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Overexpression of Vimentin Contributes to Prostate Cancer Invasion and Metastasis Via Src Regulation

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Overexpression of Vimentin Contributes to Prostate Cancer Invasion and Metastasis Via Src Regulation ANTICANCER RESEARCH 28: 327-334 (2008) Overexpression of Vimentin Contributes to Prostate Cancer Invasion and Metastasis via Src Regulation JUNCHENG WEI*, GANG XU*, MINGFU WU, YONGTAO ZHANG, QIONG LI, PING LIU, TAO ZHU, ANPING SONG, LIANGPIN ZHAO, ZHIQIANG HAN, GANG CHEN, SHIXUAN WANG, LI MENG, JIANFENG ZHOU, YUNPING LU, SHIXUAN WANG and DING MA Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China Abstract. A significant proportion of prostate cancer patients Prostate carcinoma is the second leading cause of cancer- treated with curative intent develop advanced disease. At a related death in the United States and Europe (1), mainly fundamental biological level, very little is known about what due to its high potential for bone metastasis. However, the makes the disease aggressive and metastatic. Observational molecular mechanisms of prostate cancer metastasis are not pathology reports and experimental data suggest that an well understood. Among the currently available techniques, epithelial-mesenchymal transition (EMT) is involved in prostate cancer proteomics permits the analysis of thousands of cancer invasiveness. The mechanism by which vimentin modified or unmodified proteins simultaneously and has promotes prostate cancer cell invasion and metastasis was become increasingly popular for identifying biomarkers for examined. The highly metastatic human prostate epithelial cell the early detection, classification and prognosis of tumors, line PC-3M-1E8 (1E8-H) and the low metastatic line PC-3M- as well as for pinpointing molecular targets for improving 2B4 (2B4-L) were used for comparative proteomic analysis by treatment outcomes (2). two-dimensional gel electrophoresis, followed by matrix-assisted It is known that tumor progression to malignancy requires laser desorption/time of flight mass spectrometry (MALDI- a change from an epithelial phenotype to a fibroblast or TOF-MS). A transwell assay was performed to test cell mesenchymal phenotype, a re-programming known as an migration and invasion and immunoblotting was used to epithelial-mesenchymal transition (EMT) (3-6), which is analyze the relative expression of proteins. High vimentin characterized by the acquisition of an invasive, mesenchymal expression was detected in 1E8-H compared to 2B4-L cells. phenotype by epithelial cells that enables their migration into Down-regulation of vimentin in 1E8-H by antisense-vimentin a new microenvironment. E-cadherin is a prototypic type I transfection led to a significant inhibition of invasiveness, and cadherin that forms homophilic interactions through its selective stimulation of vimentin activity in 2B4-L by delivery extracellular immunoglobulin (Ig) domain, which connects to of recombinant vimentin promoted cell invasiveness. Vimentin actin filaments indirectly via ·-catenin and ‚-catenin through activity was associated with C-src, ‚-catenin and E-cadherin their cytoplasmic domains (7-9). The appropriate expression expression. PP2, a specific inhibitor of src family kinases, of E-cadherin on the plasma membrane is essential for cells reduced phospho-‚-catenin expression and induce E-cadherin to retain an epithelial morphology. Evidence from previous expression. Vimentin promotes tumor cell invasiveness and the studies has shown a correlation between loss of cell–cell targeting of vimentin/C-src may be a promising strategy for adhesion during EMT and decreased E-cadherin function, preventing or blocking prostate cancer metastasis. resulting in the aggressiveness, de-differentiation and metastasis of many carcinomas (10-17). The transgenic mouse model generated by Perl et al. has demonstrated that the loss of E-cadherin-mediated intercellular adhesion is a rate- *Both authors contributed equally to this manuscript. limiting step in the progression from adenoma to invasive carcinoma in vivo (18). The intermediate filament protein Correspondence to: Dr. Ding Ma, Cancer Biology Research Center, (IFP) vimentin, expressed in mesenchymal cells, is a well- Tongji Hospital, Tongji Medical College, Huazhong University of known marker for EMT (3). Vimentin expression and Science and Technology, Wuhan, Hubei 430030, P.R. China. Fax: 86-27-83662779 e-mail: [email protected] perturbation of E-cadherin-mediated cell adhesion are therefore both regarded as hallmarks of EMT-associated Key Words: Vimentin, prostate cancer, invasion, C-src, E- events. The heterogeneity and reversibility of E-cadherin cadherin/‚-catenin. protein production also remains a subject of controversy (19). 0250-7005/2008 $2.00+.40 327 ANTICANCER RESEARCH 28: 327-334 (2008) Src is a signal-transducing protein kinase that plays 2-DE and image analysis. The 2-DE was performed as previously central roles in the control of cell growth and described (25) using the PROTEAN IEF and PROTEAN Plus differentiation. Overexpression and aberrant activation of Dodeca cell systems (Bio-Rad). For preparative 2-DE, the total proteins (260 Ìg/185 Ìl) premixed with re-hydration buffer were src family kinases have been identified in various human run in an isoelectric focusing (IEF) system using a pre-cast 11 cm cancer cells (20, 21). It has been demonstrated that C-src immobilized pH gradient (IPG) strip with pI ranges of 4-7 (Bio- kinases are enriched at the cell-cell adherent junctions of Rad). The total Vh was 52,000-69,000. SDS-PAGE gels were run various types of cells, including hepatocytes (22). at constant voltages of 150 V for 45 minutes and 200 V for 6 h until Transfection of a V-src mutant into Madin-Darby canine the bromophenol blue dye reached the bottom of the gels. After kidney cells led to increased association of E-cadherin with electrophoresis in the second dimension, the gels were stained with ‚-catenin, which caused a disruption of intercellular “Blue Silver”, a sensitive colloidal Coomassie G-250 stain compatible with mass spectrometry (MS) analysis (26). The gels adhesion and an increase in vulnerability to invasion (23). were scanned using a GS710 imaging densitometer (Bio-Rad) and In the present study, the human prostate epithelial analyzed by PDQuest software (Bio-Rad). cancer cell line PC-3M and its clonal cells, PC-3M-1E8 (1E8-H) cells and PC-3M-2B4 (2B4-L) cells, which possess Protein identification by matrix-assisted laser desorption/time of flight opposite metastatic potentials but similar genetic mass spectrometry (MALDI-TOF-MS). Protein identification was backgrounds (24), were selected as model systems for the performed as described previously (27), with some modifications. study of the molecular events involved in prostate Briefly, spots were cut out from the gels, destained for 20 minutes in 30 mM KCN/100 mM Na S O 1:1 (v/v) and washed with MilliQ metastasis. Using a transfection approach the proteins 2 2 3 water until the gels became clear. Background gel was cut out and associated with metastasis were screened and the potential used as a negative control. Isolated gel fragments were kept in 0.2 candidates involved in regulating tumor migration and M NH4HCO3 for 20 minutes, lyophilized and digested overnight in invasion were investigated. Preliminary data showed that 12.5 ng/l trypsin containing 0.1 M NH4HCO3. The peptides were vimentin is one of the predominant overexpressed proteins extracted three times with 50% acetonitrile (CAN) containing 0.1% in the highly metastatic cell line 1E8-H compared to the trifluoroacetic acid (TFA), mixed with the hydroxycinnamic (HCCA) low metastatic potential 2B4-L cells. Our studies therefore matrix (Sigma), applied to the target and analyzed using a Bruker REFLEX III MALDI-TOF mass spectrometer (BrukerFranzen focused on the role of vimentin in mediating tumor Analytik, Bremen, Germany). Protein identification using peptide metastasis and the possibility of cross-linking between mass fingerprinting was performed by Mascot search vimentin and C-src, as well as sustained E-cadherin/‚- (http://www.matrixscience.com, MatrixScience Ltd, London, UK) catenin complex conformation. against the National Center for Biotechnology Information (NCBI) non-redundant protein database. The errors in peptide masses Materials and Methods were within the range of 0.01%-0.1%. One missed tryptic cleavage site per peptide was allowed during the search. Cysteines were Cell culture and reagents. The human prostate epithelial cancer cell carboamidomethylated and methionines were oxidated. Proteins lines 1E8-H and 2B4-L were obtained from the Peking University matching more than four peptides and with a Mascot score higher Health Science Center (Beijing, China) and were cultured in RPMI than 63 were considered significant (p<0.05). growth media supplemented with 10% fetal bovine serum (FBS), 100 Ìg/ml streptomycin and 100 U/ml penicillin. The cells were Immunofluorescence microscopy. The cells were seeded onto sterile kept at 37ÆC in a humidified atmosphere with 5% CO2. PP2 (src glass coverslips at 50% confluency. After 24 h, they were fixed at kinase inhibitor) was purchased from Calbiochem (La Jolla, CA, room temperature in 3.7% formaldehyde in PBS for 10 min and USA). A stock solution of PP2 was prepared in Me2SO (Sigma, St. then washed three times in PBS. The cells were permeabilized for Louis, MO, USA) and stored at –70ÆC. 10 min in 0.2% Triton X-100 and blocked in 1% bovine serum albumin (BSA). After a series of washes, the coverslips were Protein preparation for two-dimensional gel electrophoresis (2-DE). incubated with mouse anti-human
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