Identification of α-Enolase as an Autoantigen in Lung Cancer: Its Overexpression Is Associated with Clinical Outcomes

Abstract

Purpose: Although existence of humoral immunity has been previously shown in malignant pleural effusions, only a limited number of immunogenic tumor-associated antigens (TAA) have been identified and associated with lung cancer. In this study, we intended to identify moreTAAs in pleural effusion^derived tumor cells. Experimental Design: Using morphologically normal lung tissues as a control lysate inWestern blotting analyses, 54 tumor samples were screened with autologous effusion antibodies. Biochemical purification and mass spectrometric identification of TAAs were done using established effusion tumor cell lines as antigen sources. We identified a p48 antigen as a-enolase (ENO1). Semiquantitative immunohistochemistry was used to evaluate expression status of ENO1 in the tissue samples of 80 patients with non^small cell lung cancer (NSCLC) and then correlated with clinical variables. Results: Using ENO1-specifc antiserum, up-regulation of ENO1expression in effusion tumor cells from 11of 17 patients was clearly observed compared with human normal lung primary epithelial and non-cancer-associated effusion cells. Immunohistochemical studies consistently showed high level of ENO1 expression in all the tumors we have examined thus far. Log-rank and Cox's analyses of ENO1 expression status revealed that its expression level in primary tumors was a key factor contributing to overall-and progression-free survivals of patients (P < 0.05). The same result was also obtained in the early stage of NSCLC patients, showing that tumors expressing relatively higher ENO1level were tightly correlated with poorer survival outcomes. Conclusions: Our data strongly support a prognostic role of ENO1in determining tumor malignancy of patients with NSCLC.

Identification of salt-inducible kinase 3 as a novel tumor antigen associated with tumorigenesis of ovarian cancer

“…In this study, the ascitic tumor cells of 12 patients were first purified with Ficoll-Percoll gradient centrifugation, lyzed and immunoblotted with their ascitic autoantibodies as described previously (Chang et al, 2006). The ascitic antibodies of patient CA502 were highly immunoreactive to a major protein band with an approximate molecular mass of 150 kDa in the autologous tumor lysates (Supplementary Figure 1A).…”

“…Cell lysates were prepared as previously described (Chang et al, 2006). Briefly, cells were transfected with plasmids using Lipofectamine for HeLa cells, Lipo2000 (Invitrogen) for SK-OV3, and ExGene 500 (Fermentas, Vilnius, Lithuania) for OVCAR3 cells.…”

“…After transfection, cells were treated with or without various pharmacological inhibitors such as LY294002 for PI3K, PD98059 for mitogen-activated protein/ extracellular signal-regulated kinase 1, rapamycin for mechanistic target of rapamycin-mediated S6 kinase, staurosporine for broad-spectrum protein kinases, H89 for protein kinase A and PP2 for Src-family tyrosine kinases. Afterward, the cells were lyzed in cell lysis buffer (Chang et al, 2006), and the protein concentration was quantified using the BCA assay (Thermo Fisher). The lysates were resolved in 7.5-10% polyacrylamide gel containing sodium dodecyl sulfate, transferred to nitrocellulose membranes, and probed with purified CA502 ascitic antibody (32 mg/ml) or antibody specific to Myc tag, GST-tag (Thermo Fisher), p21 Waf/Cip1 , p27 Kip , cyclin Ds, cyclin E, pTyr 416 -Src, c-Src (Cell Signaling Technology, Beverly, MA, USA) or SIK3.…”

“…The presence of humoral response to TAAs has been well demonstrated in patients with various cancers. The responses are frequently mounted against tumor cells expressing altered or abnormally high levels of normal self-antigens with inappropriate timing (Brewer et al, 2009), cellular compartmentalization (Chang et al, 2006) or tissue expression (Old, 2008) that result in increased immunogenicity. Therefore, high levels of tumor-associated autoantibodies can be also observed in some cancer patients throughout their disease progressions (Gagnon et al, 2008), and some of them are associated with their disease outcomes (Reuschenbach et al, 2009).…”

A Panel of Sputum-Based Genomic Marker for Early Detection of Lung Cancer

“…The present observations of the genes are consistent with our earlier findings, confirming their potential role as biomarkers for early NSCLC. Of the 3 newly characterized genes, ENO1 is located at chromosome 1p36.23 that is one of the most frequent targets of genomic amplification in lung cancer (8,19,20). Racz et al (21) found that ENO1 was amplified in tumors tissues of early stage of NSCLC, especially SCC.…”