Publications by authors named "Anastasia Meshcheryakova"

21 Publications

  • Page 1 of 1

The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer.

Cancers (Basel) 2020 Oct 25;12(11). Epub 2020 Oct 25.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna 1090, Austria.

The gut-associated lymphoid tissue represents an integral part of the immune system. Among the powerful players of the mucosa-associated lymphoid tissue are isolated lymphoid structures (ILSs), which as information centers, drive the local (and systemic) adaptive immune responses. Germinal center reactions, taking place within ILSs, involve the coordinated action of various immune cell types with a central role given to B cells. In the current study, we aimed at dissecting the impact of ILSs within non-tumorous colon tissue (NT) on the pathobiology of colorectal cancer (CRC) with metastasis in the liver (CRCLM). In particular, we focused on the immune phenotypes of ILSs and ectopic lymphoid structures (ELSs), built up at matching primary and metastatic tumor sites. We implemented an integrative analysis strategy on the basis of tissue image cytometry and clonality assessment to explore the immune phenotype of ILS/ELS at three tissue entities: NT, CRC, and CRCLM (69 specimens in total). Applying a panel of lineage markers used for immunostaining, we characterized and compared the anatomical features, the cellular composition, the activation, and proliferation status of ILSs and ELSs, and assessed the clinical relevance of staining-derived data sets. Our major discovery was that ILS characteristics at the NT site predefine the immune phenotype of ELSs at CRC and CRCLM. Thereby, B-cell-enriched (CD20) and highly proliferative (Ki67) ILSs and ELSs were found to be associated with improved clinical outcome in terms of survival and enabled patient stratification into risk groups. Moreover, the data revealed a linkage between B-cell clonality at the NT site and the metastatic characteristics of the tumor in the distant liver tissue. Consolidation of immunostaining-based findings with the results of compendium-wide transcriptomic analysis furthermore proposed CD27 as a novel marker of T follicular helper cells within lymphoid structures. Overall, the study nominates the ILS immune phenotype as a novel prognostic marker for patients with metastatic CRC.
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http://dx.doi.org/10.3390/cancers12113117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692185PMC
October 2020

Metabolism of Estrogens: Turnover Differs Between Platinum-Sensitive and -Resistant High-Grade Serous Ovarian Cancer Cells.

Cancers (Basel) 2020 Jan 23;12(2). Epub 2020 Jan 23.

Division of Clinical Pharmacy and Diagnostics, Department of Pharmaceutical Chemistry, University of Vienna, 1090 Vienna, Austria.

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary response; however, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to "platinum-sensitive", exhibiting a decreased IC value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, thereby allowing more efficient interventions.
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http://dx.doi.org/10.3390/cancers12020279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072378PMC
January 2020

Interrelations of Sphingolipid and Lysophosphatidate Signaling with Immune System in Ovarian Cancer.

Comput Struct Biotechnol J 2019 10;17:537-560. Epub 2019 Apr 10.

Molecular Systems Biology and Pathophysiology Research Group, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

The sphingolipid and lysophosphatidate regulatory networks impact diverse mechanisms attributed to cancer cells and the tumor immune microenvironment. Deciphering the complexity demands implementation of a holistic approach combined with higher-resolution techniques. We implemented a multi-modular integrative approach consolidating the latest accomplishments in gene expression profiling, prognostic/predictive modeling, next generation digital pathology, and systems biology for epithelial ovarian cancer. We assessed patient-specific transcriptional profiles using the sphingolipid/lysophosphatidate/immune-associated signature. This revealed novel sphingolipid/lysophosphatidate-immune gene-gene associations and distinguished tumor subtypes with immune high/low context. These were characterized by robust differences in sphingolipid-/lysophosphatidate-related checkpoints and the drug response. The analysis also nominates novel survival models for stratification of patients with , , , , and emerging as the most prognostically important genes. Alignment of proprietary data with curated transcriptomic data from public databases across a variety of malignancies (over 600 categories; over 21,000 arrays) showed specificity for ovarian carcinoma. Our systems approach identified novel sphingolipid-lysophosphatidate-immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes. This holds great promise for delivering novel stratifying and targeting strategies.
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http://dx.doi.org/10.1016/j.csbj.2019.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479272PMC
April 2019

Resveratrol Inhibits Key Steps of Steroid Metabolism in a Human Estrogen-Receptor Positive Breast Cancer Model: Impact on Cellular Proliferation.

Front Pharmacol 2018 10;9:742. Epub 2018 Jul 10.

Division of Clinical Pharmacy and Diagnostics, Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria.

The role of resveratrol (RES) in preventing breast cancer is controversial, as low concentrations may stimulate the proliferation of estrogen-receptor alpha positive (ERα+) breast cancer cells. As metabolism is the key factor in altering cellular estrogens, thereby influencing breast tumor growth, we investigated the effects of RES on the formation of estrogen metabolites, namely 4-androstene-3,17-dione (AD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone-3--sulfate (DHEA-S), estrone (E1), estrone-3-sulfate (E1-S), 17β-estradiol (E2), 17β-estradiol-3--(β-D-glucuronide) (E2-G), 17β-estradiol-3--sulfate (E2-S), 16α-hydroxy-17β-estradiol (estriol, E3), and testosterone (T) in ERα- MDA-MB-231 and ERα+ MCF-7 cells. Incubation of both of the cell lines with the hormone precursors DHEA and E1 revealed that sulfation and glucuronidation were preferred metabolic pathways for DHEA, E1 and E2 in MCF-7 cells, compared with in MDA-MB-231 cells, as the V values were significantly higher (DHEA-S: 2873.0 ± 327.4 fmol/10 cells/h, E1-S: 30.4 ± 2.5 fmol/10 cells/h, E2-S: 24.7 ± 4.9 fmol/10 cells/h, E2-G: 7.29 ± 1.36 fmol/10 cells/h). RES therefore significantly inhibited DHEA-S, E1-S, E2-S and E2-G formation in MCF-7, but not in MDA-MB-231 cells (Ks: E2-S, 0.73 ± 0.07 μM < E1-S, 0.94 ± 0.03 μM < E2-G, 7.92 ± 0.24 μM < DHEA-S, 13.2 ± 0.2 μM). Suppression of these metabolites subsequently revealed twofold higher levels of active E2, concomitant with an almost twofold increase in MCF-7 cell proliferation, which was the most pronounced upon the addition of 5 μM RES. As the content of RES in food is relatively low, an increased risk of breast cancer progression in women is likely to only be observed following the continuous consumption of high-dose RES supplements. Further long-term human studies simultaneously monitoring free estrogens and their conjugates are therefore highly warranted to evaluate the efficacy and safety of RES supplementation, particularly in patients diagnosed with ERα+ breast cancer.
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http://dx.doi.org/10.3389/fphar.2018.00742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048268PMC
July 2018

Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis.

PLoS One 2017 24;12(7):e0180900. Epub 2017 Jul 24.

Institute of Immunology, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

A crucial role of cell metabolism in immune cell differentiation and function has been recently established. Growing evidence indicates that metabolic processes impact both, innate and adaptive immunity. Since a down-stream integrator of metabolic alterations, mammalian target of rapamycin (mTOR), is responsible for controlling the balance between pro-inflammatory interleukin (IL)-12 and anti-inflammatory IL-10, we investigated the effect of upstream interference using metabolic modulators on the production of pro- and anti-inflammatory cytokines. Cytokine release and protein expression in human and murine myeloid cells was assessed after toll-like receptor (TLR)-activation and glucose-deprivation or co-treatment with 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators. Additionally, the impact of metabolic interference was analysed in an in-vivo mouse model. Glucose-deprivation by 2-deoxy-D-glucose (2-DG) increased the production of IL-12p40 and IL-23p19 in monocytes, but dose-dependently inhibited the release of anti-inflammatory IL-10. Similar effects have been observed using pharmacological AMPK activation. Consistently, an inhibition of the tuberous sclerosis complex-mTOR pathway was observed. In line with our in vitro observations, glycolysis inhibition with 2-DG showed significantly reduced bacterial burden in a Th2-prone Listeria monocytogenes mouse infection model. In conclusion, we showed that fasting metabolism modulates the IL-12/IL-10 cytokine balance, establishing novel targets for metabolism-based immune-modulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180900PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524343PMC
September 2017

Sphingosine 1-phosphate signaling in bone remodeling: multifaceted roles and therapeutic potential.

Expert Opin Ther Targets 2017 07 7;21(7):725-737. Epub 2017 Jun 7.

a Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology , Medical University of Vienna , Vienna , Austria.

Introduction: Sphingolipids belong to a complex class of lipid molecules that are crucially involved in the regulation of important biological processes including proliferation, migration and apoptosis. Given the significant progress made in understanding the sphingolipid pathobiology of several diseases, sphingolipid-related checkpoints emerge as attractive targets. Recent data indicate the multifaceted contribution of the sphingolipid machinery to osteoclast - osteoblast crosstalk, representing one of the pivotal interactions underlying bone homeostasis. Imbalances in the interplay of osteoblasts and osteoclasts might lead to bone-related diseases such as osteoporosis, rheumatoid arthritis, and bone metastases. Areas covered: We summarize and analyze the progress made in bone research in the context of the current knowledge of sphingolipid-related mechanisms regulating bone remodeling. Particular emphasis was given to bioactive sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs). Moreover, the mechanisms of how dysregulations of this machinery cause bone diseases, are covered. Expert opinion: In the context of bone diseases, pharmacological interference with sphingolipid machinery may lead to novel directions in therapeutic strategies. Implementation of knowledge derived from in vivo animal models and in vitro studies using pharmacological agents to manipulate the S1P/S1PRs axes suggests S1PR2 and S1PR3 as potential drug targets, particularly in conjunction with technology for local drug delivery.
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http://dx.doi.org/10.1080/14728222.2017.1332180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470107PMC
July 2017

Activation of the ileal neuroendocrine tumor cell line P-STS by acetylcholine is amplified by histamine: role of H3R and H4R.

Sci Rep 2017 05 2;7(1):1313. Epub 2017 May 2.

Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, 1090, Austria.

Neuroendocrine tumors may present with pseudoallergic reactions like diarrhea and idiopathic anaphylaxis. Here we present the P-STS human ileal neuroendocrine cell line as a model cell line for these tumors. Neuroendocrine markers and changes in cytoplasmic calcium concentration ([Ca]i) in response to several possible activators of 5-hydroxytryptamine (5-HT) release were analyzed. P-STS cells still expressed chromogranin A and synaptophysin after 2 years of culture. Tryptophan hydroxylase 1 mRNA and a low amount of 5-HT were also detected. Acetylcholine (ACh) caused a rise in [Ca]i. Somatostatin inhibited, whereas histamine (HA) but not the HA receptor ligand betahistine enhanced activation by ACh. The [Ca]i response to ACh/HA was inhibited by the HA receptor H3 (H3R) agonist methimepip and by the antidepressant imipramine. Further [Ca]i response studies indicated the presence of H4Rs and of a functional calcium sensing receptor. High or low affinity IgE receptor protein or mRNA were not detected. Taken together, neuroendocrine markers and response to intestinal neurotransmitters approve the P-STS cell line as a valuable model for enterochromaffin cells. Enhancement of their ACh-induced pro-secretory response by HA, with a role for H3R and H4R, suggests an amplifying role of neuroendocrine cells in allergen-induced diarrhea or anaphylaxis.
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http://dx.doi.org/10.1038/s41598-017-01453-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430954PMC
May 2017

Human skin dendritic cell fate is differentially regulated by the monocyte identity factor Kruppel-like factor 4 during steady state and inflammation.

J Allergy Clin Immunol 2017 Jun 11;139(6):1873-1884.e10. Epub 2016 Oct 11.

Institute of Pathophysiology and Immunology, Medical University of Graz, Graz, Austria; Institute of Immunology, Medical University of Vienna, Vienna, Austria. Electronic address:

Background: Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin-resident dermal DCs (dDCs)/interstitial-type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte-affiliated markers. Inversely, LCs express various epithelial genes critical for their long-term peripheral tissue residency.

Objective: Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood.

Methods: In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification.

Results: Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel-like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte-derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt-related transcription factor 3 in response to TGF-β1, thereby allowing LC differentiation marked by a low cytokine expression profile.

Conclusion: Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4.
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http://dx.doi.org/10.1016/j.jaci.2016.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538449PMC
June 2017

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BMC Genomics 2016 08 16;17(1):643. Epub 2016 Aug 16.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Background: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells.

Results: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background.

Conclusions: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.
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http://dx.doi.org/10.1186/s12864-016-3001-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986275PMC
August 2016

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

Oncotarget 2016 Apr;7(16):22295-323

Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.

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.
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http://dx.doi.org/10.18632/oncotarget.7947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5008362PMC
April 2016

Immunology of Osteoporosis: A Mini-Review.

Gerontology 2016 17;62(2):128-37. Epub 2015 Jun 17.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University Vienna, Vienna, Austria.

Osteoporosis is a major cause of fractures and associated morbidity in the aged population. The pathogenesis of osteoporosis is multifactorial; whereas traditional pathophysiological concepts emphasize endocrine mechanisms, it has been recognized that also components of the immune system have a significant impact on bone. Since 2000, when the term 'osteoimmunology' was coined, novel insights into the role of inflammatory cytokines by influencing the fine-tuned balance between bone resorption and bone formation have helped to explain the occurrence of osteoporosis in conjunction with chronic inflammatory reactions. Moreover, the phenomenon of a low-grade, chronic, systemic inflammatory state associated with aging has been defined as 'inflamm-aging' by Claudio Franceschi and has been linked to age-related diseases such as osteoporosis. Given the tight anatomical and physiological coexistence of B cells and the bone-forming units in the bone marrow, a role of B cells in osteoimmunological interactions has long been suspected. Recent findings of B cells as active regulators of the RANK/RANKL/OPG axis, of altered RANKL/OPG production by B cells in HIV-associated bone loss or of a modulated expression of genes linked to B-cell biology in response to estrogen deficiency support this assumption. Furthermore, oxidative stress and the generation of advanced glycation end products have emerged as links between inflammation and bone destruction.
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http://dx.doi.org/10.1159/000431091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821368PMC
December 2016

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

Mol Cancer 2015 Mar 18;14:61. Epub 2015 Mar 18.

Department of Pathophysiology and Allergy Research, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.

Background: The 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 Results: In 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 (HT29(CaSR)) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29(EMP)). The HT29(CaSR) 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 HT29(CaSR) 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.

Conclusions: The 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.
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http://dx.doi.org/10.1186/s12943-015-0330-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405849PMC
March 2015

[Researcher of the month].

Wien Klin Wochenschr 2015 Feb;127(3-4):160-1

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http://dx.doi.org/10.1007/s00508-015-0763-1DOI Listing
February 2015

Vemurafenib resistance signature by proteome analysis offers new strategies and rational therapeutic concepts.

Mol Cancer Ther 2015 Mar 22;14(3):757-68. Epub 2015 Jan 22.

Department of Dermatology, Medical University of Vienna, Vienna, Austria.

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.
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http://dx.doi.org/10.1158/1535-7163.MCT-14-0701DOI Listing
March 2015

Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation.

Blood 2014 Oct 11;124(17):2713-24. Epub 2014 Sep 11.

Institute of Immunology, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria; Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University Graz, Graz, Austria;

During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.
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http://dx.doi.org/10.1182/blood-2014-07-588178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536888PMC
October 2014

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

PLoS One 2014 6;9(6):e99008. Epub 2014 Jun 6.

Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099008PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048213PMC
January 2015

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

Electrophoresis 2014 May 20;35(10):1428-38. Epub 2014 Mar 20.

Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Austria; Department of Medicine I, Institute of Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Austria.

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.
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http://dx.doi.org/10.1002/elps.201300581DOI Listing
May 2014

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

Clin Proteomics 2014 21;11(1):41. Epub 2014 Nov 21.

Faculty of Chemistry, Department of Analytical Chemistry, University of Vienna, Vienna, Austria ; Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria ; Christian Doppler Laboratory for the Diagnosis and Regeneration of Cardiac and Thoracic Diseases, Medical University Vienna, Vienna, Austria.

Background: Fibroblasts 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.

Results: In 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.

Conclusions: Inflammation-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.
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http://dx.doi.org/10.1186/1559-0275-11-41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448269PMC
June 2015

Extracellular matrix remodeling by bone marrow fibroblast-like cells correlates with disease progression in multiple myeloma.

J Proteome Res 2014 Feb 2;13(2):844-54. Epub 2013 Dec 2.

Faculty of Chemistry, Institute of Analytical Chemistry, University of Vienna , Währingerstraße 38, A-1090 Vienna, Austria.

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.
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http://dx.doi.org/10.1021/pr400881pDOI Listing
February 2014

Proteome signatures of inflammatory activated primary human peripheral blood mononuclear cells.

J Proteomics 2012 Dec 16;76 Spec No.:150-62. Epub 2012 Jul 16.

Institute of Analytical Chemistry, University of Vienna, Austria.

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, GBP1 and 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.
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http://dx.doi.org/10.1016/j.jprot.2012.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509337PMC
December 2012

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

Cancer Immunol Immunother 2012 Sep 19;61(9):1591-8. Epub 2012 Apr 19.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria.

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.
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http://dx.doi.org/10.1007/s00262-012-1255-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427704PMC
September 2012