RESUMO
Epigenetic regulation of gene expression is crucial to the determination of cell fate in development and differentiation, and the Polycomb (PcG) and Trithorax (TrxG) groups of proteins, acting antagonistically as complexes, play a major role in this regulation. Although originally identified in Drosophila, these complexes are conserved in evolution and the components are well defined in mammals. Each complex contains a protein with methylase activity (KMT), which can add methyl groups to a specific lysine in histone tails, histone 3 lysine 27 (H3K27), by PcG complexes, and H3K4 and H3K36 by TrxG complexes, creating transcriptionally repressive or active marks, respectively. Histone demethylases (KDMs), identified later, added a new dimension to histone methylation, and mutations or changes in levels of expression are seen in both methylases and demethylases and in components of the PcG and TrX complexes across a range of cancers. In this review, we focus on both methylases and demethylases governing the methylation state of the suppressive and active marks and consider their action and interaction in normal tissues and in cancer. A picture is emerging which indicates that the changes which occur in cancer during methylation of histone lysines can lead to repression of genes, including tumour suppressor genes, or to the activation of oncogenes. Methylases or demethylases, which are themselves tumour suppressors, are highly mutated. Novel targets for cancer therapy have been identified and a methylase (KMT6A/EZH2), which produces the repressive H3K27me3 mark, and a demethylase (KDM1A/LSD1), which demethylates the active H3K4me2 mark, are now under clinical evaluation.
Assuntos
Histona Desmetilases , Histona Metiltransferases , Histonas , Neoplasias , Animais , Epigênese Genética , Histona Desmetilases/metabolismo , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Neoplasias/genética , Proteínas do Grupo Polycomb/metabolismoRESUMO
Posttranslational modification of histones serve a crucial role in the control of gene transcription. Trimethylation of lysine 4 on histone 3 is associated with transcription activation. There are currently six known methylases and six known demethylases that can control the methylation status of this site. Lysine demethylase 5B (KDM5B) is one such demethylase, which can repress gene expression. In particular KDM5B has been found to be overexpressed in a number of cancer types, and smallmolecular weight inhibitors of its demethylase activity have been identified. Previous characterisation of Kdm5b knockout mice has revealed that this genotype leads to either embryonic or neonatal lethality. However, the ΔAT rich interaction domain (ΔARID)KDM5B strain of mice, which have the ARID domain and five amino acids within the Jumonji (Jmj)N domain spliced out from KDM5B, remain viable and fertile. In the present study, ΔARIDKDM5B was found to have no demethylase activity as determined by in vitro demethylase assays and by immunofluorescence in transfected Cos1 cells. Furthermore, molecular dynamic simulations revealed conformational changes within the ΔARIDKDM5B structure compared with that in WTKDM5B, particularly in the JmjC domain, which is responsible for the catalytic activity of WTKDM5B. This supports the experimental data that shows the loss of demethylase activity. Since Kdm5b knockout mice show varying degrees of lethality, these data suggest that KDM5B serves a crucial function in development in a manner that is independent of its demethylase activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Domínios Proteicos/genética , Animais , Desmetilação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Ensaios Enzimáticos , Feminino , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/ultraestrutura , Masculino , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers. Here, we show that upon EGF binding, breast cancer cells carrying different O-glycans respond by transcribing different gene expression signatures. MMP10, the principal gene upregulated when cells carrying sialylated core 1 glycans were stimulated with EGF, is also upregulated in ER-positive breast carcinoma reported to express high levels of ST3Gal1 and hence mainly core 1 sialylated O-glycans. In contrast, isogenic cells engineered to carry core 2 glycans upregulate CX3CL1 and FGFBP1 and these genes are upregulated in ER-negative breast carcinomas, also known to express longer core 2 O-glycans. Changes in O-glycosylation did not significantly alter signal transduction downstream of EGFR in core 1 or core 2 O-glycan expressing cells. However, striking changes were observed in the formation of an EGFR/galectin-3/MUC1/ß-catenin complex at the cell surface that is present in cells carrying short core 1-based O-glycans but absent in core 2 carrying cells.
Assuntos
Neoplasias da Mama/metabolismo , Mucina-1/metabolismo , Neoplasias da Mama/patologia , Receptores ErbB/metabolismo , Feminino , Glicosilação , Humanos , Receptores de Estrogênio/metabolismoRESUMO
The tumour microenvironment plays a crucial role in the growth and progression of cancer, and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies have demonstrated that TAMs display transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype that to the best of our knowledge has not previously been described. These TAMs can recruit and prolong the lifespan of neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients.
Assuntos
Macrófagos/fisiologia , Monócitos/fisiologia , Mucina-1/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mucina-1/genéticaRESUMO
Changes in mucin-type O-linked glycosylation are seen in over 90% of breast cancers where increased sialylation is often observed and a change from branched glycans to linear glycans is often seen. There are many mechanisms involved including increased/altered expression of glycosyltransferases and relocalisation to the endoplasmic reticulum of the enzymes responsible for the addition of the first sugar, N-acetyl-d-galactosamine. It is now becoming clear that these changes can contribute to tumour growth and progression by modulating the micro-environment through glycan-sensing lectins expressed on immune cells, by modulating interactions with tumour surface receptors and by binding to selectins. The understanding of how changes in mucin-type O-linked glycosylation influence tumour growth and progression reveals new potential targets for therapeutic intervention in the treatment of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Mucina-1/metabolismo , Acetilgalactosamina/metabolismo , Transporte Biológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Regulação Enzimológica da Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Complexo de Golgi/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Metástase Neoplásica , Microambiente TumoralRESUMO
Currently, there is renewed interest in attempting to recruit the host immune system to eliminate cancers, and within this renewed activity, MUC1 continues to arouse interest. MUC1 has been considered a possible therapeutic target for the past 30 years as it is up-regulated, aberrantly glycosylated and its polarization is lost in many adenocarcinomas. Moreover, MUC1 is expressed by some haematopoietic cancers, including acute myeloid leukaemia and myeloma. Although multiple clinical trials have been initiated and immune responses have been documented, effective clinical benefit worthy of approval for general application has not as yet been achieved. However, this does not appear to have quelled the interest in MUC1 as a therapeutic target, as shown by the increase in the number of MUC1-based clinical trials initiated in 2017 ( Figure 1). As with all translational studies, incorporating new relevant research findings into therapeutic strategy is difficult. Decisions are made to commit to a specific strategy based on the information and data available when the trial is initiated. However, the time required for preclinical studies and early trials can render the founding concept not always appropriate for proceeding to a larger definitive trial. Here, we summarize the attempts made, to date, to bring MUC1 into the world of cancer immunotherapy and discuss how research findings regarding MUC1 structure and function together with expanded knowledge of its interactions with the tumour environment and immune effector cells could lead to improved therapeutic approaches. ppbiost;46/3/659/BST20170400CF1F1BST-2017-0400CF1Figure 1.Number of MUC1-targeted trials initiated each year.
Assuntos
Imunoterapia , Mucina-1/imunologia , Neoplasias/terapia , Antimetabólitos Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Terapia Combinada , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Glicosilação , Humanos , Mucina-1/química , Mucina-1/fisiologia , Neoplasias/tratamento farmacológico , Microambiente Tumoral , GencitabinaAssuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Histona Desmetilases/antagonistas & inibidores , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Neoplasias/enzimologia , Neoplasias/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Proteína 2 de Ligação ao Retinoblastoma/antagonistas & inibidoresRESUMO
We read with interest the manuscript by June and colleagues published recently in Immunity in which they describe targeting of aberrantly glycosylated tumor-associated cell membrane mucin MUC1 using chimeric antigen receptor-engineered human T cells (Posey et al., 2016). In that study, the authors used a second generation 4-1BB costimulatory-molecule-based chimeric antigen receptor (CAR) (Imai et al., 2004) in which targeting was achieved using a single-chain variable fragment (scFv) derived from the 5E5 antibody. This CAR selectively binds MUC1 that carries the Tn or sialyl (S)Tn glycan. Both of these truncated glycans are aberrantly expressed on the MUC1 glycoprotein in a spectrum of malignancies and consequently represent attractive targets for immunotherapeutic exploitation.
Assuntos
Mucina-1/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias , Glicosilação , Humanos , Neoplasias/imunologiaRESUMO
Siglec-9 is a sialic-acid-binding lectin expressed predominantly on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers and results in increased sialylation. Thus, when the mucin MUC1 is expressed on cancer cells, it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). Here we found that this cancer-specific MUC1 glycoform, through engagement of Siglec-9, 'educated' myeloid cells to release factors associated with determination of the tumor microenvironment and disease progression. Moreover, MUC1-ST induced macrophages to display a tumor-associated macrophage (TAM)-like phenotype, with increased expression of the checkpoint ligand PD-L1. Binding of MUC1-ST to Siglec-9 did not activate the phosphatases SHP-1 or SHP-2 but, unexpectedly, induced calcium flux that led to activation of the kinases MEK-ERK. This work defines a critical role for aberrantly glycosylated MUC1 and identifies an activating pathway that follows engagement of Siglec-9.
Assuntos
Antígenos CD/metabolismo , Mucina-1/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Microambiente Tumoral/imunologia , Antígenos CD/genética , Biomarcadores , Diferenciação Celular , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Glicosilação , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Mieloides/citologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/patologia , Fenótipo , Ligação Proteica , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genéticaRESUMO
Emerging evidence suggests pathological and immunoregulatory functions for IgG4 antibodies and IgG4+ B cells in inflammatory diseases and malignancies. We previously reported that IgG4 antibodies restrict activation of immune effector cell functions and impair humoral responses in melanoma. Here, we investigate IgG4 as a predictor of risk for disease progression in a study of human sera (n = 271: 167 melanoma patients; 104 healthy volunteers) and peripheral blood B cells (n = 71: 47 melanoma patients; 24 healthy volunteers). IgG4 (IgG4/IgGtotal) serum levels were elevated in melanoma. High relative IgG4 levels negatively correlated with progression-free survival (PFS) and overall survival. In early stage (I-II) disease, serum IgG4 was independently negatively prognostic for progression-free survival, as was elevation of IgG4+ circulating B cells (CD45+CD22+CD19+CD3-CD14-). In human tissues (n = 256; 108 cutaneous melanomas; 56 involved lymph nodes; 60 distant metastases; 32 normal skin samples) IgG4+ cell infiltrates were found in 42.6% of melanomas, 21.4% of involved lymph nodes and 30% of metastases, suggesting inflammatory conditions that favor IgG4 at the peripheral and local levels. Consistent with emerging evidence for an immunosuppressive role for IgG4, these findings indicate association of elevated IgG4 with disease progression and less favorable clinical outcomes. Characterizing immunoglobulin and other humoral immune profiles in melanoma might identify valuable prognostic tools for patient stratification and in the future lead to more effective treatments less prone to tumor-induced blockade mechanisms.
RESUMO
Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galß1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar dead adhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Neoplasias da Mama/metabolismo , Lectinas Tipo C/metabolismo , Mucina-1/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Ligação ProteicaRESUMO
DCs are the most potent APCs and are the focus of many immunotherapeutic approaches for the treatment of cancer, although most of these approaches require the ex vivo generation and pulsing of DCs. We have targeted a subset of DCs in vivo using an Ab to DNGR-1, a C-type lectin dedicated to the cross-presentation of Ag expressed by subsets of DCs. HLA-A2 epitopes from the tumour-associated Ag, MUC1, were coupled to the anti-DNGR-1 Ab, and their efficacy in generating a Th1-cell response and inhibiting tumour growth was evaluated in a clinically relevant double transgenic mouse model expressing human MUC1 and A2K/b. Using this strategy, we demonstrate that an effective immune response to MUC1 can be generated, which results in a significant delay in the growth of MUC1-expressing tumours in both prophylactic and therapeutic settings. In addition, we also show, using PBMCs isolated from healthy volunteer blood, that target an MUC1 HLA-A2 epitope to human DNGR-1 in vitro can induce an MUC1-specific CD8(+) -T-cell response, which confirms the relevance of our in vivo murine results in the human setting.
Assuntos
Anticorpos/imunologia , Vacinas Anticâncer/imunologia , Lectinas Tipo C/imunologia , Mucina-1/imunologia , Receptores Mitogênicos/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-A2/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50-p65 and ERα induced by the E7 oncoprotein. The E7-mediated transcriptional complex also recruited the histone demethylase JARID1B and histone deacetylase HDAC1. The entire complex bound to a specific region on the TLR9 promoter, which resulted in decreased methylation and acetylation of histones upstream of the TLR9 transcriptional start site. The involvement of NF-κB and ERα in the TLR9 down-regulation by HPV16 E7 was fully confirmed in cervical tissues from human patients. Importantly, we present evidence that the HPV16-induced TLR9 down-regulation affects the interferon response which negatively regulates viral infection. Our studies highlight a novel HPV16-mediated mechanism that combines epigenetic and transcriptional events to suppress a key innate immune sensor.
Assuntos
Papillomavirus Humano 16/imunologia , Papillomavirus Humano 16/patogenicidade , Proteínas E7 de Papillomavirus/imunologia , Receptor Toll-Like 9/genética , Sequência de Bases , Linhagem Celular Tumoral , Colo do Útero/imunologia , Colo do Útero/metabolismo , Colo do Útero/virologia , Regulação para Baixo/genética , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Feminino , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Dados de Sequência Molecular , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismoRESUMO
Aberrant glycosylation is a common feature of malignant change. Changes in mucin-type O-linked glycosylation in breast cancer can result in the expression of truncated core 1-based sialylated glycans rather than the core 2-based glycans observed in normal mammary epithelium cells. This has been shown, in part, to be due to changes in the expression of glycosyltransferases, including the up-regulation of some sialyltransferases. Using the breast cancer cell line T47D, we have shown that PGE2, one of the final products of the cyclooxygenase-2 (COX-2) pathway, can induce the mRNA expression of the sialyltransferase α-2,3-sialyltransferase-3 (ST3Gal-I), resulting in increased sialyltransferase activity, demonstrated by a reduction in PNA lectin staining. Induction of COX-2 in the MDA-MB-231 breast cancer cell line also results in the increased expression of ST3Gal-I, leading to increased sialylation of the substrate of ST3Gal-I, core 1 Galß1,3GalNAc. This effect on sialylation could be reversed by the selective COX-2 inhibitor celecoxib. The use of siRNA to knock down COX-2 and overexpression of COX-2 in MDA-MD-231 cells confirmed the involvement of COX-2 in the up-regulation of ST3Gal-I. Moreover, analysis of the expression of ST3Gal-I and COX-2 by 74 primary breast cancers showed a significant correlation between the two enzymes. COX-2 expression has been associated with a number of tumors, including breast cancer, where its expression is associated with poor prognoses. Thus, these results suggest the intriguing possibility that some of the malignant characteristics associated with COX-2 expression may be via the influence that COX-2 exerts on the glycosylation of tumor cells.
Assuntos
Neoplasias da Mama/enzimologia , Ciclo-Oxigenase 2/metabolismo , Sialiltransferases/genética , Regulação para Cima , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Sialiltransferases/metabolismo , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a)) and Sialyl-Le(x) (SLe(x)) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLe(a)/SLe(x)-MUC1 and STn/SLe(a)/SLe(x)-MUC2 glycoforms in ≥50% of the cases, with a variable distribution among organs. Some new glycoforms-T/SLe(a)-MUC2, STn/T/SLe(a) SLe(x)-MUC5AC and STn/T/SLe(a)/SLe(x)-MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.
Assuntos
Biomarcadores Tumorais/análise , Mucina-5AC/análise , Mucina-1/análise , Mucina-2/análise , Mucina-6/análise , Neoplasias/diagnóstico , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Antígenos Glicosídicos Associados a Tumores/análise , Antígenos Glicosídicos Associados a Tumores/metabolismo , Mama/patologia , Antígeno CA-19-9 , Colo/patologia , Imunofluorescência , Gangliosídeos/análise , Gangliosídeos/metabolismo , Glicosilação , Humanos , Imuno-Histoquímica , Pulmão/patologia , Neoplasias/patologia , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Antígeno Sialil Lewis XRESUMO
The glycome acts as an essential interface between cells and the surrounding microenvironment. However, changes in glycosylation occur in nearly all breast cancers, which can alter this interaction. Here, we report that profiles of glycosylation vary between ER-positive and ER-negative breast cancers. We found that genes involved in the synthesis of sialyl-Lewis x (sLe(x); FUT3, FUT4, and ST3GAL6) are significantly increased in estrogen receptor alpha-negative (ER-negative) tumors compared with ER-positive ones. SLe(x) expression had no influence on the survival of patients whether they had ER-negative or ER-positive tumors. However, high expression of sLe(x) in ER-positive tumors was correlated with metastasis to the bone where sLe(x) receptor E-selectin is constitutively expressed. The ER-positive ZR-75-1 and the ER-negative BT20 cell lines both express sLe(x) but only ZR-75-1 cells could adhere to activated endothelial cells under dynamic flow conditions in a sLe(x) and E-selectin-dependent manner. Moreover, L/P-selectins bound strongly to ER-negative MDA-MB-231 and BT-20 cell lines in a heparan sulfate (HS)-dependent manner that was independent of sLe(x) expression. Expression of glycosylation genes involved in heparan biosynthesis (EXT1 and HS3ST1) was increased in ER-negative tumors. Taken together, our results suggest that the context of sLe(x) expression is important in determining its functional significance and that selectins may promote metastasis in breast cancer through protein-associated sLe(x) and HS glycosaminoglycans.
Assuntos
Neoplasias da Mama/metabolismo , Selectina E/metabolismo , Antígenos CD15/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Selectina E/genética , Feminino , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicômica/métodos , Heparitina Sulfato/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Antígenos CD15/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Metástase Neoplásica , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígeno Sialil Lewis X , Sialiltransferases/genética , Sialiltransferases/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6GalNAc-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we found that ST6GalNAc-I is weakly expressed in normal gastric mucosa, but over-expressed in intestinal metaplasia, co-localized with sialyl-Tn. In gastric carcinomas ST6GalNAc-I was also associated with sialyl-Tn, but with heterogeneous staining and partial co-localization. Our results showed ST6GalNAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues.
Assuntos
Trato Gastrointestinal/imunologia , Sialiltransferases/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos Glicosídicos Associados a Tumores , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Primers do DNA , Camundongos , Camundongos Endogâmicos BALB C , Sialiltransferases/imunologiaRESUMO
INTRODUCTION: Detection of serum biomarkers for early diagnosis of breast cancer remains an important goal. Changes in the structure of O-linked glycans occur in all breast cancers resulting in the expression of glycoproteins that are antigenically distinct. Indeed, the serum assay widely used for monitoring disease progression in breast cancer (CA15.3), detects a glycoprotein (MUC1), but elevated levels of the antigen cannot be detected in early stage patients. However, since the immune system acts to amplify the antigenic signal, antibodies can be detected in sera long before the antigen. We have exploited the change in O-glycosylation to measure autoantibody responses to cancer-associated glycoforms of MUC1 in sera from early stage breast cancer patients. METHODS: We used a microarray platform of 60mer MUC1 glycopeptides, to confirm the presence of autoantibodies to cancer associated glycoforms of MUC1 in a proportion of early breast cancer patients (54/198). Five positive sera were selected for detailed definition of the reactive epitopes using on chip glycosylation technology and a panel of glycopeptides based on a single MUC1 tandem repeat carrying specific glycans at specific sites. Based on these results, larger amounts of an extended repertoire of defined MUC1 glycopeptides were synthesised, printed on microarrays, and screened with sera from a large cohort of breast cancer patients (n = 395), patients with benign breast disease (n = 108) and healthy controls (n = 99). All sera were collected in the 1970s and 1980s and complete clinical follow-up of breast cancer patients is available. RESULTS: The presence and level of autoantibodies was significantly higher in the sera from cancer patients compared with the controls, and a highly significant correlation with age was observed. High levels of a subset of autoantibodies to the core3MUC1 (GlcNAcß1-3GalNAc-MUC1) and STnMUC1 (NeuAcα2,6GalNAc-MUC1) glycoforms were significantly associated with reduced incidence and increased time to metastasis. CONCLUSIONS: Autoantibodies to specific cancer associated glycoforms of MUC1 are found more frequently and at higher levels in early stage breast cancer patients than in women with benign breast disease or healthy women. Association of strong antibody response with reduced rate and delay in metastases suggests that autoantibodies can affect disease progression.
Assuntos
Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/imunologia , Mucina-1/imunologia , Idoso , Autoanticorpos/imunologia , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/patologia , Estudos de Coortes , Epitopos/imunologia , Feminino , Glicopeptídeos/imunologia , Humanos , Pessoa de Meia-Idade , PrognósticoRESUMO
The four members of the JARID1/KDM5 family of proteins, a sub-group of the larger ARID (AT rich DNA binding domain) family, have been shown to demethylate trimethylated lysine 4 on histone 3 (H3K4me3), a chromatin mark associated with actively transcribed genes. In some lower organisms a single homologue of JARID1 is found, and functions of the four proteins found in mice and humans may be specific or overlapping. To investigate the function of the Jarid1B protein we examined the effects of deletion of the gene in mice. Systemic knock out of Jarid1b resulted in early embryonic lethality, whereas mice not expressing the related Jarid1A gene are viable and fertile. A second mouse strain expressing a Jarid1b gene with the ARID domain deleted was viable and fertile but displayed a mammary phenotype, where terminal end bud development and side branching was delayed at puberty and in early pregnancy. Since development of terminal end buds are completely dependent on signalling from the estrogen receptor (ERα), we investigated the expression of a target gene (progesterone receptor) in the ∆ARID mouse and found levels to be reduced as compared to wild-type. JARID1B is widely expressed in ER+ breast cancers and breast cancer cell lines, and interaction with ERα was demonstrated by co-immunoprecipitations in cells transfected with tagged ERα and JARID1B genes. Down-regulation of expression of JARID1B using shRNAi in MCF-7 cells resulted in a dramatic decrease in E2 stimulated tumour growth in nude mice. The data demonstrate a specific role for Jarid1B in early embryonic development, in the development and differentiation of the normal mammary gland, and in estrogen induced growth of ER+ breast cancer.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/fisiologia , Embrião de Mamíferos/citologia , Receptor alfa de Estrogênio/análise , Histona Desmetilases com o Domínio Jumonji/fisiologia , Glândulas Mamárias Animais/citologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Animais , Neoplasias da Mama/química , Linhagem Celular Tumoral , Proliferação de Células , Embrião de Mamíferos/fisiologia , Feminino , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Repressoras/genéticaRESUMO
The nuclear protein PLU-1/JARID1B/KDM5 is widely expressed in breast cancers while showing highly restricted expression in normal adult tissues. To investigate whether JARID1B is a potential target antigen for immunotherapy of breast cancer, we have analyzed the responses of CD8(+) T cells to JARID1B HLA-A*0201 peptides in vitro and used peptide multimers to detect the presence of JARID1B reactive T cells in the circulation of breast cancer patients. Peptides were selected using two web-based algorithms: criteria for inclusion being a high score in both prediction algorithms, and nonhomology with retinoblastoma binding protein-2 (RBP2/JARID1A/KDM5A). A 65-peptide panel was selected and assayed for binding strength by competition assay to obtain the IC(50). The immunogenicity in vitro of these peptides was assessed by T cell stimulation experiments, using autologous dendritic cells as APCs in the first rounds followed by autologous lymphoblasts. Fourteen of the peptides assayed produced cultures having >2% of the CD8(+) cells being IFN-γ(+) after 3-6 rounds of stimulation. An HLA-A*0201 cell line could activate the specific T cells if pulsed with peptide, but endogenous peptide levels were insufficient for activation. Nevertheless, multimer staining of circulating T cells from breast cancer patients showed a significantly higher percentage of multimer positive CD8(+) T cells, as compared to healthy adults for two of three JARID1B epitopes tested. One of these, peptide 73 (QLYALPCVL), was analyzed for memory phenotype, and found to have a significantly higher proportion of central memory T cells than the control group, demonstrating a previous exposure to the peptide.