Abstract
Abstract Prostate cancer is still the most common and second leading cause of cancer death in men in the United States and is overwhelmingly lethal when metastatic. Evidence from histochemical analysis and experimental tumor models indicates alterations in cell-surface sialylation as a general observation associated with the metastatic potential of tumor cells. Our preliminary data from lectin staining also demonstrates changes of cell-surface sialylation in a syngeneic prostate cancer cell model, which includes two PC3 sublines, N2 (non-metastatic) and ML2 (highly metastatic). Characterization of cell-surface sialylated glycoproteins in this model system could provide new information related to the metastatic phenotype in prostate cancer. Thus, cell-surface sialoglycoproteins in the N2 and ML2 cell lines were metabolicly labeled with with an unnatural mannose analogue (AC4ManNAz); This was followed by chemoselective conjugation of the azido sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry and affinity detection and enrichment by streptavidin-conjugated beads (or fluorescein). Captured glycoproteins were separated by 1D gel electrophoresis, in-gel proteolytic digestion, and protein identification by means of tandem mass spectrometry (LC-MS/MS). The expression of the targeted sialoglycoproteins was verified in our prostate cancer cell lines and clinical samples by means of Western blot, confocal microscopy and fluorescent immunohistochemistry. Forty three cell-surface N-linked glycoproteins were identified as differentially expressed between the PC3 metastasis variants, including CUB-domain containing protein 1 (CDCP1). Western blot analysis of the cell-surface glycoprotein enriched fractions and total cell lysate confirmed that CDCP1 was over-expressed in the metastatic ML2 cells. Expression of CDCP1 is dysregulated in prostate cancer tissues, with malignant glands showing a more intense cell surface staining pattern, whereas normal glands demonstrated weak membrane and intracellular granular staining on the basal side. We conclude that glycan metabolic labeling technology combined with mass spectrometry analysis provides a powerful tool for the identification of specific cell-surface glycoproteins that may contribute to prostate cancer metastasis, like CDCP1. Further studies are warranted to evaluate the clinical value of CDCP1 and to understand its function in prostate cancer progression. 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 3307.
Published Version
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