Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5-year survival rate. a-Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to a-enolase. The present work was intended to assess the ability of a-enolase to induce antigen-specific T cell responses. We show that a-enolase-pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN-c and lyse PDAC cells but not normal cells. In vivo, a-enolase-specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to a-enolase, the existence of a-enolase-specific T cells was also demonstrated. Taken as a whole, these results indicate that a-enolase elicits a PDAC-specific, integrated humoral and cellular response. It is thus a promising and clinically relevant molecular target candidate for immunotherapeutic approaches as new adjuvants to conventional treatments in pancreatic cancer. ' 2009 UICC

See Covering the Cover synopsis on page 862. 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 ␣-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. CONCLU-SIONS: 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.

The human PVT-1 gene is located on chromosome 8 telomeric to the c-Myc gene and it is frequently involved in the translocations occurring in variant Burkitt's lymphomas and murine plasmacytomas. It has been proposed that PVT-1 regulates c-Myc gene transcription over a long distance. To get new insights into the functional relationships between the two genes, we have investigated PVT-1 and c-Myc expression in normal human tissues and in transformed cells. Our findings indicate that PVT-1 expression is restricted to a relative low number of normal tissues compared to the wide distribution of c-Myc mRNA, whereas the gene is highly expressed in many transformed cell types including neuroblastoma cells that do not express c-Myc. Reporter gene assays were used to dissect the PVT-1 promoter and to identify the region responsible for the elevated expression observed in transformed cells. This region contains two putative binding sites for Myc proteins. The results of transfection experiments in RAT1-MycER cells and chromatin immunoprecipitation (ChIP) assays in proliferating and differentiated neuroblastoma cells indicate that PVT-1 is a downstream target of Myc proteins.

Background miRNAs are master regulators of signaling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate whether the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development. Methods NHBE cells were cultured at air‐liquid interface and treated with interleukin (IL)‐13 to induce an asthma‐like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, and EV‐associated miRNAs were identified by a RT‐qPCR‐based profiling. Significant candidates were confirmed in EVs isolated by size‐exclusion chromatography from nasal lavages of children with mild‐to‐moderate (n = 8) or severe asthma (n = 9), and healthy controls (n = 9). Results NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL‐13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR‐92b, miR‐210, and miR‐34a significantly correlated with lung function parameters in children (FEV1FVC%pred and FEF25‐75%pred), thus lower sEV‐miRNA levels in nasal lavages associated with airway obstruction. Subsequent ingenuity pathway analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation. Conclusion Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR‐34a, miR‐92b, and miR‐210, might be involved in the early development of a Th2 response in the airways and asthma.