This is an author version of the contribution published on:Questa è la versione dell'autore dell'opera: J Proteome Res. 2011 Jan 7;10(1):105-12. doi: 10.1021

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

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. It is the fourth leading cause of cancer death with a 2% 5-year survival rate. Biomarkers for its early detection are lacking. This study was designed to use a proteomics-based approach as a means of identifying antigens that elicit a humoral response in PDAC patients. Antibodies against PDAC-associated antigens are useful for early cancer diagnosis and therapy. Proteins from PDAC cell lines were separated by 2-DE, and the serum IgG reactivity of 70 PDAC patients, 40 healthy subjects (HS), 30 non-PDAC tumor patients, and 15 chronic pancreatitis (CP) patients was tested by Western blot analysis. Spots specifically recognized by PDAC sera and revealed by mass spectrometry corresponded to metabolic enzymes or cytoskeletal proteins. Most were up-regulated in PDAC tissues. Thus, it seems that metabolic enzymes and cytoskeletal proteins are specific targets of the humoral response during PDAC. The results of further studies of these serological-defined antigens could be of diagnostic and therapeutic significance in PDAC.

IntroductionInterferon-␥ (IFN-␥), produced by T and natural killer (NK) cells, is considered the principal effector cytokine of cell-mediated immunity and exerts its effects on target cells through a highaffinity receptor complex linked to a specific Janus kinase (Jak)/ signal transducer and activator of transcription (STAT) signaling cascade. 1,2 The IFN-␥ receptor (IFN-␥R) complex consists of 2 chains: an IFN-␥R1 binding chain and an IFN-␥R2 signaling chain. 1 The intracellular portions of the 2 chains provide the Jak1 and Jak2 docking sites. Upon phosphorylation, Jak1 and Jak2 activate STAT1: Following phosphorylation and dimerization, STAT1 is translocated into the nucleus where it activates transcription of numerous sets of IFN-␥-inducible genes. 2 The IFN-␥/STAT1 pathway plays an essential role in controlling the expansion of normal and neoplastic cell types of different origin. Activation of this pathway switches on many proapoptotic and antiproliferative genes such as interferon regulatory factor 1 (IRF1), p21 waf/cip , Fas and FasL, and activates caspases. [3][4][5][6][7][8][9][10] However, the signals transduced by IFN-␥ do not always induce apoptosis or block proliferation, and lymphoid cells become resistant to the antiproliferative effects of IFN-␥. Resting, malignant, or normal T cells that develop toward the T helper 1 (Th1) pathway become resistant to the antiproliferative effects of the IFN-␥/STAT1 pathway 9,11,12 or rather, IFN-␥ favors their proliferation and differentiation. [13][14][15] The refractoriness of T cells to the IFN-␥/STAT1 pathway has been attributed mainly to down-regulation of the IFN-␥R chains, especially IFN-␥R2, which protects these cells from the antiproliferative/proapoptotic effects of [16][17][18][19] Both IFN-␥-dependent and -independent mechanisms have been reported to downregulate IFN-␥R2 expression in T lymphocytes. During murine Th cell differentiation, IFN-␥ itself induces IFN-␥ resistance by down-regulating IFN-␥R2, 16 whereas in human T lymphocytes, IFN-␥R2 internalization occurs mostly in clathrin-coated pits independently from IFN-␥ 17 and is selectively induced by insulinlike growth factor 1 (IGF1). 20 Since the IFN-␥/STAT1 pathway is usually down-regulated in T lymphocytes, information on the mechanisms that maintain low IFN-␥R2 expression in these cells might prove useful for devising therapeutic approaches centered on selectively reinstating the IFN-␥/STAT1 apoptotic signaling pathway in autoreactive or neoplastic T cells.Besides T-cell receptor (TCR) engagement, 9,12 up-regulation of surface IFN-␥R2 in T cells may also be induced by serum deprivation, 5 exposure to nitric oxide (NO), 21 Among the plethora of factors present in serum, iron has profound effects on numerous critical cell functions, such as electron and oxygen transport, mitochondrial energy metabolism, and detoxification, thus requiring tight homeostatic regulation. 24 Iron binds to cytoplasmic iron regulatory protein 1 (IRP1) and IRP2 which in turn regulate expression of proteins such as fe...

We have demonstrated that sera from pancreatic ductal adenocarcinoma (PDAC) patients contain IgG autoantibodies to α-enolase, a glycolytic enzyme that also works as surface plasminogen receptor. As this implies that α-enolase elicits a B-cell dependent humoral response in vivo in PDAC patients, its ability to induce a T cell mediated response to PDAC was investigated. Here we show that α-enolase-pulsed dendritic cells were capable to specifically stimulate healthy autologous T cells to proliferate, secrete IFN-γ and lyse PDAC cells. In vivo, α-enolase-specific T cells completely inhibited the growth of PDAC cells in immunodeficient mice. In PDAC patients, and in particular in those with circulating IgG autoantibodies to α-enolase, the existence of memory T cells to α-enolase was also demonstrated. As a whole, these results indicate that α-enolase elicits a PDAC-specific humoral and cellular responses. The sum of its features makes α-enolase a promising candidate for new immunotherapeutic strategies in the cure of PDAC that lacks until now of efficacious therapies to associate the conventional ones.