One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and se-

Purpose Using prostatic fluids rich in glycoproteins like prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) , the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging mass spectrometry-based glycopeptide analysis platforms. Experimental Design A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides. Results Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data. Conclusion and clinical relevance Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis.

Prostate cancer is a clinically heterogeneous disease, ranging from indolent asymptomatic disease to very aggressive metastatic and life threatening forms of the disease. Distant metastasis represents the major lethal cause of prostate cancer. The most critical clinical challenge in the management of the patients is identifying those individuals at risk of developing metastatic disease. To understand the molecular mechanisms of prostate cancer metastasis and identify markers with metastatic potential, we have analyzed protein expression in two syngeneic prostate cancer cells lines PC3-N2 and PC3-ML2 using isobaric tags for relative and absolute quantitation labeling and multi-dimensional protein identification technology liquid chromatography matrix assisted laser desorption ionization tandem mass spectrometry. PC3-N2 is lowly metastatic while PC3-ML2 highly metastatic. A total of 1,756 proteins were identified in the analyses with 130 proteins showing different expression levels (p<0.01) in the two cell lines. Out of these, 68 proteins were found to be significantly up-regulated while 62 are significantly down-regulated in PC3-ML2 cells compared with PC3-N2 cells. The upregulation of plectin and vimentin which were the most significantly differentially expressed were validated by Western blot and their functional relevance with respect to invasion and migration was determined by siRNA gene silencing. To our knowledge, this study is the first to demonstrate that up-regulation of vimentin and plectin expression positively correlates with the invasion and metastasis of androgen-independent PCA.

Introduction: Prostate cancer (PCa) is the most prevalent cancer among men and the third most common cause of cancer related-deaths. Distant metastasis represents the major lethal cause of prostate cancer. The most critical clinical challenge in the management of the patients is identifying those individuals at risk of developing metastatic disease. Our long term objective is to identify the molecules and characterize the mechanisms that are involved in PCa metastasis. The identification of biomarkers for predicting the susceptibility of men to develop metastasis and understanding the mechanisms of metastasis will potentially better discriminate the more aggressive metastatic forms of the disease and provide better treatment and clinical management opportunities. Methods: We have analyzed protein expression in two syngeneic prostate cancer cells lines with different metastatic potentials (PC3-N2, lowly metastatic; PC3-ML2, highly metastatic) using high throughput quantitative mass spectrometry. Validations of the most significantly differentially expressed proteins were done by western blot, qRT-PCR and characterization of the target proteins was carried out by performing siRNA knockdowns, proliferation assays and invasion assays, flow cytometry western blot and Immunofluorescence assays. Results: We identified 130 proteins that were differentially expressed between PC3-N2 and PC3-ML2 cell lines. Out of these, 68 proteins were found to be significantly up-regulated while 62 are significantly down-regulated in PC3-ML2 cells compared with PC3-N2 cells. Plectin was the most significantly upregulated protein in the proteomics experiments and this was validated by Western blot, confocal microscopy and immunohistochemistry. SiRNA knockdown of plectin suppresses the invasive and migratory capacity of the highly metastatic PC3-ML2 cells and results in the modulation of various signal transduction pathways. To our knowledge, this study is the first to demonstrate that up-regulation of plectin positively correlates with the invasion and metastasis of androgen-independent PCa cells. Conclusions: We have identified several proteins including plectin that are differentially expressed in prostate cancer cell lines with different metastatic potentials. Furthermore, these results suggest that plectin plays a role in the progression and metastasis of PCa. Plectin regulates protein kinase activation, as well as receptor signaling cascades, and recently plectin was identified as a putative biomarker in pancreatic cancer. These findings are currently being validated using disease stratified tissue microarrays to determine the expression patterns in PCa patients. Further studies are focusing on uncovering the mechanisms responsible for progression and metastasis of PCa that are modulated by plectin. These could assist in the development of novel diagnostic and therapeutic strategies for the disease. Citation Format: Tanya C. Burch, Megan T. Watson, Julius O. Nyalwidhe. Plectin modulates invasion and metastasis in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 414. doi:10.1158/1538-7445.AM2013-414