Protein profiles associated with survival in lung adenocarcinoma

Abstract

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.

Fig. 1.

Two-dimensional PAGE image and 2D Western blots for selected survival-related proteins. (A) Two-dimensional gel image showing protein separation by molecular mass (MW) and isoelectric point (pI). (B) Two-dimensional separation of the regions that include GRP78, GRK4, PGAM1, and PGK1 isoforms. (C) Two-dimensional Western blot showing GRP78, PGAM1, GRK4, or PGK1 immunoreactive protein spots. The same isoforms for each protein are indicated with arrows for B and C.(D) Tandem mass spectrometry (ESI MS/MS) confirmation for the PGK1 protein spot shown in B.

Fig. 2.

Protein expression profiles and patient survival. (A) Kaplan-Meier survival plots showing the relationship between patient survival and the risk index based on the leave-one-out cross-validation procedure using the top 20 survival-associated proteins among all 682 proteins using all 90 tumors. The high- and low-risk groups differ significantly (P = 0.005). (B) Relationship between patient survival and the risk index based on the leave-one-out cross-validation procedure using the top 20 survival-associated proteins among the 62 stage I tumors. The high- and low-risk groups differ significantly (P = 0.01). (C) Relationship between patient survival and PGK1 protein expression in an independent validation set of 90 lung adenocarcinomas. PGK1 immunohistochemical analysis of a tissue array indicates that increased PGK1 is associated with a reduced survival (P = 0.04). (D) Relationship between patient survival and serum PGK1 levels (ratio of PGK1/total serum protein) by using ELISA analysis with 107 lung adenocarcinomas (P = 0.004).

Fig. 3.

Immunohistochemical analysis of survival-associated proteins PGK1, GRK4, GRP78, and PGAM1. (A) PGK1 expression in normal lung. Low-level staining of alveloar macrophages (arrow) and parenchymal lung cells is observed. (B) An adenocarcinoma demonstrating low-level staining of PGK1 (arrow). (C) Adenocarcinoma showing abundant cytoplasmic and/or nuclear staining of PGK1 within the tumor cells (arrow). (D) Relatively abundant nuclear localization (arrow) of PGK1 immunoreactivity in an adenocarcinoma. (E) Both cytoplasmic and nuclear PGK1 staining of a squamous cell carcinoma of the lung. (F) GRK4 expression in normal lung is primarily observed in neutrophils as shown located within a large blood vessel (arrow). (G) Abundant GRK4 staining in neutrophils within an adenocarcinoma. (H) GRK4 staining in neutrophils and in the tumor cells (arrow) in a squamous lung carcinoma. (I) GRP78 positive staining in an adenocarcinoma (arrow). (J) PGAM1 positive staining (arrow) in an adenocarcinoma. All original magnifications, ×200.

Fig. 4.

Proteins and mRNAs significantly correlated with patient survival in lung adenocarcinomas representing components in the glycolysis pathway. Increased expression in the glycolysis pathway enzymes is shown for tumors showing poor patient outcome. The figure is based on results using genmapp (www.GenMAPP.org).