Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells.

Dysfunction of vascular barriers is a critical step in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1, also known as ARHGEF2) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2; also known as MYL2) phosphorylation by localising GEF-H1 to cell junctions. By using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function during disease.

Molecular Profiling of Thymoma and Thymic Carcinoma: Genetic Differences and Potential Novel Therapeutic Targets.

Thymoma and thymic carcinoma are thymic epithelial tumors (TETs). We performed a molecular profiling to investigate the pathogenesis of TETs and identify novel targets for therapy. We analyzed 37 thymomas (18 type A, 19 type B3) and 35 thymic carcinomas. The sequencing of 50 genes detected nonsynonymous mutations in 16 carcinomas affecting ALK, ATM, CDKN2A, ERBB4, FGFR3, KIT, NRAS and TP53. Only two B3 thymomas had a mutation in noncoding regions of the SMARCB1 and STK11 gene respectively. Three type A thymomas harbored a nonsynonymous HRAS mutation. Fluorescence in situ hybridization detected in 38 % of carcinomas a CDKN2A, in 32 % a TP53 and in 8 % an ATM gene deletion, whereas only one B3 thymoma exhibited a CDKNA deletion, and none of the type A thymomas showed a gene loss. Sequencing of the total miRNA pool of 5 type A thymomas and 5 thymic carcinomas identified the C19MC miRNA cluster as highly expressed in type A thymomas, but completely silenced in thymic carcinomas. Furthermore, the miRNA cluster C14MC was downregulated in thymic carcinomas. Among non-clustered miRNAs, the upregulation of miR-21, miR-9-3 and miR-375 and the downregulation of miR-34b, miR-34c, miR-130a and miR-195 in thymic carcinomas were most significant. The expression of ALK, HER2, HER3, MET, phospho-mTOR, p16INK4A, PDGFRA, PDGFRB, PD-L1, PTEN and ROS1 was investigated by immunohistochemistry. PDGFRA was increased in thymic carcinomas and PD-L1 in B3 thymomas and thymic carcinomas. In summary, our results reveal genetic differences between thymomas and thymic carcinomas and suggest potential novel targets for therapy.

Use of high-throughput protein array for profiling of differentially expressed proteins in normal and malignant breast tissue.

cDNA arrays provide a powerful tool to identify gene expression pattern that are potentially associated with tumor invasion and metastasis. However, genes work at the protein level and, since the transcriptional activity of a gene does not necessarily reflect cellular protein expression, the identification and quantification of proteins is essential for the understanding of molecular events leading to malignant transformation. We have therefore employed a high-throughput protein microarray system which contains 378 well-characterized monoclonal antibodies in order to compare the gene expression pattern of malignant and adjacent normal breast tissue in a patient with primary breast cancer. Using this technique, we have identified a number of proteins that show increased expression levels in malignant breast tissues such as casein kinase Ie, p53, annexin XI, CDC25C, eIF-4E and MAP kinase 7. The expression of other proteins, such as the multifunctional regulator 14-3-3e was found to be decreased in malignant breast tissue, whereas the majority of proteins remained unchanged when compared to the corresponding non-malignant samples. The protein expression pattern was confirmed by immunohistochemistry, in which antibodies against 8 representative proteins known to be involved in carcinogenesis were employed in paraffin-embedded normal and malignant tissue sections deriving from the same patient. In each case, the results obtained by IHC matched the data obtained by antibody microarray system. Taken together, we have described for the first time a tumor cell specificity protein expression pattern by use of a novel commercially available antibody microarray system. We have thus demonstrated the feasibility of high-throughput protein arrays in the proteomic analysis of human breast tissue. We hypothesize that the use of protein arrays will not only increase our understanding of the molecular events, but could prove useful in evaluating prognosis and in determining optimal antineoplastic therapy.