Abstract
Abstract Changes in the pattern of protein expression by tumor cells reflect the stage of tumor progression, the potential for invasion and metastasis, and drug resistance. Objectives: To use proteomic analysis to detect differences in protein expression that serve as biomarkers of tumor cell properties and to translate the results into diagnostic and prognostic tools based on immuno-cytological staining and immunoassay. Methods: Two-dimensional isoelectric focusing and gel electrophoresis (2-DG) followed by mass spectrometry were used to detect, measure, and identify changes in protein expression. MTC is a non-metastatic cell clone derived from a primary rat mammary adenocarcinoma. MTLn2 and MTLn3 are low and high metastatic potential cell clones derived from lung metastases of the primary tumor. 2-DG was performed simultaneously on samples of 3 extracts from cultured cells of each of the 3 clones. The 9 gels were stained with SYPRO Ruby. Matching of protein spots, densitometric analysis, and statistical analysis were performed using PDQuest 7.1 software. Excised proteins were identified by comparing the mass and sequence of tryptic peptides with those of all known rat proteins using liquid chromatography electrospray-ionization quadrupole ion trap mass spectrometry, SEQUEST software, and the NCBI protein database. Results: A total of 1500 proteins were detected. The patterns of protein expression of MTLn2 and MTLn3 cells were similar. Only 5 spots had a greater than 3-fold difference in staining intensity that was statistically significant (p<0.05), whereas 48 spots differed between MTC and MTLn3 cells. Twenty spots were selected for further study, 10 that had a positive correlation of staining intensity with metastatic potential and 10 that had a negative correlation. Of the 17 unique proteins that were identified, five were of special interest because they are often cited as potential tumor biomarkers. These included the positive biomarkers nucleophosmin (NPM) and protein 14-3-3 sigma and the negative biomarkers raf kinase inhibitor protein (RKIP), peroxiredoxin-2, and galectin-1. Immuno-cytological staining and ELISA confirmed increased NPM and protein 14-3-3 sigma expression and decreased galectin-1 expression. The results suggest a role for stromal elements in biomarker expression. Conclusions: Increased metastatic potential is associated with positive and negative changes in protein expression. Proteins that are positively correlated with metastatic potential may be more useful as clinical biomarkers, whereas those with negative correlations may provide information about underlying mechanisms of metastatic spread and possible stromal influences. Changes in protein expression with differences in tumor properties will provide a consensus panel of biomarkers for diagnostic and prognostic tests. Supported in part by the Sam Mount, Jr. Endowment for Oral Cancer Research. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3301.
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