Cappello, Paola

Novelli, Francesco

Principe, Moitza

English

Institutional Research Information System University of Turin

Blockade of Surface Alpha-Enolase (ENO1) as a Novel ImmunotherapeuticApproach in Pancreatic CancerMoitza Principe1,2, Paola Cappello1,2,3 and Francesco Novelli1,2,3,4*1Center for Experimental Research and Medical Studies (CeRMS), Azienda Universitaria Ospedaliera Città della Salute e della Scienza di Torino, Turin, Italy2Department of Molecular Biotechnology and Health Sciences, University of Turin, Italy3Molecular Biotechnology Center, University of Turin, Italy4Imunogenetics and Transplantation Service, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Italy*Corresponding author: Francesco Novelli, Center for Experimental Research and Medical Studies (CeRMS), Azienda Universitaria Ospedaliera Città della Salute edella Scienza di Torino, Turin, Italy, Tel: +390116336886; E-mail: franco.novelli@unito.itReceived date: January 11, 2016; Accepted date: February 15, 2016; Published date: February 20, 2016Copyright: © 2016 Principe M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.Keywords: Pancreatic cancer; ENO1; Antibody; ImmunotherapyIntroductionIn 2010 in Italy, there were 175,046 cancer deaths, with totalmortality rates, respectively for men and women, of 138 and83/100,000 [1]. Total cancer mortality in Italy has been declining sincethe 1980s in men and earlier in women [1], similarly to most Europeancountries [2] and the United States [3]. However, some rising trendshave also been observed, notably for female lung cancer and pancreaticcancer in both sexes [1]. Although the incidence of pancreatic cancer isrelative low compared to other tumor types such as breast, lung andprostate cancer, in pancreatic cancer the mortality is almost equal tothe incidence level, with 41,780 and 40,560 deaths predicted in Europeand USA respectively [2,4]. For this reason, PDA is the 4th leadingcause of cancer deaths in the Western Countries [1,2,4]. Despite theefforts of clinicians and scientists, pancreatic cancer has the worstprognosis of all major malignancies, with a 5-year survival rate of 8%[4].Pancreatic ductal adenocarcinomas (PDA), originate from theexocrine pancreas, account for 95% of pancreatic cancers and ischaracterized by rapid progression, invasiveness and resistance to bothradiation and chemotherapy [5]. Therefore, surgery provides the onlypossibility for cure, but most patients present metastatic PDA uponinitial diagnosis, and less than 20% of patients are suitable candidatesat the time of diagnosis and 5-years survival of completely resectedpatients is only up to 25% [5]. The major hurdle for PDA is the lack ofmarkers for early diagnosis, along with few efficacious therapies.Alpha-Enolase (Eno1) and Anti-Eno1 Antibody-BasedImmunotherapyIn recent years, targeted therapies have been tested for treatingmetastatic pancreatic cancer, for example Erlotinib, an inhibitor of theepidermal growth factor receptor (EGFR) (ClinicalTrials.gov numberNCT01608841), is a successful example of how using an antibody, incombination with gemcitabine-based chemotherapy, is effective inimproving overall and progression-free survival, as well as responserates in metastatic PDA patients [6].Immunotherapy, by both passive and active approaches, can beconsidered as a kind of “targeted therapy” in terms of activating theimmune system against a specific target, or nullifying it through anantibody. We have previously identified novel PDA-associated antigensby detecting autoantibodies in the sera of PDA patients, which can beconsidered as “spies” of the activation of the immune system [7]. Someof these antigens have been characterized as suitable therapeutictargets, such as α-Enolase (ENO1) [8]. ENO1 is a multi-functionalprotein, acting as both a glycolytic enzyme and a plasminogen receptorexpressed on the cell surface of tumor cells, thus playing a critical rolein metastasis and spreading [9]. As glycolytic enzyme is ubiquitouslyexpressed in almost normal tissues (see website www.proteinatlas.org)including liver, brain, spleen, adipose tissue, kidney [10], reproductivetract [11] and pancreas [8]. By promoting plasminogen activation intoplasmin, a serine-protease involved in extracellular matrixdegradation, ENO1 favors cell invasion and metastasis (Figure1 upperpanel) [9]. The plasminogen system is involved in tumor growth,invasion and metastasis [12]. Several proteins of this system have beendemonstrated to have a clinical value as diagnostic and prognosticmarkers in cancer. In particular, overexpression of plasminogenreceptors has been associated with poor prognosis, shorter survivaland resistance to chemotherapy [12].We first characterized ENO1 as a plasminogen receptor; it isexpressed on the surface of human PDA cells lines and at higher levelsin metastatic cell lines, whereas it is absent or expressed at lower levelsin primary tumor-derived cell lines [13]. Ex vivo analysis of a livermetastasis-derived cell line (PANC-1/M) revealed an increased cellsurface expression of ENO1 compared to the parental cell line(PANC-1/P) from the primary tumor. This observation suggests thatspreading and invasion of PDA cells is strictly related to the high cellsurface expression of ENO1, which, in turn, facilitates binding ofelevated concentrations of plasminogen at the cell surface.Plasminogen is cleaved into plasmin by uPA (urokinase plasminogenactivator), resulting in an increased ability of PDA cells to degrade theECM [12]. We targeted surface ENO1 by using a mouse monoclonalantibody (mAb) that blocks the plasminogen-ENO1 interaction [13].The anti-human ENO1 mAb inhibited plasminogen-dependent cellmigration through Matrigel in vitro. This invasion requiressimultaneous expression of surface ENO1 and uPAR (receptor foruPA), as well as expression of uPA. Although uPA and uPAR were notexpressed in PDA cells, in vivo they can be expressed by tumor stromalcells that are abundantly present in the PDA microenvironment, or up-regulated in cancer cells by growth factors (such as EGF and HGF) orcytokines (such as TNF-α, TGF-β) [14-17]. In fact, exposure of ENO1-expressing PDA cells to TGF-β induced the up-regulation of uPA anduPAR, which renders the cells sensitive to plasminogen in terms ofincreased invasion. In vivo, immune-compromised mice treated withanti-ENO1 mAb displayed a reduced number of metastasis after xeno-transplantation of PDA cells compared to control IgG-treated miceChemotherapy:Open Access Principe, et al., Chemo Open Access 2016, 5:2http://dx.doi.org/10.4172/2167-7700.1000188Commentary Open AccessChemo Open AccessISSN:2167-7700 CMT, an Open Access Journal Volume 5 • Issue 2 • 1000188[13]. These data indicate that ENO1 is involved in the PDA invasionprocess, suggesting that interfering with the ENO1-plasminogeninteraction could be useful in inhibiting this invasion. The anti-ENO1mAb effect is strictly related to this interaction, as demonstrated by invitro and in vivo experiments, using PDA cells expressing a triple-mutated plasminogen binding site of ENO1 (CFPAC-1 TM). Thesecells were no longer able to invade in response to plasminogen.An innovative Adeno Associated Viral Vector (AAVV)-basedtherapeutic strategy has been adopted to increase the efficacy of anti-ENO1 mAb treatment in a metastatic mouse model. This approachexploited the innovative technology of antibody gene transfer [18-19].Passive immunization involves the delivery of purified antibodies tothe host with a resulting short-lived immunity, necessitating numerousinoculations to produce an appreciable effect. By contrast, when themAb gene is packaged into an (AAVV) vector and delivered by directintramuscular injection, the mAb molecules are endogenouslysynthesized and passively distributed to the circulatory system, thusproviding long-term protection. This strategy has been proved to besuccessful. After injection of the AAVV expressing anti-ENO1 mAb(Figure 1 left panel), the presence of the mAb in the mouse seraprogressively increased from 1 to 4 weeks after injection. Thiscontinuous, long-lasting and sustained production of circulating anti-ENO1 mAb resulted in the observation of a significant decrease in lungmetastases compared to control mice, producing a more pronouncedeffect compared to biweekly inoculation of anti-ENO1 mAb (Figure 1left panel)[13].Taken together, these findings strongly suggest that blocking theENO1/plasminogen interaction through the injection of AAVV-anti-ENO1 mAb could provide a new therapeutic approach for treatingmetastatic PDA patients.Natural Anti-ENO1 Antibodies Elicited By ENO1VaccinationWe have also demonstrated that ENO1 is able to induce bothhumoral and T cell specific responses in PDA patients [20].Autoantibodies against ENO1 are detected in more than 60% of PDApatients, and their presence correlates with a longer survival [7-8]. Thisprompted us to propose ENO1 as a therapeutic candidate owing to itsability to induce an integrated humoral and cellular response. Wedeveloped a DNA vaccination to ENO1 in genetically engineered mice(GEM) that spontaneously develop autochthonous, lethal pancreaticcancer [21], and we showed that the vaccine significantly induced aspecific immune response against pancreatic tumors, which efficientlyprolonged mouse survival. We demonstrated that ENO1 DNAvaccination induced a specific antibody as well as a cellular response inmice. Notably, DNA vaccination elicited anti-ENO1 IgG binding at thesurface of GEM-derived PDA cells, which killed them by inducing acomplement-dependent cytotoxicity (Figure 1 central panel) [21].Furthermore, ENO1 DNA vaccination reduced the numbers ofperipheral and intratumoral myeloid-derived suppressor cells (MDSC)and T-regulatory cells and increased T-helper 1 (Th1) and Th17responses. Due to the ENO1 cell surface expression on MDSC, and itsenhancement by inflammation, we also investigated if anti-ENO1antibodies interfered with MDSC invasion in pancreatic tumors [22].In vitro-generated MDSC treated with anti-ENO1 antibodies were lessable to adhere to pancreatic endothelial cells and invade them or theMatrigel (Figure1 right panel). In addition, there was significantly lessmigration of MDSC treated with anti-ENO1 to the lymph node afterfoot-pad injection. Finally, ENO1-vaccinated mice showing anti-ENO1specific antibodies, displayed a lower percentage of MDSC in thetumor compared to that evaluated in empty-plasmid vaccinated mice[22].Despite ENO1 is found in almost all human tissues [23], theincreased expression of ENO1 and its cell surface localization is aunique feature of cancer cells. ENO1-specific T cells spared normalkeratinocytes in in vitro cytotoxic assay [20] and no autoimmunityevent nor side effects were detected after DNA-vaccination [21] oranti-ENO1 mAb treatment [13] in mice. This suggests that bothENO1-DNA vaccine and anti-ENO1 mAb therapy are safe.Figure 1: Anti-ENO1 antibody immunotherapy. The plasminogenreceptor ENO1 causes plasminogen to localize to the extracellularsurface, allowing its cleavage into plasmin, catalyzed by uPA boundto its surface receptor uPAR. Plasmin is a serine protease thatdegrades extracellular matrix (ECM) proteins and helps the invasiveprocess. The anti-ENO1 antibodies, produced using the AAVconstruct or elicited by the vaccination, inhibit the binding ofplasminogen to ENO1 or binding to the endothelial cells, blockinginvasion of both tumor cells and MDSC. Anti-ENO1 treated MDSCalso favor Th1 and Th17 cell responses. In addition, anti-ENO1 Absmediate complement-dependent cytotoxicity and PDA cell killing.ConclusionMetastasis is one of the greatest clinical challenges in modernoncology; it is, in fact, the disseminated disease that causes the deathof cancer patients. This is especially true for PDA, which is one of themost aggressive human cancers, due to its high potential of localinvasion and metastasis [5]. Despite many studies, metastasis remainslargely a terra incognita waiting to be explored. Our recent findingshighlight how the anti-ENO1 antibodies inhibit PDA cells, as well assuppressor immune cells, making the immunotherapy effective (Figure1). Taken together, these results once again define ENO1 as apromising molecular and immunological target for the realization ofnew personalized therapies for PDA patients.Citation: Principe M, Cappello P, Novelli F (2016) Blockade of Surface Alpha-Enolase (ENO1) as a Novel Immunotherapeutic Approach inPancreatic Cancer. Chemo Open Access 5: 188. doi:10.4172/2167-7700.1000188Page 2 of 3Chemo Open AccessISSN:2167-7700 CMT, an Open Access Journal Volume 5 • Issue 2 • 1000188AcknowledgementsWe would like to thank Dr. Radhika Srinivasan for critically readingthe manuscript. This work was partly supported by grants from theAssociazione Italiana Ricerca sul Cancro (Special Program 5 x milleno. 12182 to FN and IG no. 15257 to FN); University of Turin-ProgettiAteneo 2014-Compagnia di San Paolo (PC-METAIMMUNOTHER toFN; PANTHER to PC); Ministero della Salute, Progetti di RicercaSanitaria Finalizzata 2015 (RF-2013-02354892); Fondazione RicercaMolinette; MP is recipient of a fellowship from Fondazione UrsulaGiorgio Cytron.References1. Rosso T, Bertuccio P, La Vecchia C, Negri E, Malvezzi M, et al. (2015)Cancer mortality trend analysis in Italy, 1980-2010, and predictions for2015. Tumori 101: 664-675.2. Malvezzi M, Bertuccio P, Rosso T, Rota M, Levi F, et al. (2015) Europeancancer mortality predictions for the year 2015: does lung cancer have thehighest death rate in EU women? Ann Oncol 26: 779-786.3. Chatenoud L, Bertuccio P, Bosetti C, Malvezzi M, Levi F, et al. (2014)Trends in mortality from major cancers in the Americas: 1980-2010. AnnOncol 25: 1843-1853.4. Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer JClin 66: 7-30.5. Kamisawa T, Wood LD, Itoi T, Takaori K (2016) Pancreatic cancer.Lancet .6. Wang JP, Wu CY, Yeh YC, Shyr YM, Wu YY, et al. (2015) Erlotinib iseffective in pancreatic cancer with epidermal growth factor receptormutations: a randomized, open-label, prospective trial. Oncotarget 6:18162-18173.7. Tomaino B, Cappello P, Capello M, Fredolini C, Ponzetto A, et al. (2007)Autoantibody signature in human ductal pancreatic adenocarcinoma. JProteome Res 6: 4025-4031.8. Tomaino B, Cappello P, Capello M, Fredolini C, Sperduti I, et al. (2011)Circulating autoantibodies to phosphorylated α-enolase are a hallmark ofpancreatic cancer. J Proteome Res 10: 105-112.9. Capello M, Ferri-Borgogno S, Cappello P, Novelli F (2011) α-enolase: apromising therapeutic and diagnostic tumor target. FEBS J 278:1064-1074.10. Pancholi V (2001) Multifunctional alpha-enolase: its role in diseases. CellMol Life Sci 58: 902-920.11. Souza CE, Rego JP, Lobo CH, Oliveira JT, Nogueira FC, et al. (2012)Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams. J Proteomics 75: 4436-4456.12. Ceruti P, Principe M, Capello M, Cappello P, Novelli F (2013) Three arebetter than one: plasminogen receptors as cancer theranostic targets. ExpHematol Oncol 2: 12.13. Principe M, Ceruti P, Shih NY, Chattaragada MS, Rolla S, et al. (2015)Targeting of surface alpha-enolase inhibits the invasiveness of pancreaticcancer cells. Oncotarget 6: 11098-11113.14. Hildenbrand R, Schaaf A, Dorn-Beineke A, Allgayer H, Sütterlin M, et al.(2009) Tumor stroma is the predominant uPA-, uPAR-, PAI-1-expressingtissue in human breast cancer: prognostic impact. Histol Histopathol 24:869-877.15. Ulisse S, Baldini E, Sorrenti S, D'Armiento M (2009) The urokinaseplasminogen activator system: a target for anti-cancer therapy. CurrCancer Drug Targets 9: 32-71.16. Bauer TW, Liu W, Fan F, Camp ER, Yang A et al. (2005) Targeting ofurokinase plasminogen activator receptor in human pancreaticcarcinoma cells inhibits c-Met- and insulin-like growth factor-I receptor-mediated migration and invasion and orthotopic tumor growth in mice.Cancer Res 65: 7775-7781.17. Lund LR, Ellis V, Rønne E, Pyke C, Danø K (1995) Transcriptional andpost-transcriptional regulation of the receptor for urokinase-typeplasminogen activator by cytokines and tumour promoters in the humanlung carcinoma cell line A549. Biochem J 310 : 345-352.18. Schnepp BC, Johnson PR (2014) Vector-Mediated In Vivo AntibodyExpression. Microbiol Spectr 2: AID-0016-2014.19. Schnepp BC, Johnson PR (2014) Adeno-associated virus delivery ofbroadly neutralizing antibodies. Curr Opin HIV AIDS 9: 250-256.20. Cappello P, Tomaino B, Chiarle R, Ceruti P, Novarino A et al. (2009) Anintegrated humoral and cellular response is elicited in pancreatic cancerby alpha-enolase, a novel pancreatic ductal adenocarcinoma-associatedantigen. Int J Cancer 125: 639-648.21. Cappello P, Rolla S, Chiarle R, Principe M, Cavallo F, et al. (2013)Vaccination with ENO1 DNA prolongs survival of genetically engineeredmice with pancreatic cancer. Gastroenterology 144: 1098-1106.22. Cappello P, Tonoli E, Curto R, Giordano D, Giovarelli M, et al. (2015)Anti-a-enolase antibody limits the invasion of myeloid-derivedsuppressor cells and attenuates their restraining effector T cell response.OncoImmunology23. Díaz-Ramos A, Roig-Borrellas A, García-Melero A, López-Alemany R(2012) α-Enolase, a multifunctional protein: its role onpathophysiological situations. J Biomed Biotechnol 2012: 156795. Citation: Principe M, Cappello P, Novelli F (2016) Blockade of Surface Alpha-Enolase (ENO1) as a Novel Immunotherapeutic Approach inPancreatic Cancer. Chemo Open Access 5: 188. doi:10.4172/2167-7700.1000188Page 3 of 3Chemo Open AccessISSN:2167-7700 CMT, an Open Access Journal Volume 5 • Issue 2 • 1000188

BLOCKADE OF SURFACE ALPHA-ENOLASE (ENO1) AS A NOVEL IMMUNOTHERAPEUTIC APPROACH IN PANCREATIC CANCER

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BLOCKADE OF SURFACE ALPHA-ENOLASE (ENO1) AS A NOVEL IMMUNOTHERAPEUTIC APPROACH IN PANCREATIC CANCER

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