Publications by authors named "Georgios T Stathopoulos"

94 Publications

Immune Resistance in Lung Adenocarcinoma.

Cancers (Basel) 2021 Jan 21;13(3). Epub 2021 Jan 21.

Comprehensive Pneumology Center (CPC), Institute for Lung Biology and Disease (iLBD), Helmholtz Center Munich-German Research Center for Environmental Health, Member of the German Center for Lung Research, 81377 Munich, Germany.

Lung cancer is the leading cancer killer worldwide, imposing grievous challenges for patients and clinicians. The incidence of lung adenocarcinoma (LUAD), the main histologic subtype of lung cancer, is still increasing in current-, ex-, and even non-smokers, whereas its five-year survival rate is approximately 15% as the vast majority of patients usually present with advanced disease at the time of diagnosis. The generation of novel drugs targeting key disease driver mutations has created optimism for the treatment of LUAD, but, as these mutations are not universal, this therapeutic line benefits only a subset of patients. More recently, the advent of targeted immunotherapies and their documented clinical efficacy in many different cancers, including LUAD, have started to change cancer management. Immunotherapies have been developed in order to overcome the cancer's ability to develop mechanisms of immune resistance, i.e., to adapt to and evade the host inflammatory and immune responses. Identifying a cancer's immune resistance mechanisms will likely advance the development of personalized immunotherapies. This review examines the key pathways of immune resistance at play in LUAD and explores therapeutic strategies which can unleash potent antitumor immune responses and significantly improve therapeutic efficacy, quality of life, and survival in LUAD.
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http://dx.doi.org/10.3390/cancers13030384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864325PMC
January 2021

Engineered versus hybrid cellular vesicles as efficient drug delivery systems: a comparative study with brain targeted vesicles.

Drug Deliv Transl Res 2021 Jan 20. Epub 2021 Jan 20.

Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, 26510, Rio, Greece.

Herein we elaborated on methods to load cellular vesicles (CVs) and to incorporate cholesterol (Chol) and PEG lipids in their membrane, for enhancing the potential of such engineered CVs (e-CVs) as drug carriers. Hybrids formed by fusion between PEGylated liposomes (PEG-LIP) and CVs were evaluated as alternatives to e-CV, for the first time. Freeze-thawing cycles (FT) and incubation protocols were tested, and vesicle fusion was monitored by FRET dilution. B16F10, hCMEC/D3, and LLC cells were used for e-CV or hybrid development, and FITC-dextran as a model hydrophilic drug. Results show that dehydration rehydration vesicle (DRV) method is optimal for highest CV loading and integrity, while optimal protocols for Chol/PEG enrichment were identified. FT was found to be more efficient than incubation for hybrid formation. Interestingly, despite their high Chol content, CVs had very low integrity that was not increased by enrichment with Chol, but only after PEG coating; e-CVs demonstrated higher integrity than hybrids. Vesicle uptake by hCMEC cells is in the order: LIP < e-CVs < Hybrids ≤ CVs (verified by confocal microscopy); the higher PEG content of e-CVs is possibly the reason for their reduced cell uptake. While CV and hybrid uptake are highly caveolin-dependent, e-CVs mostly follow clathrin-dependent pathways. In vivo and ex vivo results show that brain accumulation of hybrids is only slightly higher that of CVs, indicating that the surface PEG content of hybrids is not sufficient to prevent uptake by macrophages of the reticuloendothelial system. Taking together with the fact that subjection of CVs to FT cycles reduced their cellular uptake, it is concluded that PEGylated e-CVs are better than hybrids as brain-targeted drug carriers.
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http://dx.doi.org/10.1007/s13346-021-00900-1DOI Listing
January 2021

Interferon Regulatory Factor 9 Promotes Lung Cancer Progression via Regulation of Versican.

Cancers (Basel) 2021 Jan 8;13(2). Epub 2021 Jan 8.

Max Planck Institute for Heart and Lung Research, Member of the German Center for Lung Research (DZL), Member of the Cardio-Pulmonary Institute (CPI), 61231 Bad Nauheim, Germany.

Transcription factors can serve as links between tumor microenvironment signaling and oncogenesis. Interferon regulatory factor 9 (IRF9) is recruited and expressed upon interferon stimulation and is dependent on cofactors that exert in tumor-suppressing or oncogenic functions via the JAK-STAT pathway. IRF9 is frequently overexpressed in human lung cancer and is associated with decreased patient survival; however, the underlying mechanisms remain to be elucidated. Here, we used stably transduced lung adenocarcinoma cell lines (A549 and A427) to overexpress or knockdown . Overexpression led to increased oncogenic behavior in vitro, including enhanced proliferation and migration, whereas knockdown reduced these effects. These findings were confirmed in vivo using lung tumor xenografts in nude mice, and effects on both tumor growth and tumor mass were observed. Using RNA sequencing, we identified versican () as a novel downstream target of IRF9. Indeed, and expression levels were found to be correlated. We showed for the first time that IRF9 binds at a newly identified response element in the promoter region of to regulate its transcription. Using an siRNA approach, VCAN was found to enable the oncogenic properties (proliferation and migration) of IRF9 transduced cells, perhaps with involvement. The targeted inhibition of IRF9 in lung cancer could therefore be used as a new treatment option without multimodal interference in microenvironment JAK-STAT signaling.
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http://dx.doi.org/10.3390/cancers13020208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827113PMC
January 2021

Patient-derived malignant pleural mesothelioma cell cultures: a tool to advance biomarker-driven treatments.

Thorax 2020 11 17;75(11):1004-1008. Epub 2020 Sep 17.

Oxford Centre for Respiratory Medicine, Churchill Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom.

Malignant pleural mesothelioma (MPM) is an aggressive cancer, associated with poor prognosis. We assessed the feasibility of patient-derived cell cultures to serve as an ex vivo model of MPM. Patient-derived MPM cell cultures (n=16) exhibited stemness features and reflected intratumour and interpatient heterogeneity. A subset of the cells were subjected to high-throughput drug screening and coculture assays with cancer-specific cytotoxic T cells and showed diverse responses. Some of the biphasic MPM cells were capable of processing and presenting the neoantigen SSX-2 endogenously. In conclusion, patient-derived MPM cell cultures are a promising and faithful ex vivo model of MPM.
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http://dx.doi.org/10.1136/thoraxjnl-2020-215027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569377PMC
November 2020

MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.

Nat Commun 2020 09 10;11(1):4527. Epub 2020 Sep 10.

Department of Medicine III, Klinikum rechts der Isar, TUM School of Medicine, Technical University of Munich, Munich, Germany.

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.
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http://dx.doi.org/10.1038/s41467-020-18372-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484793PMC
September 2020

Socioeconomic correlates of SARS-CoV-2 and influenza H1N1 outbreaks.

Eur Respir J 2020 09 17;56(3). Epub 2020 Sep 17.

Comprehensive Pneumology Center (CPC) and Institute for Lung Biology and Disease (iLBD), Helmholtz Center Munich for Environmental Health (Helmholtz Zentrum München für Gesundheit und Umwelt, HMGU); and German Center for Lung Research (Deutsches Zentrum für Lungenforschung, DZL), Neuherberg, Germany

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http://dx.doi.org/10.1183/13993003.01400-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366181PMC
September 2020

Integrin-linked kinase (ILK) regulates KRAS, IPP complex and Ras suppressor-1 (RSU1) promoting lung adenocarcinoma progression and poor survival.

J Mol Histol 2020 Aug 22;51(4):385-400. Epub 2020 Jun 22.

Department of Anatomy-Histology-Embryology, Medical School, University of Patras, 26500, Patras, Greece.

Integrin-linked kinase (ILK) forms a heterotrimeric protein complex with PINCH and PARVIN (IPP) in Focal Adhesions (FAs) that acts as a signaling platform between the cell and its microenvironment regulating important cancer-related functions. We aimed to elucidate the role of ILK in lung adenocarcinoma (LUADC) focusing on a possible link with KRAS oncogene. We used immunohistochemistry on human tissue samples and KRAS-driven LUADC in mice, analysis of large scale publicly available RNA sequencing data, ILK overexpression and pharmacological inhibition as well as knockdown of KRAS in lung cancer cells. ILK, PINCH1 and PARVB (IPP) proteins are overexpressed in human LUADC and KRAS-driven LUADC in mice representing poor prognostic indicators. Genes implicated in ILK signaling are significantly enriched in KRAS-driven LUADC. Silencing of KRAS, as well as, overexpression and pharmacological inhibition of ILK in lung cancer cells provide evidence of a two-way association between ILK and KRAS. Upregulation of PINCH, PARVB and Ras suppressor-1 (RSU1) expression was demonstrated in ILK overexpressing lung cancer cells in addition to a significant positive correlation between these factors in tissue samples, while KRAS silencing downregulates IPP and RSU1. Pharmacological inhibition of ILK in KRAS mutant lung cancer cells suppresses cell growth, migration, EMT and increases sensitivity to platinum-based chemotherapy. ILK promotes an aggressive lung cancer phenotype with prognostic and therapeutic value through functions that involve KRAS, IPP complex and RSU1, rendering ILK a promising biomarker and therapeutic target in lung adenocarcinoma.
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http://dx.doi.org/10.1007/s10735-020-09888-3DOI Listing
August 2020

Biologically based models of cancer risk in radiation research.

Int J Radiat Biol 2021 16;97(1):2-11. Epub 2020 Jul 16.

Comprehensive Pneumology Center, Helmholtz Zentrum München, Munich, Germany.

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http://dx.doi.org/10.1080/09553002.2020.1784490DOI Listing
July 2020

Reprogramming of tumor-associated macrophages by targeting β-catenin/FOSL2/ARID5A signaling: A potential treatment of lung cancer.

Sci Adv 2020 Jun 5;6(23):eaaz6105. Epub 2020 Jun 5.

Max Planck Institute for Heart and Lung Research, Member of the German Center for Lung Research (DZL), Member of the Cardio-Pulmonary Institute (CPI), Bad Nauheim 61231, Germany.

Tumor-associated macrophages (TAMs) influence lung tumor development by inducing immunosuppression. Transcriptome analysis of TAMs isolated from human lung tumor tissues revealed an up-regulation of the Wnt/β-catenin pathway. These findings were reproduced in a newly developed in vitro "trained" TAM model. Pharmacological and macrophage-specific genetic ablation of β-catenin reprogrammed M2-like TAMs to M1-like TAMs both in vitro and in various in vivo models, which was linked with the suppression of primary and metastatic lung tumor growth. An in-depth analysis of the underlying signaling events revealed that β-catenin-mediated transcriptional activation of FOS-like antigen 2 (FOSL2) and repression of the AT-rich interaction domain 5A (ARID5A) drive gene regulatory switch from M1-like TAMs to M2-like TAMs. Moreover, we found that high expressions of β-catenin and FOSL2 correlated with poor prognosis in patients with lung cancer. In conclusion, β-catenin drives a transcriptional switch in the lung tumor microenvironment, thereby promoting tumor progression and metastasis.
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http://dx.doi.org/10.1126/sciadv.aaz6105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274802PMC
June 2020

Role of exosomal microRNAs in lung cancer biology and clinical applications.

Cell Prolif 2020 Jun 11;53(6):e12828. Epub 2020 May 11.

Department of Respiratory Medicine, Xiangya Hospital, Central South University, Changsha, China.

Exosomes, small extracellular vesicles ranging from 30 to 150 nm, are secreted by various cell types, including tumour cells. Recently, microRNAs (miRNAs) were identified to be encapsulated and hence protected from degradation within exosomes. These exosomal miRNAs can be horizontally transferred to target cells, in which they subsequently modulate biological processes. Increasing evidence indicates that exosomal miRNAs play a critical role in modifying the microenvironment of lung cancers, possibly facilitating progression, invasion, angiogenesis, metastasis and drug resistance. In this review, we summarize the novel findings on exosomal miRNA functions during lung cancer initiation and progression. In addition, we highlight their potential role and challenges as biomarkers in lung cancer diagnosis, prognosis and drug resistance and as therapeutic agents.
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http://dx.doi.org/10.1111/cpr.12828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309943PMC
June 2020

Effects of Inhaled Tobacco Smoke on the Pulmonary Tumor Microenvironment.

Adv Exp Med Biol 2020 ;1225:53-69

Comprehensive Pneumology Center (CPC) and Institute for Lung Biology and Disease (iLBD), University Hospital, Ludwig-Maximilians University and Helmholtz Center Munich, Member of the German Center for Lung Research (DZL), Munich, Bavaria, Germany.

Tobacco smoke is a multicomponent mixture of chemical, organic, and inorganic compounds, as well as additive substances and radioactive materials. Many studies have proved the carcinogenicity of various of these compounds through the induction of DNA adducts, mutational potential, epigenetic changes, gene fusions, and chromosomal events. The tumor microenvironment plays an important role in malignant tumor formation and progression through the regulation of expression of key molecules which mediate the recruitment of immune cells to the tumor site and subsequently regulate tumor growth and metastasis. In this chapter, we discuss the effects of inhaled tobacco smoke in the tumor microenvironment of the respiratory tract. The mechanisms underlying these effects as well as their link with tumor progression are analyzed.
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http://dx.doi.org/10.1007/978-3-030-35727-6_4DOI Listing
February 2020

Anti-neuroinflammatory, protective effects of the synthetic microneurotrophin BNN-20 in the advanced dopaminergic neurodegeneration of "weaver" mice.

Neuropharmacology 2020 03 23;165:107919. Epub 2019 Dec 23.

Department of Physiology, School of Medicine, University of Patras, Patras, 26500, Greece. Electronic address:

BNN-20 is a synthetic microneurotrophin, long-term (P1-P21) administration of which exerts potent neuroprotective effect on the "weaver" mouse, a genetic model of progressive, nigrostriatal dopaminergic degeneration. The present study complements and expands our previous work, providing evidence that BNN-20 fully protects the dopaminergic neurons even when administration begins at a late stage of dopaminergic degeneration (>40%). Since neuroinflammation plays a critical role in Parkinson's disease, we investigated the possible anti-neuroinflammatory mechanisms underlying the pharmacological action of BNN-20. The latter was shown to be microglia-mediated, at least in part. Indeed, BNN-20 induced a partial, but significant, reversal of microglia hyperactivation, observed in the untreated "weaver" mouse. Furthermore, it induced a shift in microglia polarization towards the neuroprotective M2 phenotype, suggesting a possible beneficial shifting of microglia activity. This observation was further supported by morphometric measurements. Moreover, BDNF levels, which were severely reduced in the "weaver" mouse midbrain, were restored to normal even after short-term BNN-20 administration. Experiments in "weaver"/NGL (dual GFP/luciferase-NF-κВ reporter) mice using bioluminescence after a short BNN-20 treatment (P60-P74), have shown that the increase of BDNF production was specifically mediated through the TrkB-PI3K-Akt-NF-κB signaling pathway. Interestingly, long-term BNN-20 treatment (P14-P60) significantly increased dopamine levels in the "weaver" striatum, which seems to be associated with the improved motor activity observed in the treated mutant animals. In conclusion, our findings suggest that BNN-20 may serve as a lead molecule for new therapeutic compounds for Parkinson's disease, combining strong anti-neuroinflammatory and neuroprotective properties, leading to elevated dopamine levels and improved motor activity.
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http://dx.doi.org/10.1016/j.neuropharm.2019.107919DOI Listing
March 2020

Osteopontin drives KRAS-mutant lung adenocarcinoma.

Carcinogenesis 2020 Aug;41(8):1134-1144

Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Achaia, Greece.

Increased expression of osteopontin (secreted phosphoprotein 1, SPP1) is associated with aggressive human lung adenocarcinoma (LADC), but its function remains unknown. Our aim was to determine the role of SPP1 in smoking-induced LADC. We combined mouse models of tobacco carcinogen-induced LADC, of deficiency of endogenous Spp1 alleles, and of adoptive pulmonary macrophage reconstitution to map the expression of SPP1 and its receptors and determine its impact during carcinogenesis. Co-expression of Spp1 and mutant KrasG12C in benign cells was employed to investigate SPP1/KRAS interactions in oncogenesis. Finally, intratracheal adenovirus encoding Cre recombinase was delivered to LSL.KRASG12D mice lacking endogenous or overexpressing transgenic Spp1 alleles. SPP1 was overexpressed in experimental and human LADC and portended poor survival. In response to two different smoke carcinogens, Spp1-deficient mice developed fewer and smaller LADC with decreased cellular survival and angiogenesis. Both lung epithelial- and macrophage-secreted SPP1 drove tumor-associated inflammation, while epithelial SPP1 promoted early tumorigenesis by fostering the survival of KRAS-mutated cells. Finally, loss and overexpression of Spp1 was, respectively, protective and deleterious for mice harboring KRASG12D-driven LADC. Our data support that SPP1 is functionally involved in early stages of airway epithelial carcinogenesis driven by smoking and mutant KRAS and may present an important therapeutic target.
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http://dx.doi.org/10.1093/carcin/bgz190DOI Listing
August 2020

DNA Replication Inhibitor Geminin and Retinoic Acid Signaling Participate in Complex Interactions Associated With Pluripotency.

Cancer Genomics Proteomics 2019 Nov-Dec;16(6):593-601

Department of Physiology, Medical School, University of Patras, Patras, Greece

Background/aim: Several links between DNA replication, pluripotency and development have been recently identified. The involvement of miRNA in the regulation of cell cycle events and pluripotency factors has also gained attention.

Materials And Methods: In the present study, we used the g:Profiler platform to analyze transcription factor binding sites, miRNA networks and protein-protein interactions to identify novel links among the aforementioned processes.

Results And Conclusion: A complex circuitry between retinoic acid signaling, SWI/SNF components, pluripotency factors including Oct4, Sox2 and Nanog and cell cycle regulators was identified. It is suggested that the DNA replication inhibitor geminin plays a central role in this circuitry.
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http://dx.doi.org/10.21873/cgp.20162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885373PMC
March 2020

Osteopontin as a Link between Inflammation and Cancer: The Thorax in the Spotlight.

Cells 2019 08 2;8(8). Epub 2019 Aug 2.

Comprehensive Pneumology Center and Institute for Lung Biology and Disease, University Hospital, Ludwig-Maximilians University of Munich and Helmholtz Center Munich, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Bavaria, Germany.

The glycoprotein osteopontin (OPN) possesses multiple functions in health and disease. To this end, osteopontin has beneficial roles in wound healing, bone homeostasis, and extracellular matrix (ECM) function. On the contrary, osteopontin can be deleterious for the human body during disease. Indeed, osteopontin is a cardinal mediator of tumor-associated inflammation and facilitates metastasis. The purpose of this review is to highlight the importance of osteopontin in malignant processes, focusing on lung and pleural tumors as examples.
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http://dx.doi.org/10.3390/cells8080815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721491PMC
August 2019

Elimination of KLK5 inhibits early skin tumorigenesis by reducing epidermal proteolysis and reinforcing epidermal microstructure.

Biochim Biophys Acta Mol Basis Dis 2019 11 2;1865(11):165520. Epub 2019 Aug 2.

Department of Pharmacy, School of Health Sciences, University of Patras, 265 04 Rio, Greece. Electronic address:

Epidermal desquamation involves a finely-tuned proteolytic cascade ensuring the regulated cleavage of desmosomes that releases old stratum corneum outermost layers. Although the roles of desmosomes in normal physiology are well-established, their putative involvement in cancer remains unexplored. The KLK5 protease is thought of having fundamental roles in epidermal proteolysis and homeostasis, and its aberrant activity has been linked to skin pathologies. We found that deletion of Klk5 results in significantly higher numbers of lengthier desmosomes and enhanced skin strength. Klk5 mice retained normal skin barrier function and are resistant to chemically-induced skin tumorigenesis. The resistance to tumorigenesis was not due to inhibition of inflammation, and on the contrary, absence of Klk5 increased the TPA-induced inflammatory skin response. We found that increased desmosomes and reduced proteolysis prevent oncogenic signaling by capturing β-catenin into the cytoplasm and facilitate epidermal keratinocyte apoptosis, thus, inhibiting tumor initiation. We highlight that the skin ultrastructure affects early neoplastic transformation by modulating intracellular signaling and suggest that tissue reinforcement provides a novel mode of tumor suppression.
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http://dx.doi.org/10.1016/j.bbadis.2019.07.014DOI Listing
November 2019

Interleukin-1β provided by KIT-competent mast cells is required for -mutant lung adenocarcinoma.

Oncoimmunology 2019;8(7):1593802. Epub 2019 Apr 11.

Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Achaia, Greece.

Mast cells (MC) have been identified in human lung adenocarcinoma (LADC) tissues, but their functional role has not been investigated . For this, we applied three mouse models of mutant LADC to two different MC-deficient mouse strains ( and ). Moreover, we derived MC gene signatures from murine bone marrow-derived MC and used them to interrogate five human cohorts of LADC patients. Tumor-free and mice were deficient in alveolar and skin KIT-dependent (KIT+) MC, but mice retained normal KIT-independent (KIT-) MC in the airways. Both KIT+ and KIT- MC infiltrated murine LADC to varying degrees, but KIT+ MC were more abundant and promoted LADC initiation and progression through interleukin-1β secretion. KIT+ MC and their transcriptional signature were significantly enriched in human LADC compared to adjacent normal tissue, especially in the subset of patients with mutations. Importantly, MC density increased with tumor stage and high overall expression of the KIT+ MC signature portended poor survival. Collectively, our results indicate that KIT+ MC foster LADC development and represent marked therapeutic targets.
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http://dx.doi.org/10.1080/2162402X.2019.1593802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527299PMC
April 2019

Club cells form lung adenocarcinomas and maintain the alveoli of adult mice.

Elife 2019 05 29;8. Epub 2019 May 29.

Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease.
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http://dx.doi.org/10.7554/eLife.45571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606035PMC
May 2019

Wnt1 silences chemokine genes in dendritic cells and induces adaptive immune resistance in lung adenocarcinoma.

Nat Commun 2019 03 29;10(1):1405. Epub 2019 Mar 29.

Division of Immunology, Biomedical Sciences Research Center Alexander Fleming, Vari-Athens, 16672, Greece.

Lung adenocarcinoma (LUAD)-derived Wnts increase cancer cell proliferative/stemness potential, but whether they impact the immune microenvironment is unknown. Here we show that LUAD cells use paracrine Wnt1 signaling to induce immune resistance. In TCGA, Wnt1 correlates strongly with tolerogenic genes. In another LUAD cohort, Wnt1 inversely associates with T cell abundance. Altering Wnt1 expression profoundly affects growth of murine lung adenocarcinomas and this is dependent on conventional dendritic cells (cDCs) and T cells. Mechanistically, Wnt1 leads to transcriptional silencing of CC/CXC chemokines in cDCs, T cell exclusion and cross-tolerance. Wnt-target genes are up-regulated in human intratumoral cDCs and decrease upon silencing Wnt1, accompanied by enhanced T cell cytotoxicity. siWnt1-nanoparticles given as single therapy or part of combinatorial immunotherapies act at both arms of the cancer-immune ecosystem to halt tumor growth. Collectively, our studies show that Wnt1 induces immunologically cold tumors through cDCs and highlight its immunotherapeutic targeting.
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http://dx.doi.org/10.1038/s41467-019-09370-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441097PMC
March 2019

Risk of lung adenocarcinoma from smoking and radiation arises in distinct molecular pathways.

Carcinogenesis 2019 Oct;40(10):1240-1250

Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine; University of Patras; Rio, Achaia, Greece.

KRAS mutations of lung adenocarcinoma (LADC) are associated with smoking but little is known on other exposure-oncogene associations. Hypothesizing that different inciting agents may cause different driver mutations, we aimed to identify distinct molecular pathways to LADC, applying two entirely different approaches. First, we examined clinicopathologic features and genomic signatures of environmental exposures in the large LADC Campbell data set. Second, we designed a molecular mechanistic risk model of LADC (M3LADC) that links environmental exposure to incidence risk by mathematically emulating the disease process. This model was applied to incidence data of Japanese atom-bomb survivors which contains information on radiation and smoking exposure. Grouping the clinical data by driver mutations revealed two main distinct molecular pathways to LADC: one unique to transmembrane receptor-mutant patients that displayed robust signatures of radiation exposure and one shared between submembrane transducer-mutant patients and patients with no evident driver mutation that carried the signature of smoking. Consistently, best fit of the incidence data was achieved with a M3LADC with two pathways: in one LADC risk increased with radiation exposure and in the other with cigarette consumption. We conclude there are two main molecular pathways to LADC associated with different environmental exposures. Future molecular measurements in lung cancer tissue of atom-bomb survivors may allow to further test quantitatively the M3LADC-predicted link of radiation to transmembrane receptor mutations. Moreover, the developed molecular mechanistic model showed that for low doses, as relevant e.g. for medical imaging, smokers have the same radiation risk compared with never smokers.
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http://dx.doi.org/10.1093/carcin/bgz036DOI Listing
October 2019

Tobacco chemical-induced mouse lung adenocarcinoma cell lines pin the prolactin orthologue proliferin as a lung tumour promoter.

Carcinogenesis 2019 Nov;40(11):1352-1362

Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Achaia, Greece.

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro, and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers' LADC, including carcinogen-specific Kras point mutations (KrasQ61R in urethane- and KrasQ61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease.
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http://dx.doi.org/10.1093/carcin/bgz047DOI Listing
November 2019

Comprehensive clinical profiling of the Gauting locoregional lung adenocarcinoma donors.

Cancer Med 2019 04 25;8(4):1486-1499. Epub 2019 Feb 25.

Comprehensive Pneumology Center and Institute for Lung Biology and Disease, University Hospital, Ludwig-Maximilians University of Munich (LMU) and Helmholtz Center Munich, Member of the German Center for Lung Research (DZL), Munich, Bavaria, Germany.

A comprehensive characterization of lung adenocarcinoma (LADC) clinical features is currently missing. We prospectively evaluated Caucasian patients with early-stage LADC. Patients with LADC diagnosed between 2011 and 2015 were prospectively assessed for lung resection with curative intent. Fifty clinical, pathologic, radiologic, and molecular variables were recorded. Patients were followed till death/study conclusion. The main findings were compared to a separate cohort from France. Of 1943 patients evaluated, 366 were enrolled (18.8%; 181 female; 75 never-smokers; 28% of registered Bavarian cases over the study period). Smoking and obstruction were significantly more prevalent in GLAD compared with adult Bavarians (P < 0.0001). Ever-smoker tumors were preferentially localized to the upper lobes. We observed 120 relapses and 74 deaths over 704 cumulative follow-up years. Median overall and disease-free survival were >7.5 and 3.6 years, respectively. Patients aged <45 or >65 years, resected >60 days postdiagnosis, with abnormal FVC/DL V , N2/N3 stage, or solid histology had significantly decreased survival estimates. These were fit into a weighted locoregional LADC death risk score that outperformed pTNM7 in predicting survival in the GLAD and in our second cohort. We define the clinical gestalt of locoregional LADC and provide a new clinical tool to predict survival, findings that may aid future management and research design.
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http://dx.doi.org/10.1002/cam4.2031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488114PMC
April 2019

Inhibition of B cell-dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation.

JCI Insight 2019 02 7;4(3). Epub 2019 Feb 7.

Comprehensive Pneumology Center, Member of the German Center for Lung Research, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Ludwig-Maximilians University Munich, Munich Germany.

Lung transplantation (LTx) is the only therapeutic option for many patients with chronic lung disease. However, long-term survival after LTx is severely compromised by chronic rejection (chronic lung allograft dysfunction [CLAD]), which affects 50% of recipients after 5 years. The underlying mechanisms for CLAD are poorly understood, largely due to a lack of clinically relevant animal models, but lymphocytic bronchiolitis is an early sign of CLAD. Here, we report that lymphocytic bronchiolitis occurs early in a long-term murine orthotopic LTx model, based on a single mismatch (grafts from HLA-A2:B6-knockin donors transplanted into B6 recipients). Lymphocytic bronchiolitis is followed by formation of B cell-dependent lymphoid follicles that induce adjacent bronchial epithelial cell dysfunction in a spatiotemporal fashion. B cell deficiency using recipient μMT-/- mice prevented intrapulmonary lymphoid follicle formation and lymphocytic bronchiolitis. Importantly, selective inhibition of the follicle-organizing receptor EBI2, using genetic deletion or pharmacologic inhibition, prevented functional and histological deterioration of mismatched lung grafts. In sum, we provided what we believe to be a mouse model of chronic rejection and lymphocytic bronchiolitis after LTx and identified intrapulmonary lymphoid follicle formation as a target for pharmacological intervention of long-term allograft dysfunction after LTx.
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http://dx.doi.org/10.1172/jci.insight.123971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413786PMC
February 2019

Geminin ablation in vivo enhances tumorigenesis through increased genomic instability.

J Pathol 2018 10 21;246(2):134-140. Epub 2018 Aug 21.

Department of Physiology, Medical School, University of Patras, Patras, Greece.

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham-treated controls in urethane-induced lung and azoxymethane/dextran sodium sulfate-induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non-monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5128DOI Listing
October 2018

Development and validation of response markers to predict survival and pleurodesis success in patients with malignant pleural effusion (PROMISE): a multicohort analysis.

Lancet Oncol 2018 07 13;19(7):930-939. Epub 2018 Jun 13.

Oxford Centre for Respiratory Medicine, Churchill Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK; Laboratory of Pleural and Lung Cancer Translational Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Oxford Respiratory Trials Unit, Nuffield Department of Medicine, University of Oxford, Oxford, UK; National Institute for Health Research Oxford Biomedical Research Centre, University of Oxford, Oxford, UK.

Background: The prevalence of malignant pleural effusion is increasing worldwide, but prognostic biomarkers to plan treatment and to understand the underlying mechanisms of disease progression remain unidentified. The PROMISE study was designed with the objectives to discover, validate, and prospectively assess biomarkers of survival and pleurodesis response in malignant pleural effusion and build a score that predicts survival.

Methods: In this multicohort study, we used five separate and independent datasets from randomised controlled trials to investigate potential biomarkers of survival and pleurodesis. Mass spectrometry-based discovery was used to investigate pleural fluid samples for differential protein expression in patients from the discovery group with different survival and pleurodesis outcomes. Clinical, radiological, and biological variables were entered into least absolute shrinkage and selection operator regression to build a model that predicts 3-month mortality. We evaluated the model using internal and external validation.

Findings: 17 biomarker candidates of survival and seven of pleurodesis were identified in the discovery dataset. Three independent datasets (n=502) were used for biomarker validation. All pleurodesis biomarkers failed, and gelsolin, macrophage migration inhibitory factor, versican, and tissue inhibitor of metalloproteinases 1 (TIMP1) emerged as accurate predictors of survival. Eight variables (haemoglobin, C-reactive protein, white blood cell count, Eastern Cooperative Oncology Group performance status, cancer type, pleural fluid TIMP1 concentrations, and previous chemotherapy or radiotherapy) were validated and used to develop a survival score. Internal validation with bootstrap resampling and external validation with 162 patients from two independent datasets showed good discrimination (C statistic values of 0·78 [95% CI 0·72-0·83] for internal validation and 0·89 [0·84-0·93] for external validation of the clinical PROMISE score).

Interpretation: To our knowledge, the PROMISE score is the first prospectively validated prognostic model for malignant pleural effusion that combines biological and clinical parameters to accurately estimate 3-month mortality. It is a robust, clinically relevant prognostic score that can be applied immediately, provide important information on patient prognosis, and guide the selection of appropriate management strategies.

Funding: European Respiratory Society, Medical Research Funding-University of Oxford, Slater & Gordon Research Fund, and Oxfordshire Health Services Research Committee Research Grants.
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http://dx.doi.org/10.1016/S1470-2045(18)30294-8DOI Listing
July 2018

RANK-c attenuates aggressive properties of ER-negative breast cancer by inhibiting NF-κB activation and EGFR signaling.

Oncogene 2018 09 29;37(37):5101-5114. Epub 2018 May 29.

Clinical and Molecular Oncology Laboratory, Division of Oncology, Department of Medicine, University of Patras, Patras, Greece.

The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression, and metastasis. RANK-c is a RANK receptor isoform produced through alternative splicing of the TNFRSF11A (RANK) gene and a dominant-negative regulator of RANK-induced nuclear factor-κB (NF-κB) activation. Here we report that RANK-c transcript is expressed in 3.2% of cases in The Cancer Genome Atlas breast cancer cohort evenly between ER-positive and ER-negative cases. Nevertheless, the ratio of RANK to RANK-c (RANK/RANK-c) is increased in ER-negative breast cancer cell lines compared to ER-positive breast cancer cell lines. In addition, forced expression of RANK-c in ER-negative breast cancer cell lines inhibited stimuli-induced NF-κB activation and attenuated migration, invasion, colony formation, and adhesion of cancer cells. Further, RANK-c expression in MDA-MB-231 cells inhibited lung metastasis and colonization in vivo. The RANK-c-mediated inhibition of cancer cell aggressiveness and nuclear factor-κB (NF-κB) activation in breast cancer cells seems to rely on a RANK-c/TNF receptor-associated factor-2 (TRAF2) protein interaction. This was further confirmed by a mutated RANK-c that is unable to interact with TRAF2 and abolishes the ability to attenuate NF-κB activation, migration, and invasion. Additional protein interaction characterization revealed epidermal growth factor receptor (EGFR) as a novel interacting partner for RANK-c in breast cancer cells with a negative effect on EGFR phosphorylation and EGF-dependent downstream signaling pathway activation. Our findings further elucidate the complex molecular biology of the RANKL/RANK system in breast cancer and provide preliminary data for RANK-c as a possible marker for disease progression and aggressiveness.
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http://dx.doi.org/10.1038/s41388-018-0324-yDOI Listing
September 2018

The Autotaxin-Lysophosphatidic Acid Axis Promotes Lung Carcinogenesis.

Cancer Res 2018 07 3;78(13):3634-3644. Epub 2018 May 3.

Division of Immunology, Biomedical Sciences Research Center "Alexander Fleming," Greece.

Pathogenesis and progression of lung cancer are governed by complex interactions between the environment and host genetic susceptibility, which is further modulated by genetic and epigenetic changes. Autotaxin (ATX, ENPP2) is a secreted glycoprotein that catalyzes the extracellular production of lysophosphatidic acid (LPA), a growth-factor-like phospholipid that is further regulated by phospholipid phosphatases (PLPP). LPA's pleiotropic effects in almost all cell types are mediated through at least six G-protein coupled LPA receptors (LPAR) that exhibit overlapping specificities, widespread distribution, and differential expression profiles. Here we use both preclinical models of lung cancer and clinical samples (from patients and healthy controls) to investigate the expression levels, activity, and biological role of the above components of the ATX/LPA axis in lung cancer. was genetically altered in 8% of patients with lung cancer, whereas increased ATX staining and activity were detected in patient biopsies and sera, respectively. Moreover, expression was consistently downregulated in patients with lung cancer. Comparable observations were made in the two most widely used animal models of lung cancer, the carcinogen urethane-induced and the genetically engineered -driven models, where genetic deletion of or resulted in disease attenuation, thus confirming a procarcinogenic role of LPA signaling in the lung. Expression profiling data analysis suggested that metabolic rewiring may be implicated in the procarcinogenic effects of the ATX/LPA axis in - -driven lung cancer pathogenesis. These findings establish the role of ATX/LPA in lung carcinogenesis, thus expanding the mechanistic links between pulmonary fibrosis and cancer. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3797DOI Listing
July 2018

p52 expression enhances lung cancer progression.

Sci Rep 2018 04 17;8(1):6078. Epub 2018 Apr 17.

Department of Cancer Biology, Vanderbilt University, Nashville, TN, 37232, USA.

While many studies have demonstrated that canonical NF-κB signaling is a central pathway in lung tumorigenesis, the role of non-canonical NF-κB signaling in lung cancer remains undefined. We observed frequent nuclear accumulation of the non-canonical NF-κB component p100/p52 in human lung adenocarcinoma. To investigate the impact of non-canonical NF-κB signaling on lung carcinogenesis, we employed transgenic mice with doxycycline-inducible expression of p52 in airway epithelial cells. p52 over-expression led to increased tumor number and progression after injection of the carcinogen urethane. Gene expression analysis of lungs from transgenic mice combined with in vitro studies suggested that p52 promotes proliferation of lung epithelial cells through regulation of cell cycle-associated genes. Using gene expression and patient information from The Cancer Genome Atlas (TCGA) database, we found that expression of p52-associated genes was increased in lung adenocarcinomas and correlated with reduced survival, even in early stage disease. Analysis of p52-associated gene expression in additional human lung adenocarcinoma datasets corroborated these findings. Together, these studies implicate the non-canonical NF-κB component p52 in lung carcinogenesis and suggest modulation of p52 activity and/or downstream mediators as new therapeutic targets.
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http://dx.doi.org/10.1038/s41598-018-24488-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904214PMC
April 2018

IκB Kinase α Is Required for Development and Progression of -Mutant Lung Adenocarcinoma.

Cancer Res 2018 06 27;78(11):2939-2951. Epub 2018 Mar 27.

Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB-activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in -mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of B, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic -mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage and IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against -mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in -mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease. These findings report a novel requirement for IKKα in mutant lung tumor formation, with potential therapeutic applications. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-1944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485619PMC
June 2018