Department of Molecular Biology and Genetics

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Open Access
15-Lipoxygenase-1 re-expression in colorectal cancer alters endothelial cell features through enhanced expression of TSP-1 and ICAM-1
(Elsevier, 2017-11) Tunçer, S.; Keşküş, A. G.; Çolakoğlu, M.; Çimen, I.; Yener, C.; Konu, Ö.; Banerjee, S.
15-lipoxygenase-1 (15-LOX-1) oxygenates linoleic acid to 13(S)-hydroxyoctadecadienoic acid (HODE). The enzyme is widely suppressed in different cancers and its re-expression has tumor suppressive effects. 15-LOX-1 has been shown to inhibit neoangiogenesis in colorectal cancer (CRC); in the present study we confirm this phenomenon and describe the mechanistic basis. We show that re-expression of 15-LOX-1 in CRC cell lines resulted in decreased transcriptional activity of HIF1α and reduced the expression and secretion of VEGF in both normoxic and hypoxic conditions. Conditioned medium (CM) was obtained from CRC or prostate cancer cell lines re-expressing 15-LOX-1 (15-LOX-1CM). 15-LOX-1CM treated aortic rings from 6-week old C57BL/6 mice showed significantly less vessel sprouting and more organized structure of vascular network. Human umbilical vein endothelial cells (HUVECs) incubated with 15-LOX-1CM showed reduced motility, enhanced expression of intercellular cell adhesion molecule (ICAM-1) and reduced tube formation but no change in proliferation or cell-cycle distribution. HUVECs incubated with 13(S)-HODE partially phenocopied the effects of 15-LOX-1CM, i.e., showed reduced motility and enhanced expression of ICAM-1, but did not reduce tube formation, implying the importance of additional factors. Therefore, a Proteome Profiler Angiogenesis Array was carried out, which showed that Thrombospondin-1 (TSP-1), a matrix glycoprotein known to strongly inhibit neovascularization, was expressed significantly more in HUVECs incubated with 15-LOX-1CM. TSP-1 blockage in HUVECs reduced the expression of ICAM-1 and enhanced cell motility, thereby providing a mechanism for reduced angiogenesis. The anti-angiogenic effects of 15-LOX-1 through enhanced expressions of ICAM-1 and TSP-1 are novel findings and should be explored further to develop therapeutic options.
Open Access
2012 William allan award introduction: Uta Francke
(Elsevier, 2013-03-07) Özçelik, T.
Open Access
3-Propionyl-thiazolidine-4-carboxylic acid ethyl esters: A family of antiproliferative thiazolidines
(Royal Society of Chemistry, 2015) Önen-Bayram F.E.; Buran, K.; Durmaz I.; Berk, B.; Cetin-Atalay, R.
Cancer results from unregulated cell growth. Reactivating the process of the programmed cell death, i.e. apoptosis, is a classical anticancer therapeutic strategy. The apoptosis-inducing property of the (2RS,4R)-2-phenyl-3-propionyl-thiazolidine-4-carboxylic acid ethyl ester (ALC 67) molecule has recently been discovered. We analyzed in this study the impact of the phenyl moiety of this molecule on its biological activity by synthesizing and evaluating analogues where this substituent was replaced by a series of aromatic and aliphatic groups. The results demonstrated that the molecule's antiproliferative property resisted such modifications. Thus, in addition to developing a family of thiazolidine compounds with promising anticancer properties; our investigation revealed that the second position of the thiazolidine ring can be used either to tune the physicochemical properties of ALC67 or to introduce a fluorescent tag to the structure in order to track it in cells and determine its exact molecular mechanism of action. © 2015 The Royal Society of Chemistry.
Open Access
3D-MSCs A151 ODN-loaded exosomes are immunomodulatory and reveal a proteomic cargo that sustains wound resolution
(Elsevier B.V., 2022-11) Camões, Sérgio P.; Bulut, Özlem; Yazar, Volkan; Gaspar, Maria M.; Simões, Sandra; Ferreira, Rita; Vitorino, Rui; Santos, Jorge M.; Gürsel, İhsan; Miranda, Joana P.
Introduction: Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. Objectives: To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. Methods: MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. Results: Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro, with upregulation of IL-1α and VEGF-α or increased secretion of TGF-β, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-β, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. Conclusion: Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines. © 2022
Open Access
A candidate sex determination locus in amphibians which evolved by structural variation between X- and Y-chromosomes
(NATURE PORTFOLIO, 2024-06-05) Kuhl, Heiner; Tan, Wen Hui; Klopp, Christophe; Kleiner, Wibke; Koyun, Baturalp; Ciorpac, Mitica; Feron, Romain; Knytl, Martin; Kloas, Werner; Schartl, Manfred; Winkler, Christoph; Stoeck, Matthia
Most vertebrates develop distinct females and males, where sex is determined by repeatedly evolved environmental or genetic triggers. Undifferentiated sex chromosomes and large genomes have caused major knowledge gaps in amphibians. Only a single master sex-determining gene, the dmrt1-paralogue (dm-w) of female-heterogametic clawed frogs (Xenopus; ZW female/ZZ male), is known across >8740 species of amphibians. In this study, by combining chromosome-scale female and male genomes of a non-model amphibian, the European green toad, Bufo(tes) viridis, with ddRAD- and whole genome pool-sequencing, we reveal a candidate master locus, governing a male-heterogametic system (XX female/XY male). Targeted sequencing across multiple taxa uncovered structural X/Y-variation in the 5 '-regulatory region of the gene bod1l, where a Y-specific non-coding RNA (ncRNA-Y), only expressed in males, suggests that this locus initiates sex-specific differentiation. Developmental transcriptomes and RNA in-situ hybridization show timely and spatially relevant sex-specific ncRNA-Y and bod1l-gene expression in primordial gonads. This coincided with differential H3K4me-methylation in pre-granulosa/pre-Sertoli cells, pointing to a specific mechanism of amphibian sex determination.
Open Access
A common form of dominant human IFNAR1 deficiency impairs IFN-α and -ω but not IFN-β-dependent immunity
(Rockefeller University Press, 2024-12-16) Qureshah, Fahd Al; Pen, Jérémie Le; Weerd, Nicole A. de; Moncada-Velez, Marcela; Materna, Marie; Lin, Daniel C.; Milisavljevic, Baptiste; Vianna, Fernanda; Bizien, Lucy; Lorenzo, Lazaro; Lecuit, Marc; Pommier, Jean-David; Keles, Sevgi; Özçelik, Tayfun; Pedraza-Sanchez, Sigifredo; Prost, Nicolas de; Zein, Loubna El; Hammoud, Hassan; Ng, Lisa F.P.; Halwani, Rabih; Sharif-Askari, Narjes Saheb; Lau, Yu Lung; Tam, Anthony R.; Singh, Neha; Bhattad, Sagar; Berkun, Yackov; Chantratita, Wasun; Aguilar-López, Raúl; Shahrooei, Mohammad; Abel, Laurent; Bastard, Paul; Jouanguy, Emmanuelle; Béziat, Vivien; Zhang, Peng; Rice, Charles M.; Cobat, Aurélie; Zhang, Shen-Ying; Hertzog, Paul J.; Casanova, Jean-Laurent; Zhang, Qian
Autosomal recessive deficiency of the IFNAR1 or IFNAR2 chain of the human type I IFN receptor abolishes cellular responses to IFN-α, -β, and -ω, underlies severe viral diseases, and is globally very rare, except for IFNAR1 and IFNAR2 deficiency in Western Polynesia and the Arctic, respectively. We report 11 human IFNAR1 alleles, the products of which impair but do not abolish responses to IFN-α and -ω without affecting responses to IFN-β. Ten of these alleles are rare in all populations studied, but the remaining allele (P335del) is common in Southern China (minor allele frequency ≈2%). Cells heterozygous for these variants display a dominant phenotype in vitro with impaired responses to IFN-α and -ω, but not -β, and viral susceptibility. Negative dominance, rather than haploinsufficiency, accounts for this dominance. Patients heterozygous for these variants are prone to viral diseases, attesting to both the dominance of these variants clinically and the importance of IFN-α and -ω for protective immunity against some viruses.
Open Access
A novel gene list identifies tumors with a stromal-mesenchymal phenotype and worse prognosis in gastric cancer
(MDPI, 2023-06-02) Demirkol Canlı, Seçil; Üner, M.; Küçükkaraduman, Barış; Karaoğlu, D. A.; Işık, A.; Turhan, N.; Akyol, A.; Gömceli, I.; Güre, A. O. A
Background: Molecular biomarkers that predict disease progression can help identify tumor subtypes and shape treatment plans. In this study, we aimed to identify robust biomarkers of prognosis in gastric cancer based on transcriptomic data obtained from primary gastric tumors. Methods: Microarray, RNA sequencing, and single-cell RNA sequencing-based gene expression data from gastric tumors were obtained from public databases. Freshly frozen gastric tumors (n = 42) and matched FFPE (formalin-fixed, paraffin-embedded) (n = 40) tissues from a Turkish gastric cancer cohort were used for quantitative real-time PCR and immunohistochemistry-based assessments of gene expression, respectively. Results: A novel list of 20 prognostic genes was identified and used for the classification of gastric tumors into two major tumor subgroups with differential stromal gene expression (“Stromal-UP” (SU) and “Stromal-DOWN” (SD)). The SU group had a more mesenchymal profile with an enrichment of extracellular matrix-related gene sets and a poor prognosis compared to the SD group. Expression of the genes within the signature correlated with the expression of mesenchymal markers ex vivo. A higher stromal content in FFPE tissues was associated with shorter overall survival. Conclusions: A stroma-rich, mesenchymal subgroup among gastric tumors identifies an unfavorable clinical outcome in all cohorts tested.
Open Access
A novel heterozygous NFKB2 variant in a multiplex family with common variable immune deficiency and autoantibodies against type I IFNs
(Springer New York LLC, 2024-11-23) Baran, Alperen; Lülecioğlu, Aysima Atılgan; Gao, Liwei; Yazıcı, Yılmaz Yücehan; Demirel, Fevzi; Metin, Ayşe; Casanova, Jean-Laurent; Pue, Anne; Le Voyer, Tom; Beyaz, Şengül; Belkaya, Serkan
We studied a family with three male individuals across two generations affected by common variable immune deficiency (CVID). We identified a novel missense heterozygous variant (c.2602T>A:p.Y868N) of NFKB2 in all patients and not in healthy relatives. Functional studies of the mutant allele in an overexpression system and of the patients’ cells confirmed the deleteriousness of the NFKB2 variant and genotype, respectively, on the activation of the non-canonical NF-κB signaling pathway. Impaired processing of p100 into p52 underlies p100 accumulation, which results in gain-of-function (GOF) of IκBδ inhibitory activity and loss-of-function (LOF) of p52 transcriptional activity. The three patients’ plasma contained autoantibodies that neutralized IFN-α2 and/or IFN-ω, accounting for the severe or recurrent viral diseases of the patients, including influenza pneumonia in one sibling, and severe COVID-19 and recurrent herpes labialis in another. Our results confirm that NFKB2 alleles that are IκBδ GOF and p52 LOF can underlie CVID and drive the production of autoantibodies neutralizing type I IFNs, thereby predisposing to severe viral diseases.
Embargo
A novel injectable nanotherapeutic platform increasing the bioavailability and anti-tumor efficacy of Arachidonylcyclopropylamide on an ectopic non-small cell lung cancer xenograft model: A randomized controlled trial
(Elsevier BV, 2024-12-31) Boyacıoğlu, Özge; Varan, Cem; Bilensoy, Erem; Aykut, Zaliha Gamze; Recber, Tuba; Nemutlu, Emirhan; Kılıç, Nedret; Korkusuz, Petek
Rapid progressing non-small cell lung adenocarcinoma (NSCLC) decreases treatment success. Cannabinoids emerge as drug candidates for NSCLC due to their anti-tumoral capabilities. We previously reported the controlled release of Arachidonylcyclopropylamide (ACPA) selectively targeting cannabinoid 1 (CB1) receptor in NSCLC cells in vitro. Hydrophobic polymers like polycaprolactone (PCL) offer prolonged circulation time and slower drug clearance which is suitable for hydrophobic molecules like ACPA. Thus, the extended circulation time with enhanced bioavailability and half-life of nanoparticular ACPA is crucial for its therapeutic performance in the tumor area. We assumed that a novel high technology-controlled release system increasing the bioavailability of ACPA compared to free ACPA could be transferred to the clinic when validated in vivo. Plasma profile of ACPA and ACPA-loaded PCL-based nanomedicine by LC-MS/MS and complete blood count (CBC) was assessed in wild-type Balb/c mice. Tumor growth in nanomedicine-applied NSCLC-induced athymic nude mice was assessed using bioluminescence imaging (BLI) and caliper measurements, histomorphometry,immunohistochemistry, TUNEL assay, and Western blot on days 7-21. Injectable NanoACPA increased its systemic exposure to tissues 5.5 times and maximum plasma concentration 6 times higher than free ACPA by substantially improving bioavailability. The potent effect of NanoACPA lasted for at least two days on ectopic NSCLC model through Akt/PI3K, Ras/MEK/Erk, and JNK pathways that diminished Ki-67 proliferative and promoted TUNEL apoptotic cell scores on days 7-21. The output reveals that NanoACPA platform could be a chemotherapeutic for NSCLC in the clinic following scale-up GLP/GMP-based phase trials, owing to therapeutic efficacy at a safe low dose window.
Open Access
A small non-interface surface epitope in human IL18 mediates the dynamics and self-assembly of IL18-IL18BP heterodimers
(Elsevier, 2023-07-01) Yazıcı, Yılmaz Yücehan; Belkaya, Serkan; Timuçin, E.
Interleukin 18 (IL18) is a pro-inflammatory cytokine that modulates innate and adaptive immune responses. IL18 activity is tightly controlled by the constitutively secreted IL18 binding protein (IL18BP). PDB structures of human IL18 showed that a short stretch of amino acids between 68 and 81 adopted a disordered conformation in all IL18-IL18BP complexes while adopting a 310 helical structure in other IL18 structures, including the receptor complexes. The C74 of human IL18, which was reported to form a novel intermolecular disulfide bond in the human tetrameric assembly, is also located in this short epitope. These observations reflected the importance of this short surface epitope for the structure and dynamics of the IL18-IL18BP heterodimers. We have analyzed all known IL18-IL18BP complexes in the PDB by all-atom MD simulations. The analysis also included two computed complex models adopting a helical structure for the surface epitope. Heterodimer simulations showed a stabilizing impact of the small surface region at the helical form by reducing flexibility of the complex backbone. Analysis of the symmetry-related human IL18-IL18BP tetramer showed that the unfolding of this small surface region also contributed to the IL18-IL18BP stability through a completely exposed C74 sidechain to form an intermolecular disulfide bond in the self-assembled human IL18-IL18BP dimer. Our findings showed how the conformation of the short IL18 epitope between amino acids 68 and 81 would affect IL18 activity by mediating the intermolecular interactions of IL18.
Open Access
The ability to generate senescent progeny as a mechanism underlying breast cancer cell heterogeneity
(Public Library of Science, 2010) Mumcuoğlu, Mine; Bağışlar, Sevgi; Yüzügüllü, Haluk; Alotaibi, Hani; Şentürk, Şerif; Telkoparan, Pelin; Gür-Dedeoğlu, Bala; Cingöz, Burcu; Bozkurt, B.; Tazebay, Uygar H.; Yuluğ, Işık G.; Akçalı, Kamil Can; Öztürk, Mehmet
Background Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. Methodology/Principal Findings A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21Cip1 correlated with senescence in these cell lines. p21Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. Conclusions/Significance Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential.
Open Access
Abnormal subcortical activity in congenital mirror movement disorder with RAD51 mutation
(Turkish Society of Radiology, 2018) Demirayak, Pınar; Onat, Onur Emre; Gevrekci, A. Ö.; Gülsüner, S.; Uysal, H.; Bilgen, R.; Doerschner, Katja; Özçelik, Tayfun; Boyacı, Hüseyin
PURPOSE Congenital mirror movement disorder (CMMD) is characterized by unintended, nonsuppressible, homologous mirroring activity contralateral to the movement on the intended side of the body. In healthy controls, unilateral movements are accompanied with predominantly contralateral cortical activity, whereas in CMMD, in line with the abnormal behavior, bilateral cortical activity is observed for unilateral motor tasks. However, task-related activities in subcortical structures, which are known to play critical roles in motor actions, have not been investigated in CMMD previously. METHODS We investigated the functional activation patterns of the motor components in CMMD patients. By using linkage analysis and exome sequencing, common mutations were revealed in seven affected individuals from the same family. Next, using functional magnetic resonance imaging (fMRI) we investigated cortical and subcortical activity during manual motor actions in two right-handed affected brothers and sex, age, education, and socioeconomically matched healthy individuals. RESULTS Genetic analyses revealed heterozygous RAD51 c.401C>T mutation which cosegregated with the phenotype in two affected members of the family. Consistent with previous literature, our fMRI results on these two affected individuals showed that mirror movements were closely related to abnormal cortical activity in M1 and SMA during unimanual movements. Furthermore, we have found previously unknown abnormal task-related activity in subcortical structures. Specifically, we have found increased and bilateral activity during unimanual movements in thalamus, striatum, and globus pallidus in CMMD patients. CONCLUSION These findings reveal further neural correlates of CMMD, and may guide our understanding of the critical roles of subcortical structures for unimanual movements in healthy individuals.
Open Access
Accumulation of nonylphenol in gold fish and suckermouth catfish in the semi static aquarium system
(Indian Veterinary Association, 2004) Uguz, C.; Dagcan, A. T.; Iscan, M.; Togan, I.
[No abstract available]
Open Access
Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells
(Nature Publishing Group, 2001) Sayan, A. E.; Sayan, B. S.; Findikli, N.; Ozturk, M.
p53 and p73 proteins activate similar target genes and induce apoptosis and cell cycle arrest. However, p53, but not p73 is considered a tumour-suppressor gene. Unlike p53, p73 deficiency in mice does not lead to a cancer-prone phenotype, and p73 gene is not mutated in human cancers, including hepatocellular carcinoma. Here we report that normal liver cells express only ΔN-p73 transcript forms giving rise to the synthesis of N-terminally truncated, transcriptionally inactive and dominant negative p73 proteins. In contrast, most hepatocellular carcinoma cells express TA-p73 transcript forms encoding full-length and transcriptionally active p73 proteins, in addition to ΔN-p73. We also show that together with the acquired expression of TA-p73, the 'retinoblastoma pathway' is inactivated, and E2F1-target genes including cyclin E and p14ARF are activated in hepatocellular carcinoma. However, there was no full correlation between 'retinoblastoma pathway' inactivation and TA-p73 expression. Most TA-p73-expressing hepatocellular carcinoma cells have also lost p53 function either by lack of expression or missense mutations. The p73 gene, encoding only ΔN-p73 protein, may function as a tumour promoter rather than a tumour suppressor in liver tissue. This may be one reason why p73 is not a mutation target in hepatocellular carcinoma.
Open Access
Acquired tolerance of hepatocellular carcinoma cells to selenium deficiency: a selective survival mechanism?
(American Association for Cancer Research, 2003) Irmak, M. B.; Ince, G.; Ozturk, M.; Cetin Atalay, R.
Selenium is essential to human health, and its deficiency is associated with different diseases including liver necrosis. Selenium is protective against viral hepatitis and hepatocellular carcinoma (HCC). The underlying molecular mechanisms of selenium effects are not well known. In this study, in vitro response of HCC-derived cell lines to selenium deficiency is examined alone or in conjunction with Vitamin E and copper/zinc. Here, we show that itt vitro selenium deficiency in a subset of HCC-derived cell lines causes oxidative stress and cytochrome c release with subsequent cell death by apoptosis. The oxidative stress and consequent cell death induced by selenium deficiency on these cells are reverted by the antioxidant effect of Vitamin E. However, most HCC cell lines (10 of 13) tolerate selenium deficiency. Consequently, they escape apoptosis. Moreover, nine of these tolerant cell lines have integrated hepatitis B Virus (HBV) DNA in their genomes, and some display p53-249 mutation, indicating past exposure to HBV or aflatoxins, established factors for oxidative stress and cancer risk in liver. An HBV-transfected clone (2.2.15) of the sensitive HepG2 cell line has gained tolerance to selenium deficiency. Our findings indicate that selenium deficiency induces apoptosis in some "hepatocyte-like" cells. However, most HCC cells, particularly HBV-related ones, tolerate selenium deficiency and escape its deadly consequences. Thus, as demonstrated by the gain of survival capacity of apoptosis-sensitive cell lines with Vitamin E, such malignant cells have acquired a selective survival advantage that is prominent under selenium-deficient and oxidative-stress conditions.
Open Access
Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment
(American Association for Cancer Research Inc., 2014) Cekic, C.; Linden J.
Adenosine A2A receptor (A2AR) blockade enhances innate and adaptive immune responses. However, mouse genetic studies have shown that A2AR deletion does not inhibit the growth of all tumor types. In the current study, we showed that growth rates for ectopic melanoma and bladder tumors are increased in Adora2a-/- mice within 2 weeks of tumor inoculation. A2AR deletion in the host reduced numbers of CD8+ T cells and effector-memory differentiation of all T cells. To examine intrinsic functions in T cells, we generated mice harboring a T-cell-specific deletion of A2AR. In this host strain, tumor-bearing mice displayed increased growth of ectopic melanomas, decreased numbers of tumor-associated T cells, reduced effector-memory differentiation, and reduced antiapoptotic IL7Rα (CD127) expression on antigen-experienced cells. Intratumoral pharmacologic blockade similarly reduced CD8+ T-cell density within tumors in wild-type hosts. We found that A2AR-proficient CD8+ T cells specific for melanoma cells displayed a relative survival advantage in tumors. Thus, abrogating A2AR signaling appeared to reduce IL7R expression, survival, and differentiation of T cells in the tumor microenvironment. One implication of these results is that the antitumor effects of A2AR blockade that can be mediated by activation of cytotoxic T cells may be overcome in some tumor microenvironments as a result of impaired T-cell maintenance and effector-memory differentiation. Thus, our findings imply that the efficacious application of A2AR inhibitors for cancer immunotherapy may require careful dose optimization to prevent activation-induced T-cell death in tumors. ©2014 AACR.
Open Access
Adenosine receptor signaling targets both PKA and epac pathways to polarize dendritic cells to a suppressive phenotype
(American Association of Immunologists, 2019) Kayhan, Merve; Koyaş, Altay; Akdemir, İmran; Savaş, Ali Can; Çekiç, Çağlar
Extracellular adenosine accumulates in tumors and causes suppression of immune cells. Suppressive adenosine signaling is achieved through adenosine A2A and A2B receptors, which are Gs coupled, and their activation elevates cAMP levels. Gs-coupled GPCR signaling causes cAMP accumulation, which plays an anti-inflammatory role in immune cells. Protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) are two intracellular receptors of cAMP. In this study we showed that adenosine receptor signaling polarizes activated murine dendritic cells (DCs) into a tumor-promoting suppressive phenotype. Adenosine receptor signaling activates cAMP pathway and upregulates the negative regulators of NF-κB but does not influence phosphorylation of immediate inflammatory signaling molecules downstream of TLR signaling. Pharmacologic activation of both PKA and Epac pathways by specific cAMP analogues phenocopied the effects of adenosine signaling on murine DCs, such as suppression of proinflammatory cytokines, elevation of anti-inflammatory IL-10, increased expression of regulators of NF-κB pathway, and finally suppression of T cell activation. Inhibition of effector cytokine, IL-12p40 production, and increased immunosuppressive IL-10 production by adenosine signaling is significantly reversed only when both PKA and Epac pathways were inhibited together. Adenosine signaling increased IL-10 secretion while decreasing IL-12p40 secretion in human monocyte-derived DCs. Stimulation of both PKA and Epac pathways also caused combinatorial effects in regulation of IL-12p40 secretion in human monocyte-derived DCs. Interestingly, PKA signaling alone caused similar increase in IL-10 secretion to that of adenosine signaling in human monocyte-derived DCs. Our data suggest adenosine/cAMP signaling targets both PKA/Epac pathways to fully differentiate DCs into a suppressive phenotype.
Open Access
Adjuvant autologous melanoma vaccine for macroscopic stage III disease: survival, biomarkers, and improved response to CTLA-4 blockade
(Hindawi Limited, 2016) Lotem, M.; Merims, S.; Frank, S.; Hamburger, T.; Nissan, A.; Kadouri, L.; Cohen, J.; Straussman, R.; Eisenberg, G.; Frankenburg, S.; Carmon, E.; Alaiyan, B.; Shneibaum, S.; Ayyildiz, Z. O.; Isbilen, M.; Senses, K. M.; Ron, I.; Steinberg, H.; Smith, Y.; Shiloni, E.; Gure, A. O.; Peretz, T.
Background. There is not yet an agreed adjuvant treatment for melanoma patients with American Joint Committee on Cancer stages III B and C. We report administration of an autologous melanoma vaccine to prevent disease recurrence. Patients and Methods. 126 patients received eight doses of irradiated autologous melanoma cells conjugated to dinitrophenyl and mixed with BCG. Delayed type hypersensitivity (DTH) response to unmodified melanoma cells was determined on the vaccine days 5 and 8. Gene expression analysis was performed on 35 tumors from patients with good or poor survival. Results. Median overall survival was 88 months with a 5-year survival of 54%. Patients attaining a strong DTH response had a significantly better (p = 0.0001) 5-year overall survival of 75% compared with 44% in patients without a strong response. Gene expression array linked a 50-gene signature to prognosis, including a cluster of four cancer testis antigens: CTAG2 (NY-ESO-2), MAGEA1, SSX1, and SSX4. Thirty-five patients, who received an autologous vaccine, followed by ipilimumab for progressive disease, had a significantly improved 3-year survival of 46% compared with 19% in nonvaccinated patients treated with ipilimumab alone (p = 0.007). Conclusion. Improved survival in patients attaining a strong DTH and increased response rate with subsequent ipilimumab suggests that the autologous vaccine confers protective immunity.
Open Access
Administration of bone marrow derived mesenchymal stem cells modulate tlr expression during liver regeneration
(Trakya Üniversitesi, 2019) Koçak, H.; Tokçaer Keskin, Z.; İnsal, B.; Gürsel, İhsan; Akçalı, K. C.
Liver cell transplantation is a powerful alternative to orthotopic cell transplantation in the treatment of liver failures. Recently, considerable effort is being channeled to understand the nature and kinetics of directing stem cells to effectively accumulate at the regenerating liver site. Mesenchymal stem cells are one of the promising cell sources modulating liver regeneration process. The present was designed to study how mesenchymal stem cells might modulate liver immune behaviors by changing Toll-like receptor (TLR) expression and increase regenerative potential during liver regeneration in rats. Normal and partially hepatectomized rats were treated with mesenchymal stem cells isolated and expanded from rat bone marrows. Accumulation of mesenchymal stem cells was confirmed by Real Time-Polymerase Chain Reaction (RT-PCR), Fluorescence-Activated Cell Sorting (FACS), and Immunofluorescence Staining (IFS). Student's t-test analysis was used to evaluate the significance of differences between sham and partially hepatectomized rat groups. Our results showed that mesenchymal stem cells expressed several TLRs, and their accumulation during regeneration was depended on the timing of injury. Mesenchymal stem cells isolated from bone marrow of normal rats were observed at the injured liver 3 days after the injection. There were no labeled mesenchymal stem cells in the liver sections of the uninjured animals. Mesenchymal stem cell administration significantly altered the expression of TLR2, 3 and 9 while retaining their migration potential to regenerating liver. Our findings implicated that mesenchymal stem cell administration during liver regeneration modulate the immune response through changing the expression of the TLRs in the remaining liver parts into which the cells are recruited or infused. This alteration may contribute to the regeneration process following partial hepatectomy.
Open Access
Advanced preclinical models for evaluation of drug-induced liver injury – consensus statement by the European Drug-Induced Liver Injury Network [PRO-EURO-DILI-NET]
(Elsevier, 2021-06-24) Fernandez-Checa, J. C.; Bagnaninchi, P.; Ye, H.; Sancho-Bru, P.; Falcon-Perez, J. M.; Royo, F.; Garcia-Ruiz, C.; Konu, Özlen; Miranda, J.; Lunov, O.; Dejneka, A.; Elfick, A.; McDonald, A.; Sullivan, G. J.; Aithal, G. P.; Lucena, M. I.; Andrade, R. J.; Fromenty, B.; Kranendonk, M.; Cubero, F. J.; Nelson, L. J.
Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue for which there is a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause of ALF because of its extensive worldwide use as an analgesic. Despite intensive efforts utilising current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful discoveries by detailing the requirements for more realistic human-based systems to assess hepatotoxicity and guide future drug safety testing. We present novel insights and discuss major players in APAP pathophysiology, and describe emerging in vitro and in vivo pre-clinical models, as well as advanced imaging and in silico technologies, which may improve prediction of clinical outcomes of DILI.