Modulation Of Ifn-γ Receptor1Expression By AP-2α Influences Ifn-γ Sensitivity Of Cancer Cells2.All-trans-retinoic Acid Modulates ICAM-1N-glycan Composition By Influencing GnT-III Levels And Inhibits Cell Adhesion And Trans-endothelial Migration

Part1. Modulation of IFN-y Receptor1Expression by AP-2α Influences IFN-y Sensitivity of Cancer CellsIFN-y belongs to type II interferon and plays crucial roles in regulating both innate and adaptive immunity. IFN-y is an important mediator of the immune response against some virus, intracellular bacteria and microbial infection and also involved in anti-tumor immunity. Binding of IFN-y to IFNGR1initiates autophosphorylation and trans-phosphorlation and activation of the Janus-activated kinsae (JAKs). Phosphorylation the intracellular domain of IFNGR1leads to the recruitment of Statl proteins. The phosphorylated Statl proteins, forming homodimers, dissociate from the receptor complex, translocate to the nucleus and regulate the transcription of IFN-y-inducible genes. Studies using mice with the genetic deficiencies of the components involved in the IFN-y signaling have demonstrated that IFNGR1, JAK1, JAK2and Statl are unequvivocally required for IFN-y-induced biological responses. Investigations on human tumor cells revealed that the resistance of a variety of tumor cell lines to IFN-y is owing to the defect in IFNGR1expression, underexpression of JAK1and Stat1, and production of abnormal JAK2generation. Clinical studies have underpinned the importance of IFNGR1expression in esophageal and ovarian cancers. The reduced IFNGR1expression was tightly associated with clinicopathologic feature of esophageal cancer and prognosis of ovarian cancer. However, the mechanisms of IFNGR1down-regulation are poorly undersrood.The expression of IFNGR1is usually in surplus and can be further up-regulated at the transcripton level in certain setting. Proinflammatort cytokines, such as TNF-a, IL-1, IL-6and IFN-y, stimulate the expression of IFNGR1in mRNA and protein. The transcriptional activation of IFNGR1was postulated to depend on the transcription factor nuclear factor-KB (NF-κB). Specificity protein1(SP-1) was suggested to regulate TPA-induced transcription of IFNGR1gene through binding to TPA response element (TRE) within IFNGR1promoter. How the expression of IFNGR1is controlled either positively or negatively is basically unknown.In the present study, we demonstrated that IFNGR1expression was reduced or lost in breast cancer. Heterogeneous IFNGR1immunoreactivity appeared to be associated with the morphological heterogeneity of breast cancer and loss of IFNGR1expression was predominantly observed in poorly differentiated areas. We identified the functional AP-2and SP-1sites within IFNGR1promoter. Ectopic expression ofAP-2α drastically repressed the expression of IFNGR1and hindered IFN-γ signaling,whereas AP-2α gene silencing could elevate IFNGR1level. Overexpression of SP-1effectively antagonized the repressive effects of AP-2α. Simultaneous recruitment ofboth transcription factors to the AP-2and SP-1motifs in IFNGR1promoter wasdemonstrated, implying that AP-2α and SP-1may synergistically modulate IFNGR1transcription. Moreover, the AP-2α overexpression in AP-2deficient SW480cellsremarkably inhibited Stat1phosphorylation and antiproliferative effects of IFN-γ,whereas knock-down of the AP-2α expression dramatically enhanced the sensitivitiesof HeLa cells highly expressing AP-2to IFN-γ, indicating that dysregulation of theAP-2α expression is associated with impaired IFN-γ actions in cancer cells. Furtherexploration on the regulatory network composed of AP-2and its interacting proteinsmay help to fully elucidate the physiological and pathological significance of AP-2and provide the novel target for tumor diagnosis, prognosis and treatment. Themolecular mechanism of IFNGR1expresion regulation under pathological conditionremains to be further investigated. Intercellular adhesion molecule-1(ICAM-1) belongs to the immunoglobulinsuperfamily of adhesion molecules. The well-known function of ICAM-1is defined topromote intercellular contact/adhesion and trans-endothelial migration of leukocytes.ICAM-1also acts as a co-stimulatory molecule and signal transducer to triggerintracellular signals, ultimately leading to activation of lymphocytes, cytokinesecretion and proinflammatory cascades. ICAM-1is a heavily N-glycosylatedtransmembrane protein with a molecular weight of80–114kDa, depending on itslevel of glycosylation. The various functions of ICAM-1appear to be differentiallyregulated by N-linked glycosylation. Alterations in oligosaccharide structure (glycan)of ICAM-1may significantly influence cell proliferation, differentiation, adhesion,migration and tumor invasion and metastases.N-glycan processing or remodeling reactions are catalyzed by the actions ofseveral glycosyltransferases, which generate highly diverse N-linked glycans(N-glycans). Among the glycosyltransferases, two of major glycosyltransferasesparticipating in the processing of N-glycans during the synthesis of glycoproteins areGolgi N-acetylglucosaminyltransferase III (GnT-III) and V (GnT-V). GnT-IIIcatalyzes the formation of a bisecting β1,4-GlcNAc structure, whereas GnT-Vpromotes the formation of β1,6-branching GlcNAc structure. GnT-III is considered asa key glycosyltransferase in N-glycan biosynthetic pathway since the introduction ofthe bisecting GlcNAc residue suppresses further processing and elongation ofN-glycans catalyzed by GnT-V, which is unable to utilize the bisected oligosaccharideas a substrate.Retinoids, a family of retinol metabolites and synthetic derivatives, exert diversefunctions, including regulation of growth and differentiation in different cell types,inhibition of malignant cell proliferation and modulation of the expression of vascularcell adhesion molecule-1(VCAM-1) and ICAM-1. The application of retinoids tocancer chemoprevention has had a long history. Retinoic acid (RA) and its analogueshave also been used in topical and systemic treatment of inflammatory skin diseases,since the regulated expression of cell adhesion molecules is critical for leukocyte recognition and transmigration through the endothelium in inflammatory responses.In the present study we show that all-trans-retinoic acid (ATRA) modulates theN-glycan composition of intercellular adhesion molecule-1(ICAM-1) bymanipulating the expression of two N-acetylglucosaminyltransferases GnT-III andGnT-V via ERK signaling pathway. Exposure of various cells to ATRA caused aremarkable gel mobility down-shift of ICAM-1. By treatment with PNGase F weconfirm that the reduction of the ICAM-1molecular mass is attributed to thedecreased complexity of N-glycans. We noticed that the expression of the mRNAencoding GnT-III that stops branching was significantly enhanced in response toATRA induction. In contrast, the level of the mRNA encoding GnT-V that promotesbranching was reduced. The expression pattern of these two enzymes reflects theN-glycan composition of ICAM-1. Silencing of GnT-III prevented the molecularweight shift of ICAM-1.Changes in the expression level of glycosyltransferases directly influence theoligosaccharide structures and conformations of cell surface glycoproteins, andconsequently cellular phenotype transition and biological behaviors. The extracellulardomain of ICAM-1is essential for intercellular adhesion and transendothelialmigration of leukocytes. The changes of the N-glycan profile of ICAM-1on the cellmembrane are expected to interfere with ICAM-1-mediated cellular functions, such ascell adhesion and migration. When SW480cells were treated with ATRA, celladhesion to the HUVEC monolayer was greatly inhibited. Knock-down of GnT-IIIexpression with the specific siRNA effectively restored cell adhesion function.Treatment with ATRA also dramatically reduced the trans-endothelial migration ofU937cells. These data indicate that the changes of ICAM-1N-glycan structures byATRA-induced GnT-III activities interfered with the efficacy of cell adhesion and cellmigration simultaneously. It is interesting to illustrate how the expression andN-glycosylation of ICAM-1are regulated by ATRA in different types of cells. Also,understanding the underlying mechanisms will shed a new light on the functionalversatility of ICAM-1modulated by glycosyltransferases in tumor progression andexploring new type antitumor drug.