RESUMEN
Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular "nest" structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1+ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1+ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8+ T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy.
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Neoplasias , Ratones , Animales , Neoplasias/patología , Macrófagos/metabolismoRESUMEN
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.
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Histona Demetilasas , Histona Metiltransferasas , Histonas , Neoplasias , Animales , Epigénesis Genética , Histona Demetilasas/metabolismo , Histona Metiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Neoplasias/genética , Proteínas del Grupo Polycomb/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis. This is attributed to the disease already being advanced at presentation and having a particularly aggressive tumor biology. The PDAC tumor microenvironment (TME) is characterized by a dense desmoplastic stroma, dominated by cancer-associated fibroblasts (CAF), extracellular matrix (ECM) and immune cells displaying immunosuppressive phenotypes. Due to the advanced stage at diagnosis, the depletion of immune effector cells and lack of actionable genomic targets, the standard treatment is still apoptosis-inducing regimens such as chemotherapy. Paradoxically, it has emerged that the direct induction of apoptosis of cancer cells may fuel oncogenic processes in the TME, including education of CAF and immune cells towards pro-tumorigenic phenotypes. The direct effect of cytotoxic therapies on CAF may also enhance tumorigenesis. With the awareness that CAF are the predominant cell type in PDAC driving tumorigenesis with various tumor supportive functions, efforts have been made to try to target them. However, efforts to target CAF have, to date, shown disappointing results in clinical trials. With the help of sophisticated single cell analyses it is now appreciated that CAF in PDAC are a heterogenous population with both tumor supportive and tumor suppressive functions. Hence, there remains a debate whether targeting CAF in PDAC is a valid therapeutic strategy. In this review we discuss how cytotoxic therapies and the induction of apoptosis in PDAC fuels oncogenesis by the education of surrounding stromal cells, with a particular focus on the potential pro-tumorigenic outcomes arising from targeting CAF. In addition, we explore therapeutic avenues to potentially avoid the oncogenic effects of apoptosis in PDAC CAF.
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Apoptosis , Fibroblastos Asociados al Cáncer/patología , Neoplasias Pancreáticas/patología , Microambiente Tumoral , Carcinogénesis/patología , Humanos , Modelos BiológicosRESUMEN
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.
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Neoplasias de la Mama/metabolismo , Mucina-1/metabolismo , Neoplasias de la Mama/patología , Receptores ErbB/metabolismo , Femenino , Glicosilación , Humanos , Receptores de Estrógenos/metabolismoRESUMEN
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.
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Macrófagos/fisiología , Monocitos/fisiología , Mucina-1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mucina-1/genéticaRESUMEN
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.
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Neoplasias de la Mama/metabolismo , Mucina-1/metabolismo , Acetilgalactosamina/metabolismo , Transporte Biológico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Regulación Enzimológica de la Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Glicosilación , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Metástasis de la Neoplasia , Microambiente TumoralRESUMEN
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.
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Inmunoterapia , Mucina-1/inmunología , Neoplasias/terapia , Antimetabolitos Antineoplásicos/uso terapéutico , Ensayos Clínicos como Asunto , Terapia Combinada , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Glicosilación , Humanos , Mucina-1/química , Mucina-1/fisiología , Neoplasias/tratamiento farmacológico , Microambiente Tumoral , GemcitabinaRESUMEN
Purpose: Unprecedented clinical outcomes have been achieved in a variety of cancers by targeting immune checkpoint molecules. This preclinical study investigates heme oxygenase-1 (HO-1), an immunosuppressive enzyme that is expressed in a wide variety of cancers, as a potential immune checkpoint target in the context of a chemotherapy-elicited antitumor immune response. We evaluate repurposing tin mesoporphyrin (SnMP), which has demonstrated safety and efficacy targeting hepatic HO in the clinic for the treatment of hyperbilirubinemia, as an immune checkpoint blockade therapy for the treatment of cancer.Experimental Design: SnMP and genetic inactivation of myeloid HO-1 were evaluated alongside 5-fluorouracil in an aggressive spontaneous murine model of breast cancer (MMTV-PyMT). Single-cell RNA sequencing analysis, tumor microarray, and clinical survival data from breast cancer patients were used to support the clinical relevance of our observations.Results: We demonstrate that SnMP inhibits immune suppression of chemotherapy-elicited CD8+ T cells by targeting myeloid HO-1 activity in the tumor microenvironment. Microarray and survival data from breast cancer patients reveal that HO-1 is a poor prognostic factor in patients receiving chemotherapy. Single-cell RNA-sequencing analysis suggests that the myeloid lineage is a significant source of HO-1 expression, and is co-expressed with the immune checkpoints PD-L1/2 in human breast tumors. In vivo, we therapeutically compare the efficacy of targeting these two pathways alongside immune-stimulating chemotherapy, and demonstrate that the efficacy of SnMP compares favorably with PD-1 blockade in preclinical models.Conclusions: SnMP could represent a novel immune checkpoint therapy, which may improve the immunological response to chemotherapy. Clin Cancer Res; 24(7); 1617-28. ©2018 AACR.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Metaloporfirinas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Modelos Animales de Enfermedad , Femenino , Fluorouracilo/farmacología , Hemo-Oxigenasa 1/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
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.
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Antígenos CD/metabolismo , Mucina-1/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Microambiente Tumoral/inmunología , Antígenos CD/genética , Biomarcadores , Diferenciación Celular , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Glicosilación , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células Mieloides/citología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/patología , Fenotipo , Unión Proteica , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genéticaRESUMEN
Distant metastases account for the majority of cancer-related deaths in breast cancer. The rate and site of metastasis differ between estrogen receptor (ER)-negative and ER-positive tumours, and metastatic fate can be very diverse even within the ER-negative group. Characterisation of new pro-metastatic markers may help to identify patients with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metastasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist in carbohydrate molecules, such as sialyl-Lewis x antigen (sLex), borne by glycoproteins or glycolipids on the cancer cell surface. We have previously demonstrated that the molecular scaffold presenting sLex to selectins (e.g. glycolipid vs. glycoproteins) was crucial for these interactions to occur. Moreover, we reported that detection of sLex alone in breast carcinomas was only of limited prognostic value. However, since sLex was found to be carried by several glycoproteins in cancer cells, we hypothesized that the combination of the carbohydrate with its carriers could be more relevant than each marker independently. In this study, we addressed this question by analysing sLex expression together with two glycoproteins (BST-2 and LGALS3BP), shown to interact with E-selectin in a carbohydrate-dependent manner, in a cohort of 249 invasive breast cancers. We found both glycoproteins to be associated with distant metastasis risk and poorer survival. Importantly, concomitant high expression of BST-2 with sLex defined a sub-group of patients with ER-negative tumours displaying higher risks of liver and brain metastasis and a 3-fold decreased survival rate.
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Antígenos CD/biosíntesis , Antígenos de Neoplasias/biosíntesis , Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/genética , Proteínas Portadoras/biosíntesis , Selectina E/biosíntesis , Glicoproteínas/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/genética , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Supervivencia sin Enfermedad , Selectina E/genética , Receptor alfa de Estrógeno/genética , Femenino , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Humanos , Antígeno Lewis X/genética , Ligandos , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Antígeno Sialil Lewis XRESUMEN
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.
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Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Neoplasias de la Mama/metabolismo , Lectinas Tipo C/metabolismo , Mucina-1/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Unión ProteicaRESUMEN
Tumor-associated glycoproteins are a group of antigens with high immunogenic interest: The glycoforms generated by the aberrant glycosylation are tumor-specific and the novel glycoepitopes exposed can be targets of tumor-specific immune responses. The MUC1 antigen is one of the most relevant tumor-associated glycoproteins. In cancer, MUC1 loses polarity and becomes overexpressed and hypoglycosylated. Changes in glycan moieties contribute to MUC1 immunogenicity and can modify the interactions of tumor cells with antigen-presenting cells such as dendritic cells that would affect the overall antitumor immune response. Here, we show that the form of the MUC1 antigen, i.e., soluble or as microvesicle cargo, influences MUC1 processing in dendritic cells. In fact, MUC1 carried by microvesicles translocates from the endolysosomal/HLA-II to the HLA-I compartment and is presented by dendritic cells to MUC1-specific CD8(+) T cells stimulating IFN-γ responses, whereas the soluble MUC1 is retained in the endolysosomal/HLA-II compartment independently by the glycan moieties and by the modality of internalization (receptor-mediated or non-receptor mediated). MUC1 translocation to the HLA-I compartment is accompanied by deglycosylation that generates novel MUC1 glycoepitopes. Microvesicle-mediated transfer of tumor-associated glycoproteins to dendritic cells may be a relevant biologic mechanism in vivo contributing to define the type of immunogenicity elicited. Furthermore, these results have important implications for the design of glycoprotein-based immunogens for cancer immunotherapy.
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Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Mucina-1/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Vesículas Citoplasmáticas/inmunología , Femenino , Glicosilación , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interferón gamma/biosíntesis , Lisosomas/inmunología , Neoplasias/inmunología , Neoplasias Ováricas/inmunología , Proteínas Recombinantes/inmunología , Solubilidad , Células Tumorales CultivadasRESUMEN
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.
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Anticuerpos/inmunología , Vacunas contra el Cáncer/inmunología , Lectinas Tipo C/inmunología , Mucina-1/inmunología , Receptores Mitogénicos/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Antígeno HLA-A2/inmunología , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.
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Neoplasias de la Mama , Investigación , Investigación Biomédica Traslacional , Animales , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/etiología , Neoplasias de la Mama/terapia , Femenino , HumanosRESUMEN
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.
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Neoplasias de la Mama/enzimología , Ciclooxigenasa 2/metabolismo , Sialiltransferasas/genética , Regulación hacia Arriba , Neoplasias de la Mama/genética , Línea Celular Tumoral , Ciclooxigenasa 2/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Sialiltransferasas/metabolismo , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
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.
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Biomarcadores de Tumor/análisis , Mucina 5AC/análisis , Mucina-1/análisis , Mucina 2/análisis , Mucina 6/análisis , Neoplasias/diagnóstico , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patología , Antígenos de Carbohidratos Asociados a Tumores/análisis , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Mama/patología , Antígeno CA-19-9 , Colon/patología , Técnica del Anticuerpo Fluorescente , Gangliósidos/análisis , Gangliósidos/metabolismo , Glicosilación , Humanos , Inmunohistoquímica , Pulmón/patología , Neoplasias/patología , Oligosacáridos/análisis , Oligosacáridos/metabolismo , Antígeno Sialil Lewis XRESUMEN
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.
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Neoplasias de la Mama/metabolismo , Selectina E/metabolismo , Antígeno Lewis X/metabolismo , Neoplasias Hormono-Dependientes/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Selectina E/genética , Femenino , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glicómica/métodos , Heparitina Sulfato/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Antígeno Lewis X/genética , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Metástasis de la Neoplasia , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Antígeno Sialil Lewis X , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
Chinese hamster ovary (CHO) cells are widely used for the production of recombinant proteins for clinical use as well as academic research. They are particularly important for the production of glycoproteins where bacteria cannot be used. TGFß1 is a potent cytokine highly conserved across species with multiple immunological and non-immunological effects. We have discovered that CHOK1, the CHO clone most commonly used by the pharmaceutical industry, constitutively secretes latent TGFß1 and that this hamster TGFß1 is active on human cells inducing profound immunological effects. As far as we are aware, the production of TGFß1 by CHOK1 cells has not been reported before in the literature. As TGFß1 exerts powerful and pleiotropic effects on diverse cell types, and as CHO cells are used to produce a large number of clinical and non-clinical products, our findings are highly relevant to studies that rely on recombinant proteins.
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Factores Inmunológicos/metabolismo , Factores Inmunológicos/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Factores Inmunológicos/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
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.