Anastasia Meshcheryakova

Proteome signatures of inflammatory activated primary human peripheral blood mononuclear cells

Proteome profiling is the method of choice to identify marker proteins whose expression may be characteristic for certain diseases. The formation of such marker proteins results from disease-related pathophysiologic processes. In healthy individuals, peripheral blood mononuclear cells (PBMCs) circulate in a quiescent cell state monitoring potential immune-relevant events, but have the competence to respond quickly and efficiently in an inflammatory manner to any invasion of potential pathogens. Activation of these cells is most plausibly accompanied by characteristic proteome alterations. Therefore we investigated untreated and inflammatory activated primary human PBMCs by proteome profiling using a ‘top down’ 2D-PAGE approach in addition to a ‘bottom up’ LC–MS/MS-based shotgun approach. Furthermore, we purified primary human T-cells and monocytes and activated them separately. Comparative analysis allowed us to characterize a robust proteome signature including NAMPT and PAI2 which indicates the activation of PBMCs. The T-cell specific inflammation signature included IRF-4, GBP1and the previously uncharacterized translation product of GBP5; the corresponding monocyte signature included PDCD5, IL1RN and IL1B. The involvement of inflammatory activated PBMCs in certain diseases as well as the responsiveness of these cells to anti-inflammatory drugs may be evaluated by quantification of these marker proteins. This article is part of a Special Issue entitled: Integrated omics.

Activation-induced cytidine deaminase (AID) linking immunity, chronic inflammation, and cancer.

Activation-induced cytidine deaminase (AID) is critically involved in class switch recombination and somatic hypermutation of Ig loci resulting in diversification of antibodies repertoire and production of high-affinity antibodies and as such represents a physiological tool to introduce DNA alterations. These processes take place within germinal centers of secondary lymphoid organs. Under physiological conditions, AID is expressed predominantly in activated B lymphocytes. Because of the mutagenic and recombinogenic potential of AID, its expression and activity is tightly regulated on different levels to minimize the risk of unwanted DNA damage. However, chronic inflammation and, probably, combination of other not-yet-identified factors are able to create a microenvironment sufficient for triggering an aberrant AID expression in B cells and, importantly, in non-B-cell background. Under these circumstances, AID may target also non-Ig genes, including cancer-related genes as oncogenes, tumor suppressor genes, and genomic stability genes, and modulate both genetic and epigenetic information. Despite ongoing progress, the complete understanding of fundamental aspects is still lacking as (1) what are the crucial factors triggering an aberrant AID expression/activity including the impact of Th2-driven inflammation and (2) to what extent may aberrant AID in human non-B cells lead to abnormal cell state associated with an increased rate of genomic alterations as point mutations, small insertions or deletions, and/or recurrent chromosomal translocations during solid tumor development and progression.

B cells and ectopic follicular structures: novel players in anti-tumor programming with prognostic power for patients with metastatic colorectal cancer.

Remarkably limited information is available about biological mechanisms that determine the disease entity of metastatic colorectal cancer in the liver (CRCLM) with no good clinical parameters to estimate prognosis. For the last few years, understanding the relationship between tumor characteristics and local immune response has gained increasing attention. Given the multifaceted roles of B-cell-driven responses, we aimed to elucidate the immunological imprint of B lymphocytes at the metastatic site, the interrelation with macrophages, and their prognostic relevance. Here we present novel algorithm allowing to assess a link between the local patient-specific immunological capacity and clinical outcome. The microscopy-based imaging platform was used for automated scanning of large-scale tissue sections and subsequent qualitative and quantitative analyses of immune cell subtypes using lineage markers and single-cell recognition strategy. Results indicate massive infiltration of CD45-positive leukocytes confined to the metastatic border. We report for the first time the accumulation of CD20-positive B lymphocytes at the tumor – liver interface comprising the major population within the large CD45-positive aggregates. Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin. Furthermore, the CD20-based data set revealed a strong prognostic power: patients with high CD20 content and/or ectopic follicles had significantly lower risk for disease recurrence as revealed by univariate analysis (p<0.001 for both) and in models adjusted for clinicopathological variables (p<0.001 and p = 0.01, respectively), and showed prolonged overall survival. In contrast, CD68 staining-derived data set did not show an association with clinical outcome. Taken together, we nominate the magnitude of B lymphocytes, including those organized in ectopic follicles, as novel prognostic marker which is superior to clinicopathological parameters. Findings emphasize anti-tumoral role of B cell-driven mechanism(s) and thus indicate a new way of thinking about potential treatment strategies for CRCLM patients.

Extracellular Matrix Remodeling by Bone Marrow Fibroblast-like Cells Correlates with Disease Progression in Multiple Myeloma

The pathogenesis of multiple myeloma (MM) is regarded as a multistep process, in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. Molecular events characteristic for the transition from MGUS to MM are still poorly defined. We hypothesized that fibroblast-like cells in the tumor microenvironment are critically involved in the pathogenesis of MM. Therefore, we performed a comparative proteome profiling study, analyzing primary human fibroblast-like cells isolated from the bone marrow of MM, of MGUS, as well as of non-neoplastic control patients. Thereby, a group of extracellular matrix (ECM) proteins, ECM receptors, and ECM-modulating enzymes turned out to be progressively up-regulated in MGUS and MM. These proteins include laminin α4, lysyl-hydroxylase 2, prolyl 4-hydroxylase 1, nidogen-2, integrin α5β5, c-type mannose receptor 2, PAI-1, basigin, and MMP-2, in addition to PDGF-receptor β and the growth factor periostin, which are likewise involved in ECM activities. Our results indicate that ECM remodeling by fibroblast-like cells may take place already at the level of MGUS and may become even more pronounced in MM. The identified proteins which indicate the stepwise progression from MGUS to MM may offer new tools for therapeutic strategies.

Plasticity of fibroblasts demonstrated by tissue-specific and function-related proteome profiling.

BackgroundFibroblasts are mesenchymal stromal cells which occur in all tissue types. While their main function is related to ECM production and physical support, they are also important players in wound healing, and have further been recognized to be able to modulate inflammatory processes and support tumor growth. Fibroblasts can display distinct phenotypes, depending on their tissue origin, as well as on their functional state.ResultsIn order to contribute to the proteomic characterization of fibroblasts, we have isolated primary human fibroblasts from human skin, lung and bone marrow and generated proteome profiles of these cells by LC-MS/MS. Comparative proteome profiling revealed characteristic differences therein, which seemed to be related to the cell’s tissue origin. Furthermore, the cells were treated in vitro with the pro-inflammatory cytokine IL-1beta. While all fibroblasts induced the secretion of Interleukins IL-6 and IL-8 and the chemokine GRO-alpha, other inflammation-related proteins were up-regulated in an apparently tissue-dependent manner. Investigating fibroblasts from tumorous tissues of skin, lung and bone marrow with respect to such inflammation-related proteins revealed hardly any conformity but rather individual and tumor type-related variations. However, apparent up-regulation of IGF-II, PAI-1 and PLOD2 was observed in melanoma-, lung adenocarcinoma- and multiple myeloma-associated fibroblasts, as well as in hepatocellular carcinoma-associated fibroblasts.ConclusionsInflammation-related proteome alterations of primary human fibroblasts were determined by the analysis of IL-1beta treated cells. Tumor-associated fibroblasts from different tissue types hardly showed signs of acute inflammation but displayed characteristic functional aberrations potentially related to chronic inflammation. The present data suggest that the state of the tumor microenvironment is relevant for tumor progression and targeted treatment of tumor-associated fibroblasts may support anti-cancer strategies.

AllergoOncology – the impact of allergy in oncology: EAACI position paper

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE‐mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state‐of‐the‐art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE‐mediated tumour antigen cross‐presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

Vemurafenib Resistance Signature by Proteome Analysis Offers New Strategies and Rational Therapeutic Concepts

The FDA-approved BRAF inhibitor vemurafenib achieves outstanding clinical response rates in patients with melanoma, but early resistance is common. Understanding the pathologic mechanisms of drug resistance and identification of effective therapeutic alternatives are key scientific challenges in the melanoma setting. Using proteomic techniques, including shotgun analysis and 2D-gel electrophoresis, we identified a comprehensive signature of the vemurafenib-resistant M24met in comparison with the vemurafenib-sensitive A375 melanoma cell line. The resistant cells were characterized by loss of differentiation, induction of transformation, enhanced expression of the lysosomal compartment, increased potential for metastasis, migration, adherence and Ca2+ ion binding, enhanced expression of the MAPK pathway and extracellular matrix proteins, and epithelial–mesenchymal transformation. The main features were verified by shotgun analysis with QEXACTIVE orbitrap MS, electron microscopy, lysosomal staining, Western blotting, and adherence assay in a VM-1 melanoma cell line with acquired vemurafenib resistance. On the basis of the resistance profile, we were able to successfully predict that a novel resveratrol-derived COX-2 inhibitor, M8, would be active against the vemurafenib-resistant but not the vemurafenib-sensitive melanoma cells. Using high-throughput methods for cell line and drug characterization may thus offer a new way to identify key features of vemurafenib resistance, facilitating the design of effective rational therapeutic alternatives. Mol Cancer Ther; 14(3); 757–68. ©2015 AACR.

The calcium-sensing receptor suppresses epithelial-to-mesenchymal transition and stem cell- like phenotype in the colon

BackgroundThe calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown.Methods and resultsIn a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29CaSR) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29EMP). The HT29CaSR cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/β-catenin pathway as measured by a decrease in nuclear translocation of β-catenin and transcriptional regulation of genes like GSK-3β and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29CaSR cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype.ConclusionsThe results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.

Determination of cell type‐specific proteome signatures of primary human leukocytes, endothelial cells, keratinocytes, hepatocytes, fibroblasts and melanocytes by comparative proteome profiling

Cells gain their functional specialization by different protein synthesis. A lot of knowledge with respect to cell type‐specific proteins has been collected during the last thirty years. This knowledge was built mainly by using antibodies. Nowadays, modern MS, which supports comprehensive proteome analyses of biological samples, may render possible the search for cell type‐specific proteins as well. However, a therefore necessary systematic MS study comprising many different cell types has not been performed until now. Here we present a proteome analysis strategy supporting the automated and meaningful comparison of any biological samples. We have presently applied this strategy to six different primary human cell types, namely leukocytes, endothelial cells, keratinocytes, hepatocytes, fibroblasts, and melanocytes. Comparative analysis of the resulting proteome profiles allowed us to select proteins specifically identified in one of the six cell types and not in any of the five others. Based on these results, we designated cell type‐specific proteome signatures consisting each of six such characteristic proteins. These signatures independently reproduced well‐known marker proteins already established for FACS analyses in addition to novel candidate marker proteins. We applied these signatures for the interpretation of proteome profiles obtained from the analyses of hepatocellular carcinoma‐associated tissue homogenates and normal liver tissue homogenates. The identification of members of the above described signatures gave us an indication of the presence of characteristic cells in the diseased tissues and thus supported the interpretation of the proteomics data of these complex biological samples.

Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach

The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery to pathological EMT and may yield new biomarkers and therapeutic targets.