Moitza Principe

Vaccination With ENO1 DNA Prolongs Survival of Genetically Engineered Mice With Pancreatic Cancer

BACKGROUND & AIMSnPancreatic 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.nnnMETHODSnWe 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnIn 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.

Targeting the Warburg effect in cancer cells through ENO1 knockdown rescues oxidative phosphorylation and induces growth arrest

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.

Three are better than one: plasminogen receptors as cancer theranostic targets

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.

Alpha-enolase (ENO1) controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis

BackgroundWe 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.MethodsThe 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.ResultsWe 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.ConclusionThese data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.

Alpha-Enolase (ENO1), a potential target in novel immunotherapies.

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.

FAM49B, a novel regulator of mitochondrial function and integrity that suppresses tumor metastasis

Mitochondrial dysregulation plays a central role in cancers and drives reactive oxygen species (ROS)-dependent tumor progression. We investigated the pro-tumoral roles of mitochondrial dynamics and altered intracellular ROS levels in pancreatic ductal adenocarcinoma (PDAC). We identified ‘family with sequence similarity 49 member B’ (FAM49B) as a mitochondria-localized protein that regulates mitochondrial fission and cancer progression. Silencing FAM49B in PDAC cells resulted in increased fission and mitochondrial ROS generation, which enhanced PDAC cell proliferation and invasion. Notably, FAM49B expression levels in PDAC cells were downregulated by the tumor microenvironment. Overall, the results of this study show that FAM49B acts as a suppressor of cancer cell proliferation and invasion in PDAC by regulating tumor mitochondrial redox reactions and metabolism.

Abstract 1889: Can the moonlighting glycolytic enzyme α-enolase be a therapeutic target in pancreatic cancer.

Aberrant metabolism together with invasion and metastasis are hallmarks of cancer. This is particularly true for pancreatic ductal adenocarcinoma (PDAC), characterized by rapid progression, invasiveness and resistance to treatments. We have previously described α-enolase (ENOA) as a PDAC-associated antigen. It is a moonlighting protein that works both as a key metabolic enzyme and as a membrane plasminogen receptor. In order to clarify its multifunctional role in pancreatic tumorigenesis we investigated the effect of ENOA knockdown in PDAC cells. Protein expression alterations following ENOA knockdown in the human PDAC cell line CFPAC-1 were revealed by LC-MS/MS analysis. On the basis of a spectra count label-free quantitation approach a large number of proteins mainly involved in cell adhesion, metabolism and proliferation were found to be differentially expressed in ENOA silenced cells compared to the control. After ENOA silencing, PDAC cells displayed a delay in proliferation and decreased survival and colony formation capabilities, even if the pyruvate production was not affected. The growth inhibition was partially due to an increased concentration of intracellular reactive oxygen species (ROS) mainly generated through the sorbitol and NADPH oxidase pathways. Moreover in ENOA silenced cells, the in vitro plasminogen-driven invasion was abolished and the number of lung tumor masses was significantly reduced in SCID-beige mice injected with ENOA silenced cells compared to mice injected with control cells. These effects are under further confirmation in other PDAC cell lines. All together, these findings propose ENOA as a promising target for developing new therapies in pancreatic cancer management. Citation Format: Michela Capello, Moitza Principe, Michelle Samuel Chattaragada, Chiara Riganti, Weidong Zhou, Sammy Ferri-Borgogno, Simona Rolla, Lance Liotta, Emanuel Petricoin, Paola Cappello, Francesco Novelli. Can the moonlighting glycolytic enzyme α-enolase be a therapeutic target in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1889. doi:10.1158/1538-7445.AM2013-1889

Blockade of Surface Alpha-Enolase (ENO1) as a Novel ImmunotherapeuticApproach in Pancreatic Cancer

In 2010 in Italy, there were 175,046 cancer deaths, with total mortality rates, respectively for men and women, of 138 and 83/100,000 [1]. Total cancer mortality in Italy has been declining since the 1980s in men and earlier in women [1], similarly to most European countries [2] and the United States [3]. However, some rising trends have also been observed, notably for female lung cancer and pancreatic cancer in both sexes [1]. Although the incidence of pancreatic cancer is relative low compared to other tumor types such as breast, lung and prostate cancer, in pancreatic cancer the mortality is almost equal to the incidence level, with 41,780 and 40,560 deaths predicted in Europe and USA respectively [2,4]. For this reason, PDA is the 4th leading cause of cancer deaths in the Western Countries [1,2,4]. Despite the efforts of clinicians and scientists, pancreatic cancer has the worst prognosis of all major malignancies, with a 5-year survival rate of 8% [4].

Abstract A042: Targeting of alpha-enolase (ENO1) as a novel immunotherapeutical strategy for pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. By using a proteome approach, we have found that protein recognized with the highest frequency by autoantibodies in PDAC sera was the alpha-enolase (ENO1), a glycolytic enzyme that also acts as a plasminogen-binding receptor. We have found that either ENO1 silencing or treatment of the PDAC cells with single administration of Adeno-Associated Virus-expressing monoclonal antibody against human ENO1 inhibited lung metastasis in immunosuppressed mice injected with PDAC cells. In addition, PDAC ENO1-silenced cells displayed a down modulated expression of cell surface molecules that regulate the adhesion to the extracellular matrix as confirmed by their reduced binding to laminin, collagen and fibronectin. ENO1 silencing in PDAC cells increased reactive oxygen species mainly generated through the sorbitol and NADPH oxidase pathways, as well as autophagy and catabolic pathway adaptations, which together affect PDAC cell growth and induced senescence. This data indicated that ENO1 plays a critical role in PDAC progression and invasion. ENO1 possesses interesting antigenic properties as it is able to elicit T cell proliferation, activation and cytotoxic specific T lymphocytes (CTL) differentiation both in vitro and in vivo. Tumor infiltrating T lymphocytes specific for ENO1 have been identified in PDAC patients and the number of ENO1-specific T cell clones generated from peripheral blood of PDAC positively correlated with a better survival. Antibody and T cell responses to ENO1 are increase in PDAC patient underwent chemotherapy. DNA vaccination to ENO1 elicits an integrated humoral and cellular immune response that was accompanied by a reduced and regulatory T cells and MDSC that significantly extends survival of Genetically Engineered Mice that spontaneously develop PDAC. Notably, mouse anti-human ENO1 monoclonal antibody inhibits MDSC adhesion to pancreatic endothelial cells and in vitro and in vivo migration and decreases MDSC arginase activity and secretion of IL-6. As whole these data demonstrate that the self-antigen ENO1 is a promising target suitable for therapeutic purposes in PDAC. In particular, the antibodies anti-ENO1 may inhibit PDA cell and myeloid cell invasion and positively modulate T cell response, making the immunotherapy more effective. Citation Format: Francesco Novelli, Michela Capello, Michelle Chattaragada, Sammy Ferri Borgogno, Giorgia Mandili, Moitza Principe, Emanuela Mazza, Sara Bulfamante, Roberta Curto, Daniele Giordano, Paola Cappello. Targeting of alpha-enolase (ENO1) as a novel immunotherapeutical strategy for pancreatic cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A042.

Pancreatic cancer vaccine: a unique potential therapy

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Gastrointestinal Cancer: Targets and Therapy 2016:6 1–11 Gastrointestinal Cancer: Targets and Therapy Dovepress