In contrast alterations in lipid metabolism and mitochondrial function in the liver and VAT/muscle, respectively, became evident later in manifest T2D. This first multi-tissue proteomic map indicates the temporal order of tissue-specific metabolic dysregulation in T2D development.

BACKGROUND The standard therapy for glioblastoma patients is tumor resection followed by radiotherapy and temozolomide chemotherapy. Although glioblastoma has been extensively molecularly profiled along with other cancers, this knowledge has not yet been translated into improved survival outcomes. We used a bioinformatics approach to identify potential novel therapeutic strategies for glioblastoma. OBJECTIVES: Comprehensive online datasets which have assessed up to 1376 cancer cell lines in multiple ways were interrogated to identify potential drug candidates for glioblastoma. METHODS Datasets included were from the cancer cell line encyclopedia (mRNA expression), the Achilles project (cell viability following Crispr-Cas9 knockout) and PRISM (drug treatment). A t-test comparing cell viability of glioblastoma cell lines versus other cancers was used to identify potential drug candidates, followed by the use of multiple statistical tools to investigate potential mechanism of action and status of biomarkers. RESULTS Fluvastatin and pitavastatin produced the most significant effects in glioblastoma cell lines. The anti-cancer properties of statins have previously been attributed to the inhibition of HMG-Coa reductase. Here, we found their effects correlated with erastin, an enhancer of ferroptosis and with gene knockout of UBIAD1, which participates in non-mitochondrial ubiquinone synthesis. These effects were both found in glioblastoma cells and other cancers with a mesenchymal-like phenotype. CONCLUSION Statins appeared to be especially effective against glioblastoma lines and the effect could be linked to ferroptosis and inhibition of UBIAD1. In vitro validation of this finding is ongoing.

A knowledge graph platform integrates proteomics with other omics data and biomedical databases. Implementing precision medicine hinges on the integration of omics data, such as proteomics, into the clinical decision-making process, but the quantity and diversity of biomedical data, and the spread of clinically relevant knowledge across multiple biomedical databases and publications, pose a challenge to data integration. Here we present the Clinical Knowledge Graph (CKG), an open-source platform currently comprising close to 20 million nodes and 220 million relationships that represent relevant experimental data, public databases and literature. The graph structure provides a flexible data model that is easily extendable to new nodes and relationships as new databases become available. The CKG incorporates statistical and machine learning algorithms that accelerate the analysis and interpretation of typical proteomics workflows. Using a set of proof-of-concept biomarker studies, we show how the CKG might augment and enrich proteomics data and help inform clinical decision-making.

Aberrant activation of RAS oncogenes is prevalent in lung adenocarcinoma, with somatic mutation ofKRASoccurring in ∼30% of tumors. Recently, we identified somatic mutation of the RAS-family GTPaseRIT1in lung adenocarcinoma, but relatively little is known about the biological pathways regulated by RIT1 and how these relate to the oncogenic KRAS network. Here we present quantitative proteomic and transcriptomic profiles fromKRAS-mutant andRIT1-mutant isogenic lung epithelial cells and globally characterize the signaling networks regulated by each oncogene. We find that both mutant KRAS and mutant RIT1 promote S6 kinase, AKT, and RAF/MEK signaling, and promote epithelial-to-mesenchymal transition and immune evasion via HLA protein loss. However, KRAS and RIT1 diverge in regulation of phosphorylation sites on EGFR, USO1, and AHNAK proteins. The majority of the proteome changes are related to altered transcriptional regulation, but a small subset of proteins are differentially regulated by both oncoproteins at the post-transcriptional level, including intermediate filament proteins, metallothioneins, and MHC Class I proteins. These data provide the first global, unbiased characterization of oncogenic RIT1 network and identify the shared and divergent functions of oncogenic RIT1 and KRAS GTPases in lung cancer.

Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 μg of peptide derived from 0.88. The maximum deviation for the phosphoproteome coverage was 37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in −4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.

Background The cell-surface heparan sulfate proteoglycan syndecan-1 is important for tumor cell proliferation, migration, and cell cycle regulation in a broad spectrum of malignancies. Syndecan-1, however, also translocates to the cell nucleus, where it might regulate various molecular functions. Results We used a fibrosarcoma model to dissect the functions of syndecan-1 related to the nucleus and separate them from functions related to the cell-surface. Nuclear translocation of syndecan-1 hampered the proliferation of fibrosarcoma cells compared to the mutant lacking nuclear localization signal. The growth inhibitory effect of nuclear syndecan-1 was accompanied by significant accumulation of cells in the G0/G1 phase, which indicated a possible G1/S phase arrest. We implemented multiple, unsupervised global transcriptome and proteome profiling approaches and combined them with functional assays to disclose the molecular mechanisms that governed nuclear translocation and its related functions. We identified genes and pathways related to the nuclear compartment with network enrichment analysis of the transcriptome and proteome. The TGF-β pathway was activated by nuclear syndecan-1, and three genes were significantly altered with the deletion of nuclear localization signal: EGR-1 (early growth response 1), NEK11 (never-in-mitosis gene a-related kinase 11), and DOCK8 (dedicator of cytokinesis 8). These candidate genes were coupled to growth and cell-cycle regulation. Nuclear translocation of syndecan-1 influenced the activity of several other transcription factors, including E2F, NFκβ, and OCT-1. The transcripts and proteins affected by syndecan-1 showed a striking overlap in their corresponding biological processes. These processes were dominated by protein phosphorylation and post-translation modifications, indicative of alterations in intracellular signaling. In addition, we identified molecules involved in the known functions of syndecan-1, including extracellular matrix organization and transmembrane transport. Conclusion Collectively, abrogation of nuclear translocation of syndecan-1 resulted in a set of changes clustering in distinct patterns, which highlighted the functional importance of nuclear syndecan-1 in hampering cell proliferation and the cell cycle. This study emphasizes the importance of the localization of syndecan-1 when considering its effects on tumor cell fate. Electronic supplementary material The online version of this article (10.1186/s12860-017-0150-z) contains supplementary material, which is available to authorized users.

Objective: Tumor biomarkers are promising study areas for the early or differential diagnosis of malignant pleural mesothelioma (MPM). This study aimed to determine the effectiveness of analyzing serum N-ERC/mesothelin, C-ERC/mesothelin, hyaluronan, osteopontin, and syndecan-1 levels for distinguishing patients with MPM from those with metastatic malignant pleural diseases (MMPDs), benign pleural diseases (BPDs), and benign asbestos pleurisy (BAP). Methods: Tumor biomarker levels of serum samples of 230 cases were analyzed by enzyme-linked immunosorbent assays. Results: All investigated biomarkers did not reveal sufficient diagnostic information to distinguish MPM from MMPD. N-ERC/mesothelin showed moderate ability to distinguish MPM from BPDs and particularly BAP (sensitivities of 67% and 73%, respectively, and specificities of 84% and 86%, respectively). C-ERC/mesothelin had a lower efficacy than N-ERC/mesothelin, whereas osteopontin had a high specificity for distinguishing MPM from other pleural diseases (80%) but with a poor sensitivity (32%). Hyaluronan and syndecan-1 had only limited effects as individual biomarkers. However, logistic regression analysis indicated that all the studied biomarkers could contribute, and a logistic model improved their performance, with the receiver operating characteristic curve plot showing an area under the curve of 0.75. Thus, the investigated biomarkers were unable to provide sufficient sensitivity and specificity levels; however, they all may contribute as a basis for an expanded logistic multiparameter model. Conclusion: Patients with high N-ERC/mesothelin and C-ERC/mesothelin levels have a high risk for MPM; appropriate invasive procedures should be performed. The patients who have high tumor biomarker levels and undefinite histopathological investigation results at the first-line procedure, should be managed using further invasive procedures. WOS:000416688200004

Signaling pathways are orchestrated by post-translational modifications (PTMs) such as phosphorylation. However, pathway analysis of PTM data sets generated by mass spectrometry (MS)-based proteomics is typically performed at a gene-centric level because of the lack of appropriately curated PTM signature databases and bioinformatic tools that leverage PTM site-specific information. Here we present the first version of PTMsigDB, a database of modification site-specific signatures of perturbations, kinase activities and signaling pathways curated from more than 2,500 publications. We adapted the widely used single sample Gene Set Enrichment Analysis approach to utilize PTMsigDB, enabling PTM Signature Enrichment Analysis (PTM-SEA) of quantitative MS data. We used a well-characterized data set of epidermal growth factor (EGF)-perturbed cancer cells to evaluate our approach and demonstrated better representation of signaling events compared with gene-centric methods. We then applied PTM-SEA to analyze the phosphoproteomes of cancer cells treated with cell-cycle inhibitors and detected mechanism-of-action specific signatures of cell cycle kinases. We also applied our methods to analyze the phosphoproteomes of PI3K-inhibited human breast cancer cells and detected signatures of compounds inhibiting PI3K as well as targets downstream of PI3K (AKT, MAPK/ERK) covering a substantial fraction of the PI3K pathway. PTMsigDB and PTM-SEA can be freely accessed at https://github.com/broadinstitute/ssGSEA2.0.

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

Medulloblastoma (MB) is one of the most common pediatric brain tumors. Current treatment options carry substantial risk for lifelong cognitive and neurological impairment. Consequently, there is a pressing need to elucidate molecular mechanisms with the ultimate goal of developing targeted tumor-specific therapies. In a recent study on proteomes and phospho-proteomes of primary MB tumors, we identified proteomic MB subtypes that were not apparent from transcriptional or methylation data. To identify gene regulatory circuitries that drive these MB subtypes, we have now mapped accessible chromatin and 3D chromosome conformation in 23 and 13 MB tumors from the same cohort, using ATAC-seq and Hi-C respectively. We identify subtype-specific accessible regulatory chromatin regions, including a subset of regions not identifiable by the enhancer mark H3K27ac. We associate regulatory regions to their target gene promoters by correlating transcription with ATAC-seq signal strength, and confirm these relationships using the newly generated Hi-C data. Using an unpublished adaptation of the GSEA algorithm, which we tailored for an enrichment analysis of non-coding regulatory elements instead of genes, we identify transcription factors and master regulators with binding sites specific to an MB subtype. By associating these master regulators with their transcriptional targets, we have derived models of regulatory circuitry in MB which implicate central mechanisms of MB pathogenesis and may inform future therapeutic targeting. Citation Format: Owen S. Chapman, Tobias Ehrenberger, Tenley C. Archer, Maxwell P. Gold, Filip Mundt, Miriam Adam, Clarence K. Mah, Karsten Krug, Sahaana Chandran, Jesse R. Dixon, Scot L. Pomeroy, Ernest Fraenkel, Jill P. Mesirov, Lukas Chavez. Integrated omics modeling of transcriptional regulation in medulloblastoma subtypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 447.

Mantle cell lymphoma (MCL), a malignancy of B-lymphocytes, has a poor prognosis. It is thus necessary to improve the understanding of the pathobiology of MCL and identify factors contributing to its aggressiveness. Our studies, based on Affymetrix data from 17 MCL biopsies, real-time quantitative polymerase chain reaction data from 18 sorted primary MCL cells and 108 MCL biopsies compared to non-malignant tissue, reveals that GNAZ expression predicts poor clinical outcome of MCL patients (Cox regression, P = 0·014) and lymphocytosis (Mann-Whitney, P = 0·011). We show that GNAZ translates to Gα

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro. A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4–dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy. Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732–46. ©2018 AACR.

Background Britain’s native oak species are currently under threat from acute oak decline (AOD), a decline-disease where stem bleeds overlying necrotic lesions in the inner bark and larval galleries of the bark-boring beetle, Agrilus biguttatus, represent the primary symptoms. It is known that complex interactions between the plant host and its microbiome, i.e. the holobiont, significantly influence the health status of the plant. In AOD, necrotic lesions are caused by a microbiome shift to a pathobiome consisting predominantly of Brenneria goodwinii, Gibbsiella quercinecans, Rahnella victoriana and potentially other bacteria. However, the specific mechanistic processes of the microbiota causing tissue necrosis, and the host response, have not been established and represent a barrier to understanding and managing this decline. Results We profiled the metagenome, metatranscriptome and metaproteome of inner bark tissue from AOD symptomatic and non-symptomatic trees to characterise microbiota-host interactions. Active bacterial virulence factors such as plant cell wall-degrading enzymes, reactive oxygen species defence and flagella in AOD lesions, along with host defence responses including reactive oxygen species, cell wall modification and defence regulators were identified. B. goodwinii dominated the lesion microbiome, with significant expression of virulence factors such as the phytopathogen effector avrE. A smaller proportion of microbiome activity was attributed to G. quercinecans and R. victoriana. In addition, we describe for the first time the potential role of two previously uncharacterised Gram-positive bacteria predicted from metagenomic binning and identified as active in the AOD lesion metatranscriptome and metaproteome, implicating them in lesion formation. Conclusions This multi-omic study provides novel functional insights into microbiota-host interactions in AOD, a complex arboreal decline disease where polymicrobial-host interactions result in lesion formation on tree stems. We present the first descriptions of holobiont function in oak health and disease, specifically, the relative lesion activity of B. goodwinii, G. quercinecans, Rahnella victoriana and other bacteria. Thus, the research presented here provides evidence of some of the mechanisms used by members of the lesion microbiome and a template for future multi-omic research into holobiont characterisation, plant polymicrobial diseases and pathogen defence in trees. Electronic supplementary material The online version of this article (10.1186/s40168-018-0408-5) contains supplementary material, which is available to authorized users.

Malignant mesothelioma is an aggressive asbestos-induced cancer, and affected patients have a median survival of approximately one year after diagnosis. It is often difficult to reach a conclusive diagnosis, and ancillary measurements of soluble biomarkers could increase diagnostic accuracy. Unfortunately, few soluble mesothelioma biomarkers are suitable for clinical application. Here we screened the effusion proteomes of mesothelioma and lung adenocarcinoma patients to identify novel soluble mesothelioma biomarkers. We performed quantitative mass-spectrometry-based proteomics using isobaric tags for quantification and used narrow-range immobilized pH gradient/high-resolution isoelectric focusing (pH 4–4.25) prior to analysis by means of nano liquid chromatography coupled to MS/MS. More than 1,300 proteins were identified in pleural effusions from patients with malignant mesothelioma (n = 6), lung adenocarcinoma (n = 6), or benign mesotheliosis (n = 7). Data are available via ProteomeXchange with identifier PXD000531. The identified proteins included a set of known mesothelioma markers and proteins that regulate hallmarks of cancer such as invasion, angiogenesis, and immune evasion, plus several new candidate proteins. Seven candidates (aldo-keto reductase 1B10, apolipoprotein C-I, galectin 1, myosin-VIIb, superoxide dismutase 2, tenascin C, and thrombospondin 1) were validated by enzyme-linked immunosorbent assays in a larger group of patients with mesothelioma (n = 37) or metastatic carcinomas (n = 25) and in effusions from patients with benign, reactive conditions (n = 16). Galectin 1 was identified as overexpressed in effusions from lung adenocarcinoma relative to mesothelioma and was validated as an excellent predictor for metastatic carcinomas against malignant mesothelioma. Galectin 1, aldo-keto reductase 1B10, and apolipoprotein C-I were all identified as potential prognostic biomarkers for malignant mesothelioma. This analysis of the effusion proteome furthers our understanding of malignant mesothelioma, identified galectin 1 as a potential diagnostic biomarker, and highlighted several possible prognostic biomarkers of this disease.

Background Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. Methods Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR−/−) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. Results Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. Conclusions These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0748-7) contains supplementary material, which is available to authorized users.

There is a pressing need to identify therapeutic targets in tumors with low mutation rates such as the malignant pediatric brain tumor medulloblastoma. To address this challenge, we quantitatively profiled global proteomes and phospho-proteomes of 45 medulloblastoma samples. Integrated analyses revealed that tumors with similar RNA expression vary extensively at the post-transcriptional and post-translational levels. We identified distinct pathways associated with two subsets of SHH tumors, and found post-translational modifications of MYC that are associated with poor outcomes in group 3 tumors. We found kinases associated with subtypes and showed that inhibiting PRKDC sensitizes MYC-driven cells to radiation. Our study shows that proteomics enables a more comprehensive, functional readout, providing a foundation for future therapeutic strategies.

Background. The distinction between malignant and benign pleural effusions is a diagnostic challenge today and measuring soluble biomarkers could add to the diagnostic accuracy. Syndecan-1 is a proteoglycan involved in various cellular functions and is cleaved from the cell surface in a regulated manner. The shed fragment, which can be recovered in effusion supernatant and in serum, retains its binding capacities, but often with different functions and signalling properties than the cell-bound form.Aim. This study aimed to investigate the diagnostic and prognostic value of soluble syndecan-1 in pleural effusions and sera from patients with pleural malignancies.Study Design. Using two cohorts of patients, we assessed the diagnostic and prognostic value of soluble syndecan-1 in pleural effusions and sera, using enzyme-linked immunosorbent assays.Results. In pleural effusions, syndecan-1 distinguished malignant and benign diseases, with an odds ratio of 8.59 (95% CI 3.67 to 20.09). Furthermore, syndecan-1 in pleural effusions predicted a survival difference for patients with pleural metastatic disease and malignant mesothelioma of 11.2 and 9.2 months, respectively. However, no such effects were seen when syndecan-1 was measured in serum.Conclusion. Soluble syndecan-1 is a promising candidate biomarker for the cytopathological diagnosis and prognostication of malignant pleural effusions.

Mantle cell lymphoma (MCL) is a B cell Non-Hodgkin lymphoma comprising approximately 8% of malignant lymphomas. MCL is considered to be an incurable disease even though survival has improved with novel biological therapies that interrupt survival signaling from the lymphoma microenvironment. The signaling pathways that are deregulated and the factors leading to systemic dissemination of the disease are still poorly understood. There is also a clear need to identify additional druggable targets for therapy (Saba and Wiestner, Curr Opin Hematol, 2014). Druggable targets may be G-protein coupled receptors (GPCRs) and G-proteins. GPCRs belong to the family of seven transmembrane receptors, which upon activation by extracellular ligands transfer the signals inside the cell for activation of the specific pathway. The signaling involves G-proteins. G-proteins consist of the subunits: alpha, beta and gamma and their subtypes. During GPCR activation the G alpha subunit is released and the subtype of G-protein subunit determines which downstream signaling pathways (including Akt, PI3K, ERK, JNK, MAPK, NFkappaB and others) is then activated. In the immune cells different subtypes of G-proteins alpha are instrumental for leukocyte migration, architecture of B cells compartments in spleen, thymus, lymph nodes, and gastrointestinal tract (Hwang et al., PlosOne, 2013) and BCR-mediated cell death (Misra et al., J Exp Med, 2010). G alpha13 signaling is involved in development of germinal centre-derived lymphomas (Muppidi et al., Nature, 2014). In order to map the aberrant expression of GPCR and G-proteins in MCL, the expression of genes encoding for GPCRs and G-protein alpha subunits was analyzed by Affymetrix U133A Array of seventeen MCL biopsies and eight non-malignant lymph nodes. Among investigated GPCR and G-protein alpha subunit genes 25/298 were differently expressed in MCL compared to reactive lymph nodes (moderated t-test, adj. p=or< than 0.05). The four most significantly different genes expressed were: LPAR6, GPRC5C, OPN3 and GNAZ (all adj.p Disclosures No relevant conflicts of interest to declare.

About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen., Many patients with breast cancer develop resistance to the drug tamoxifen and relapse. Here Johansson et al. identify the nuclear receptor retinoic acid receptor alpha (RARA) as a marker of tamoxifen resistance and show that RARA expression correlates negatively with relapse-free survival of patients.

Background: Malignant mesothelioma cells have an epithelioid or sarcomatoid morphology, both of which may be present in the same tumor. The sarcomatoid phenotype is associated with worse prognosis and heterogeneity of mesothelioma cells may contribute to therapy resistance, which is often seen in mesothelioma. This study aimed to investigate differences in sensitivity between mesothelioma cell lines to anti-cancer drugs. We studied two novel drugs, selenite and bortezomib and compared their effect to four conventional drugs. We also investigated the immunoreactivity of potential predictive markers for drug sensitivity; Pgp, MRP-1, ERCC1, RRM1, TS, xCT and proteasome 20S subunit. Materials and methods: We treated six mesothelioma cell lines with selenite, bortezomib, carboplatin, pemetrexed, doxorubicin or gemcitabine as single agents and in combinations. Viability was measured after 24 and 48 hours. Immunocytochemistry was used to detect predictive markers. Results: As a single agent, selenite was effective on four out of six cell lines, and in combination with bortezomib yielded the greatest response in the studied mesothelioma cell lines. Cells with an epithelioid phenotype were generally more sensitive to the different drugs than the sarcomatoid cells. Extensive S-phase arrest was seen in pemetrexed-sensitive cell lines. MRP-1 predicted sensitivity of cell lines to treatment with carboplatin and xCT predicted pemetrexed effect. Conclusions: The observed heterogeneity in sensitivity of mesothelioma cell lines with different morphology highlights the need for more individualized therapy, requiring development of methods to predict drug sensitivity of individual tumors. Selenite and bortezomib showed a superior effect compared to conventional drugs, motivating clinical testing of these agents as future treatment regime components for patients with malignant mesothelioma.

Purpose: Diagnosis of malignant mesothelioma is challenging. The first available diagnostic material is often an effusion and biochemical analysis of soluble markers may provide additional diagnostic information. This study aimed to establish a predictive model using biomarkers from pleural effusions, to allow early and accurate diagnosis. Patients and Methods: Effusions were collected prospectively from 190 consecutive patients at a regional referral centre. Hyaluronan, N-ERC/mesothelin, C-ERC/mesothelin, osteopontin, syndecan-1, syndecan-2, and thioredoxin were measured using ELISA and HPLC. A predictive model was generated and validated using a second prospective set of 375 effusions collected consecutively at a different referral centre. Results: Biochemical markers significantly associated with mesothelioma were hyaluronan (odds ratio, 95% CI: 8.82, 4.82– 20.39), N-ERC/mesothelin (4.81, 3.19–7.93), CERC/mesothelin (3.58, 2.43–5.59) and syndecan-1 (1.34, 1.03–1.77). A two-step model using hyaluronan and N-ERC/mesothelin, and combining a threshold decision rule with logistic regression, yielded good discrimination with an area under the ROC curve of 0.99 (95% CI: 0.97–1.00) in the model generation dataset and 0.83 (0.74–0.91) in the validation dataset, respectively. Conclusions: A two-step model using hyaluronan and N-ERC/mesothelin predicts mesothelioma with high specificity. This method can be performed on the first available effusion and could be a useful adjunct to the morphological diagnosis of mesothelioma.

The genetic landscape of human breast cancer has been well defined in The Cancer Genome Atlas (TCGA) project. Mass spectrometry (MS)-based global proteome and phosphoproteome analyses provide a complementary, orthogonal approach to genomic studies to further improve the molecular taxonomy and biological understanding of breast cancer. We analyzed human breast cancer samples that had previously undergone comprehensive genomic and reversed phase protein array (RPPA) characterization by TCGA. Tumor samples were analyzed by global shotgun proteomics and phosphoproteomics at an unprecedented coverage of >11,000 quantified proteins and >27,000 phosphorylation sites for each tumor. We verified the translation of hundreds of genomically characterized single nucleotide and splice junction variants at the protein level. The correlation of mRNA to protein abundance was significant for 6,135 out of 9,302 protein/mRNA pairs, but differed amongst protein classes. Genes that did not correlate on the protein/mRNA level included components of basic cellular machineries such as the ribosome, RNA polymerase and spliceosome, as well as those involved in processes regulated by proteolysis. Hierarchical clustering yielded three major clusters in both the proteome and the phosphoproteome data: basal-enriched, luminal-enriched and stroma-enriched groups, the last also enriched for what have been previously designated “reactive-type” tumors by RPPA. Our deep proteome analysis promoted new insights including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. The 5q trans effects were interrogated using the Library of Integrated Network-based Cellular Signatures. Theses analyses connected the 5q genes CETN3 and SKP1 to elevated expression of EGFR, and SKP1 also to SRC. Differential phosphopeptide analyses, integrated with activity maps derived from knock-in mutated cell lines, identified multiple novel downstream effects of PIK3CA and TP53 mutation. Besides ERBB2, other amplicon-associated, highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. These and other examples demonstrate that proteogenomic analysis of breast cancer elucidates functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies potential therapeutic targets. Citation Format: Philipp Mertins, DR Mani, Kelly Ruggles, Michael Gillette, Karl Clauser, Pei Wang, Xianlong Wang, Jana Qiao, Song Cao, Francesca Petralia, Filip Mundt, Zhidong Tu, Jonathan Lei, Michael Gatza, Matthew Wilkerson, Charles Perou, Venkata Yellapantula, Kuan-lin Huang, Chenwei Lin, Michael McLellan, Ping Yan, Sherri Davies, Reid Townsend, Steven Skates, Jing Wang, Bing Zhang, Christopher Kinsinger, Mehdi Mesri, Henry Rodriguez, Li Ding, Amanda Paulovich, David Fenyo, Matthew Ellis, Steven Carr, NCI CPTAC. Proteogenomic and phosphoproteomic analysis of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA29.

AIM: The aim of this study was to identify markers that have the potential to predict BKM120 (a pan-class I PI3K inhibitor) effectiveness in triple negative breast cancer. INTRODUCTION: Triple negative breast cancer is an aggressive subtype that constitutes circa 20% of all breast cancers. This subtype of breast cancer is defined by its lack of estrogen-, progesterone- and HER2/neu expression. Since these are the operative receptors for targeted breast cancer therapies, current standard treatment instead centers on conventional cytotoxic chemotherapy. Unfortunately, the majority of triple negative breast cancer patients does not have meaningful response, and new therapeutic agents are needed. The PI3K/Akt/mTOR axis is often dysregulated in breast cancer, and clinical trials are ongoing for several inhibitors targeting this pathway. Differential analysis of the global proteome and phosphoproteome of BKM120-treated responsive and resistant tumors may identify markers, patterns and pathways associated with clinical response to support precision therapy. MATERIALS AND METHODS: We collected tumor material from triple negative breast cancer xenografts propagated in the mammary fat pads of NOD SCID mice after BKM120 treatment. Long term (∼30 days) treatment was performed to assess growth rates and sensitivity to the drug. For mass spectrometry analyses, tumor material from 6 different mouse models across 5 treatment conditions was lysed and digested into peptides. Samples were labeled with TMT6-plex reagents, and analyzed together with one reference sample in a total of 6 TMT6-plex experiments. To increase proteome coverage, sample complexity was reduced using high-pH reversed-phase separation, and phospho-peptides were enriched using immobilized metal affinity chromatography. All samples were analyzed on a Q Exactive mass spectrometer. RESULTS AND DATA ANALYSES: Cumulatively, >19,000 proteins were identified, with an average of >10,000 proteins and >21,000 phosphosites per sample. The 6 mice showed a continuum of response to the drug from highly sensitive to highly resistant. Pearson correlation analysis was used to identify proteins and phosphosites that correlate to this response gradient, while moderate t-tests were used to identify commonly regulated markers. Preliminary analyses indicate several interesting proteins and phospho-sites, some pertaining to the PI3K/Akt pathway. Further canonical pathways were detected using Gene Set Enrichment Analysis on a sample-by sample basis and additional network and pathway analyses were performed using the network analysis program GeNets, as well as Ingenuity Pathway Analysis. Citation Format: Filip O. Mundt*, Cynthia Ma*, Philipp Mertins, Zhanfang Guo, Matthew Meyer, Jana Qiao, DR Mani, Karl Clauser, Shunqiang Li, Kuan Huang, Jason Held, Sherri Davies, Kelly Ruggles, Li Ding, Michael Gillette, David Fenyo, Reid Townsend, Matthew Ellis, Steven A. Carr. Effects of PI3K/Akt pathway inhibition on global proteome levels and phosphorylation signaling in patient-derived xenograft models of triple negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C200.

BackgroundSyndecans are proteoglycans whose core proteins have a short cytoplasmic domain, a transmembrane domain and a large N-terminal extracellular domain possessing glycosaminoglycan chains. Syndecans are involved in many important cellular processes. Our recent publications have demonstrated that syndecan-1 translocates into the nucleus and hampers tumor cell proliferation. In the present study, we aimed to investigate the role of syndecan-1 in tumor cell adhesion and migration, with special focus on the importance of its distinct protein domains, to better understand the structure-function relationship of syndecan-1 in tumor progression.Methodology/principal findingsWe utilized two mesenchymal tumor cell lines which were transfected to stably overexpress full-length syndecan-1 or truncated variants: the 78 which lacks the extracellular domain except the DRKE sequence proposed to be essential for oligomerization, the 77 which lacks the whole extracellular domain, and the RMKKK which serves as a nuclear localization signal. The deletion of the RMKKK motif from full-length syndecan-1 abolished the nuclear translocation of this proteoglycan. Various bioassays for cell adhesion, chemotaxis, random movement and wound healing were studied. Furthermore, we performed gene microarray to analyze the global gene expression pattern influenced by syndecan-1. Both full-length and truncated syndecan-1 constructs decrease tumor cell migration and motility, and affect cell adhesion. Distinct protein domains have differential effects, the extracellular domain is more important for promoting cell adhesion, while the transmembrane and cytoplasmic domains are sufficient for inhibition of cell migration. Cell behavior seems to depend also on the nuclear translocation of syndecan-1. Many genes are differentially regulated by syndecan-1 and a number of genes are actually involved in cell adhesion and migration.Conclusions/significanceOur results demonstrate that syndecan-1 regulates mesenchymal tumor cell adhesion and migration, and different domains have differential effects. Our study provides new insights into better understanding of the role of syndecans in tumor progression.

Background Selenite is a promising anticancer agent which has been shown to induce apoptosis in malignant mesothelioma cells in a phenotype-dependent manner, where cells of the chemoresistant sarcomatoid phenotype are more sensitive. Methods In this paper, we investigate the apoptosis signalling mechanisms in sarcomatoid and epithelioid mesothelioma cells after selenite treatment. Apoptosis was measured with the Annexin-PI assay. The mitochondrial membrane potential, the expression of Bax, Bcl-XL, and the activation of caspase-3 were assayed with flow cytometry and a cytokeratin 18 cleavage assay. Signalling through JNK, p38, p53, and cathepsins B, D, and E was investigated with chemical inhibitors. Furthermore, the expression, nuclear translocation and DNA-binding activity of p53 was investigated using ICC, EMSA and the monitoring of p21 expression as a downstream event. Levels of thioredoxin (Trx) were measured by ELISA. Results In both cell lines, 10 μM selenite caused apoptosis and a marked loss of mitochondrial membrane potential. Bax was up-regulated only in the sarcomatoid cell line, while the epithelioid cell line down-regulated Bcl-XL and showed greater caspase-3 activation. Nuclear translocation of p53 was seen in both cell lines, but very little p21 expression was induced. Chemical inhibition of p53 did not protect the cells from apoptosis. p53 lost its DNA binding ability after selenite treatment and was enriched in an inactive form. Levels of thioredoxin decreased after selenite treatment. Chemical inhibition of MAP kinases and cathepsins showed that p38 and cathepsin B had some mediatory effect while JNK had an anti-apoptotic role. Conclusion We delineate pathways of apoptosis signalling in response to selenite, showing differences between epithelioid and sarcomatoid mesothelioma cells. These differences may partly explain why sarcomatoid cells are more sensitive to selenite.

Malignant pleural mesothelioma is a highly malignant tumor, originating from mesothelial cells of the serous cavities. In mesothelioma the expression of syndecan-1 correlates to epithelioid morphology and inhibition of growth and migration. Our previous data suggest a complex role of syndecan-1 in mesothelioma cell proliferation although the exact underlying molecular mechanisms are not completely elucidated. The aim of this study is therefore to disclose critical genes and pathways affected by syndecan-1 in mesothelioma; in order to better understand its importance for tumor cell growth and proliferation. We modulated the expression of syndecan-1 in a human mesothelioma cell line via both overexpression and silencing, and followed the transcriptomic responses with microarray analysis. To project the transcriptome analysis on the full-dimensional picture of cellular regulation, we applied pathway analysis using Ingenuity Pathway Analysis (IPA) and a novel method of network enrichment analysis (NEA) which elucidated signaling relations between differentially expressed genes and pathways acting via various molecular mechanisms. Syndecan-1 overexpression had profound effects on genes involved in regulation of cell growth, cell cycle progression, adhesion, migration and extracellular matrix organization. In particular, expression of several growth factors, interleukins, and enzymes of importance for heparan sulfate sulfation pattern, extracellular matrix proteins and proteoglycans were significantly altered. Syndecan-1 silencing had less powerful effect on the transcriptome compared to overexpression, which can be explained by the already low initial syndecan-1 level of these cells. Nevertheless, 14 genes showed response to both up- and downregulation of syndecan-1. The "cytokine - cytokine-receptor interaction", the TGF-β, EGF, VEGF and ERK/MAPK pathways were enriched in both experimental settings. Most strikingly, nearly all analyzed pathways related to cell cycle were enriched after syndecan-1 silencing and depleted after syndecan-1 overexpression. Syndecan-1 regulates proliferation in a highly complex way, although the exact contribution of the altered pathways necessitates further functional studies.