In an effort to identify a clinical biomarker for lung cancer, we used cDNA microarray and 2D protein analyses to demonstrate that increased Fas-associated death domain (FADD) mRNA and protein were significantly associated with poor survival. Analyses of copy number and sequence of the FADD gene in 24 independent tumors ruled out the existence of an amplified and/or mutated FADD gene in aggressive lung cancers. Immunohistochemistry-based tissue microarray analysis showed that nuclear localization of FADD and elevation of the phosphorylated form of FADD (p-FADD) correlated with poor outcome (P = 0.003). Tumors with increased p-FADD expression showed elevated NF-kappaB (P = 0.004) activation, a frequent molecular alteration associated with tumorigenesis and metastasis in a variety of cancers. To provide a link between p-FADD and NF-kappaB, cell culture studies demonstrated that overexpression of p-FADD leads to an increase in NF-kappaB activity and a decrease in the number of cells in the G2 phase of the cell cycle, compared with cells expressing the nonphosphorylatable form of FADD or the vector control. Furthermore, cDNA microarray analyses of lung tumor samples showed that increased levels of FADD transcripts were significantly correlated with overexpression of cyclins D1 (P < 0.01) and B1 (P < 0.01), genes that are involved in the regulation of cell cycle progression and are inducible by NF-kappaB. These studies demonstrate that induction of NF-kappaB activity and its effects on cell-cycle progression may represent a molecular basis underlying the aggressive tumor behavior associated with elevated p-FADD expression in lung adenocarcinoma.

The effects of selenium, an essential nutrient with anti‐carcinogenic properties, are mediated by selenium‐binding proteins. The protein expression status of human selenium‐binding protein 1 (SBP1) in human tumours and the exact function of this protein are not known. In this study, quantitative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used on 93 lung adenocarcinomas and ten uninvolved lung samples. Two likely isoforms of a 56 kD protein that showed a significantly decreased abundance in lung adenocarcinomas were observed. Tandem mass spectrometry and 2‐D western blot analysis identified these two proteins as human SBP1. Tumour tissue microarrays were utilized to examine the cellular expression patterns of SBP1 using immunohistochemistry. The same tissue samples were examined for SBP1 mRNA expression using oligonucleotide microarrays. Two major SBP1 isoforms were detected, with an acidic isoform (457) being significantly down‐regulated in lung adenocarcinomas compared with normal lung ( p = 0.02). Two additional more acidic SBP1 isoforms were only observed in normal lung. SBP1 protein isoforms and SBP1 mRNA levels were significantly decreased in poorly differentiated (versus moderately and well‐differentiated), T2–T4 (versus T1), and bronchus‐derived (versus bronchioloalveolar) tumours. Low levels of SBP1 protein (native form, 460) correlated significantly with poor survival ( p = 0.007). The lack of SBP1 expression was not due to gene deletion. Treatment of A549 lung adenocarcinoma cells with the methylation inhibitor 5‐azacytidine did not affect expression of the SBP1 protein. Analysis of the tumour proliferation status using Ki‐67 suggests that down‐regulated expression of SBP1 may reflect increased cell proliferation and decreased differentiation in lung adenocarcinomas. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Morphologic assessment of lung tumors is informative but insufficient to adequately predict patient outcome. We previously identified transcriptional profiles that predict patient survival, and here we identify proteins associated with patient survival in lung adenocarcinoma. A total of 682 individual protein spots were quantified in 90 lung adenocarcinomas by using quantitative two-dimensional polyacrylamide gel electrophoresis analysis. A leave-one-out cross-validation procedure using the top 20 survival-associated proteins identified by Cox modeling indicated that protein profiles as a whole can predict survival in stage I tumor patients (P = 0.01). Thirty-three of 46 survival-associated proteins were identified by using mass spectrometry. Expression of 12 candidate proteins was confirmed as tumor-derived with immunohistochemical analysis and tissue microarrays. Oligonucleotide microarray results from both the same tumors and from an independent study showed mRNAs associated with survival for 11 of 27 encoded genes. Combined analysis of protein and mRNA data revealed 11 components of the glycolysis pathway as associated with poor survival. Among these candidates, phosphoglycerate kinase 1 was associated with survival in the protein study, in both mRNA studies and in an independent validation set of 117 adenocarcinomas and squamous lung tumors using tissue microarrays. Elevated levels of phosphoglycerate kinase 1 in the serum were also significantly correlated with poor outcome in a validation set of 107 patients with lung adenocarcinomas using ELISA analysis. These studies identify new prognostic biomarkers and indicate that protein expression profiles can predict the outcome of patients with early-stage lung cancer.

The relationship between gene expression measured at the mRNA level and the corresponding protein level is not well characterized in human cancer. In this study, we compared mRNA and protein expression for a cohort of genes in the same lung adenocarcinomas. The abundance of 165 protein spots representing 98 individual genes was analyzed in 76 lung adenocarcinomas and nine non-neoplastic lung tissues using two-dimensional polyacrylamide gel electrophoresis. Specific polypeptides were identified using matrix-assisted laser desorption/ionization mass spectrometry. For the same 85 samples, mRNA levels were determined using oligonucleotide microarrays, allowing a comparative analysis of mRNA and protein expression among the 165 protein spots. Twenty-eight of the 165 protein spots (17%) or 21 of 98 genes (21.4%) had a statistically significant correlation between protein and mRNA expression (r > 0.2445; p < 0.05); however, among all 165 proteins the correlation coefficient values (r) ranged from -0.467 to 0.442. Correlation coefficient values were not related to protein abundance. Further, no significant correlation between mRNA and protein expression was found (r = -0.025) if the average levels of mRNA or protein among all samples were applied across the 165 protein spots (98 genes). The mRNA/protein correlation coefficient also varied among proteins with multiple isoforms, indicating potentially separate isoform-specific mechanisms for the regulation of protein abundance. Among the 21 genes with a significant correlation between mRNA and protein, five genes differed significantly between stage I and stage III lung adenocarcinomas. Using a quantitative analysis of mRNA and protein expression within the same lung adenocarcinomas, we showed that only a subset of the proteins exhibited a significant correlation with mRNA abundance.

Cytokeratins. (CK) are intermediate filaments whose expression is often altered in epithelial cancer. Systematic identification of lung adenocarcinoma proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry has uncovered numerous CK isoforms. In this study, 93 lung adenocarcinomas. (64 stage I and 29 stage III) and 10 uninvolved lung samples were quantitatively examined for protein expression. Fourteen of 21 isoforms of CK 7, 8, 18, 19 occurred at significantly higher levels. (P<.05) in tumors compared to uninvolved adjacent tissue. Specific isoforms of the four types of CK identified correlated with either clinical outcome or individual clinical-pathological parameters. All five of the CK7 isoforms associated with patient survival represented cleavage products. Two of five CK7 isoforms. (nos. 2165 and 2091), one of eight CK8 isoforms. (no. 439), one of three CK19 isoforms. (no. 1955) were associated with survival and significantly correlated to their mRNA levels, suggesting that transcription underlies overexpression of these CK isoforms. Our data indicate substantial heterogeneity among CK in lung adenocarcinomas resulting from posttranslational modifications, some of which correlated with patient survival and other clinical parameters. Therefore, specific isoforms of individual CK may have utility as diagnostic or predictive markers in lung adenocarcinomas.