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1.
Gastroenterology ; 144(5): 1098-106, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23333712

RESUMO

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is an aggressive tumor, and patients typically present with late-stage disease; rates of 5-year survival after pancreaticoduodenectomy are low. Antibodies against α-enolase (ENO1), a glycolytic enzyme, are detected in more than 60% of patients with PDA, and ENO1-specific T cells inhibit the growth of human pancreatic xenograft tumors in mice. We investigated whether an ENO1 DNA vaccine elicits antitumor immune responses and prolongs survival of mice that spontaneously develop autochthonous, lethal pancreatic carcinomas. METHODS: We injected and electroporated a plasmid encoding ENO1 (or a control plasmid) into Kras(G12D)/Cre (KC) mice and Kras(G12D)/Trp53(R172H)/Cre (KPC) mice at 4 weeks of age (when pancreatic intraepithelial lesions are histologically evident). Antitumor humoral and cellular responses were analyzed by histology, immunohistochemistry, enzyme-linked immunosorbent assays, flow cytometry, and enzyme-linked immunosorbent spot and cytotoxicity assays. Survival was analyzed by Kaplan-Meier analysis. RESULTS: The ENO1 vaccine induced antibody and a cellular response and increased survival times by a median of 138 days in KC mice and 42 days in KPC mice compared with mice given the control vector. On histologic analysis, the vaccine appeared to slow tumor progression. The vaccinated mice had increased serum levels of anti-ENO1 immunoglobulin G, which bound the surface of carcinoma cells and induced complement-dependent cytotoxicity. ENO1 vaccination reduced numbers of myeloid-derived suppressor cells and T-regulatory cells and increased T-helper 1 and 17 responses. CONCLUSIONS: In a genetic model of pancreatic carcinoma, vaccination with ENO1 DNA elicits humoral and cellular immune responses against tumors, delays tumor progression, and significantly extends survival. This vaccination strategy might be developed as a neoadjuvant therapy for patients with PDA.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Imunidade Celular/imunologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Fosfopiruvato Hidratase/imunologia , Vacinação/métodos , Vacinas de DNA/farmacologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Neoplasias Experimentais/genética , Neoplasias Experimentais/mortalidade , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Taxa de Sobrevida
2.
J Clin Endocrinol Metab ; 105(11)2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32785693

RESUMO

PURPOSE: Pituitary neuroendocrine tumors (PitNETs) are frequent intracranial neoplasms that present heterogenic characteristics. Little is known about the immune cell network that exists in PitNETs and its contribution to their aggressive behavior. METHODS: Here we combined flow cytometry, t-SNE analysis, and histological approaches to define the immune landscape of surgically resected PitNETs. Xenografts of rodent pituitary tumor cells and resected PitNETs were performed in Rag2KO mice, in combination with in vitro analysis aimed at dissecting the role of pituitary tumor-cells in monocyte recruitment. RESULTS: We report that gonadotroph PitNETs present an increased CD68+ macrophage signature compared to somatotroph, lactotroph, and corticotroph PitNETs. Transcriptomic and histological characterizations confirmed gonadotroph infiltrating macrophages expressed CD163, MRC-1, ARG1, and CSF1R M2 macrophage markers. Use of growth hormone (GH)3/GH4 somatotroph and LßT2/αT3.1 gonadotroph cells drove THP1 macrophage migration through respective expression of CCL5 or CSF1. Although both LßT2 and GH3 cells recruited F4/80 macrophages following their engraftment in mice, only LßT2 gonadotroph cells showed a capacity for M2-like polarization. Similar observations were performed on patient-derived xenografts from somatotroph and gonadotroph tumors. Analysis of clinical data further demonstrated a significant correlation between the percentage of CD68+ and CD163+ infiltrating macrophages and the invasive character of gonadotroph tumors. CONCLUSIONS: Gonadotroph tumor drive the recruitment of macrophages and their subsequent polarization to an M2-like phenotype. More importantly, the association between infiltrating CD68+/CD163+ macrophages and the invasiveness of gonadotroph tumors points to macrophage-targeted immunotherapies being a potent strategy to limit the progression of gonadotroph PitNETs.


Assuntos
Gonadotrofos/imunologia , Macrófagos/imunologia , Tumores Neuroendócrinos/imunologia , Hipófise/imunologia , Neoplasias Hipofisárias/imunologia , Adolescente , Adulto , Idoso , Feminino , Citometria de Fluxo , Gonadotrofos/patologia , Humanos , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/patologia , Hipófise/patologia , Neoplasias Hipofisárias/patologia , Adulto Jovem
3.
J Immunother Cancer ; 8(2)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33115943

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is an almost incurable tumor that is mostly resistant to chemotherapy (CT). Adaptive immune responses to tumor-associated antigens (TAA) have been reported, but immunotherapy (IT) clinical trials have not yet achieved any significant increase in survival, confirming the suppressive environment of PDA. As CT has immune-modulating properties, we investigated the effect of gemcitabine (GEM) in antitumor effector responses to TAA in patients with PDA. METHODS: The IgG antibody repertoire in patients with PDA before and after CT was profiled by serological proteome analysis and ELISA and their ability to activate complement-dependent cytotoxicity (CDC) was measured. Peripheral T cells were stimulated in vitro with recombinant TAA, and specific proliferation, IFN-γ/IL-10 and CD8+/Treg ratios were measured. Mice that spontaneously developed PDA were treated with GEM and inoculated with an ENO1 (α-Enolase) DNA vaccine. In some experimental groups, the effect of depleting CD4, CD8 and B cells by specific antibodies was also evaluated. RESULTS: CT increased the number of TAA recognized by IgG and their ability to activate CDC. Evaluation of the IFN-γ/IL-10 ratio and CD8+/Treg ratios revealed that CT treatment shifted T cell responses to ENO1, G3P (glyceraldheyde-3-phosphate dehydrogenase), K2C8 (keratin, type II cytoskeletal 8) and FUBP1 (far upstream binding protein 1), four of the most recognized TAA, from regulatory to effector. In PDA mice models, treatment with GEM prior to ENO1 DNA vaccination unleashed CD4 antitumor activity and strongly impaired tumor progression compared with mice that were vaccinated or GEM-treated alone. CONCLUSIONS: Overall, these data indicate that, in PDA, CT enhances immune responses to TAA and renders them suitable targets for IT.


Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Imunoterapia/métodos , Proteômica/métodos , Vacinas de DNA/uso terapêutico , Idoso , Animais , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Vacinas de DNA/farmacologia
4.
Cancer Res ; 80(16): 3359-3371, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32554750

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions. SIGNIFICANCE: This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Ativinas/biossíntese , Carcinoma Ductal Pancreático/etiologia , Senescência Celular/fisiologia , Neoplasias Pancreáticas/etiologia , Lesões Pré-Cancerosas/etiologia , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II/metabolismo , Ativinas/antagonistas & inibidores , Animais , Carcinoma Ductal Pancreático/metabolismo , Progressão da Doença , Genes ras , Humanos , Camundongos , Neoplasias Pancreáticas/metabolismo , Fosforilação , Lesões Pré-Cancerosas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ativação Transcricional
5.
Front Biosci (Landmark Ed) ; 22(5): 944-959, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27814656

RESUMO

Alpha-enolase (ENO1) is a metabolic enzyme involved in the synthesis of pyruvate. It also acts as a plasminogen receptor and mediates the activation of plasmin and extracellular matrix degradation. In tumor cells, ENO1 is up-regulated and supports the Warburg effect; it is expressed at the cell surface, where it promotes cancer invasion, and is subjected to a specific array of post-translational modifications, namely acetylation, methylation and phosphorylation. ENO1 overexpression and post-translational modifications could be of diagnostic and prognostic value in many cancer types. Information on the biochemical, proteomics and immunological characterization of ENO1, and particularly its ability to trigger a strong specific humoral and cellular immune response, make this ubiquitous protein an interesting tumor target; DNA vaccination with ENO1 in preclinical models efficiently delays the development of very aggressive tumors such as pancreatic cancer. This review aims to analyze the main stages by which the tumor associated antigen (TAA) ENO1 has become a promising target that opens potential avenues for cancer immunotherapy.


Assuntos
Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/imunologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/imunologia , Imunoterapia , Neoplasias/enzimologia , Neoplasias/terapia , Fosfopiruvato Hidratase/antagonistas & inibidores , Fosfopiruvato Hidratase/imunologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/imunologia , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Autoanticorpos/metabolismo , Biomarcadores Tumorais/metabolismo , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Neoplasias/imunologia , Fosfopiruvato Hidratase/metabolismo , Linfócitos T/imunologia , Proteínas Supressoras de Tumor/metabolismo
6.
J Hematol Oncol ; 10(1): 16, 2017 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-28086938

RESUMO

BACKGROUND: We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. METHODS: The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. RESULTS: We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. CONCLUSION: These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.


Assuntos
Biomarcadores Tumorais/fisiologia , Adesão Celular , Proteínas de Ligação a DNA/fisiologia , Integrina alfaVbeta3/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Neoplasias Pancreáticas/patologia , Fosfopiruvato Hidratase/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Forma Celular , Senescência Celular , Proteínas de Ligação a DNA/genética , Expressão Gênica , Inativação Gênica , Humanos , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Integrinas/fisiologia , Camundongos , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismo , Fosfopiruvato Hidratase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Proteínas Supressoras de Tumor/genética
7.
Oncotarget ; 7(5): 5598-612, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26734996

RESUMO

In the last 5 years, novel knowledge on tumor metabolism has been revealed with the identification of critical factors that fuel tumors. Alpha-enolase (ENO1) is commonly over-expressed in tumors and is a clinically relevant candidate molecular target for immunotherapy. Here, we silenced ENO1 in human cancer cell lines and evaluated its impact through proteomic, biochemical and functional approaches. ENO1 silencing increased reactive oxygen species that were mainly generated through the sorbitol and NADPH oxidase pathways, as well as autophagy and catabolic pathway adaptations, which together affect cancer cell growth and induce senescence. These findings represent the first comprehensive metabolic analysis following ENO1 silencing. Inhibition of ENO1, either alone, or in combination with other pathways which were perturbed by ENO1 silencing, opens novel avenues for future therapeutic approaches.


Assuntos
Autofagia , Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Fosforilação Oxidativa , Neoplasias Pancreáticas/patologia , Fosfopiruvato Hidratase/antagonistas & inibidores , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular , Proliferação de Células , Reprogramação Celular , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Camundongos , Camundongos SCID , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfopiruvato Hidratase/genética , Proteômica , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Espectrometria de Massas em Tandem , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Oncotarget ; 6(13): 11098-113, 2015 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-25860938

RESUMO

Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness and resistance to treatment. We have previously demonstrated that most PDAC patients have circulating antibodies against the glycolytic enzyme alpha-enolase (ENO1), which correlates with a better response to therapy and survival. ENO1 is a metabolic enzyme, also expressed on the cell surface where it acts as a plasminogen receptor. ENO1 play a crucial role in cell invasion and metastasis by promoting plasminogen activation into plasmin, a serine-protease involved in extracellular matrix degradation. The aim of this study was to investigate the role of ENO1 in PDAC cell invasion. We observed that ENO1 was expressed on the cell surface of most PDAC cell lines. Mouse anti-human ENO1 monoclonal antibodies inhibited plasminogen-dependent invasion of human PDAC cells, and their metastatic spreading in immunosuppressed mice was inhibited. Notably, a single administration of Adeno-Associated Virus (AAV)-expressing cDNA coding for 72/1 anti-ENO1 mAb reduced the number of lung metastases in immunosuppressed mice injected with PDAC cells. Overall, these data indicate that ENO1 is involved in PDAC cell invasion, and that administration of an anti-ENO1 mAb can be exploited as a novel therapeutic option to increase the survival of metastatic PDAC patients.


Assuntos
Anticorpos Monoclonais/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Carcinoma Ductal Pancreático/prevenção & controle , Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias Hepáticas/prevenção & controle , Neoplasias Pancreáticas/prevenção & controle , Fosfopiruvato Hidratase/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Exp Hematol Oncol ; 2(1): 12, 2013 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23594883

RESUMO

Activation of plasminogen on the cell surface initiates a cascade of protease activity with important implications for several physiological and pathological events. In particular, components of the plasminogen system participate in tumor growth, invasion and metastasis. Plasminogen receptors are in fact expressed on the cell surface of most tumors, and their expression frequently correlates with cancer diagnosis, survival and prognosis. Notably, they can trigger multiple specific immune responses in cancer patients, highlighting their role as tumor-associated antigens. In this review, three of the most characterized plasminogen receptors involved in tumorigenesis, namely Annexin 2 (ANX2), Cytokeratin 8 (CK8) and alpha-Enolase (ENOA), are analyzed to ascertain an overall view of their role in the most common cancers. This analysis emphasizes the possibility of delineating new personalized therapeutic strategies to counteract tumor growth and metastasis by targeting plasminogen receptors, as well as their potential application as cancer predictors.

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