ADAM9 is highly expressed in renal cell cancer and is associated with tumour progression

<p>Abstract</p> <p>Background</p> <p><b>A D</b>isintegrin <b>A</b>nd <b>M</b>etalloprotease (ADAM) 9 has been implicated in tumour progression of various solid tumours, however, little is known about its role in renal cell carcinoma. We evaluated the expression of ADAM9 on protein and transcript level in a clinico-pathologically characterized renal cell cancer cohort.</p> <p>Methods</p> <p>108 renal cancer cases were immunostained for ADAM9 on a tissue-micro-array. For 30 additional cases, ADAM9 mRNA of microdissected tumour and normal tissue was analyzed via quantitative RT-PCR. SPSS 14.0 was used to apply crosstables (Fisher's exact test and χ²-test), correlations and univariate as well as multivariate survival analyses.</p> <p>Results</p> <p>ADAM9 was significantly up-regulated in renal cancer in comparison to the adjacent normal tissue on mRNA level. On protein level, ADAM9 was significantly associated with higher tumour grade, positive nodal status and distant metastasis. Furthermore, ADAM9 protein expression was significantly associated with shortened patient survival in the univariate analysis.</p> <p>Conclusion</p> <p>ADAM9 is strongly expressed in a large proportion of renal cell cancers, concordant with findings in other tumour entities. Additionally, ADAM9 expression is significantly associated with markers of unfavourable prognosis. Whether the demonstrated prognostic value of ADAM9 is independent from other tumour parameters will have to be verified in larger study cohorts.</p

ADAM15 mediates upregulation of Claudin-1 expression in breast cancer cells

A Disintegrin and Metalloproteinase-15 (ADAM15) is a transmembrane protein involved in protein ectodomain shedding, cell adhesion and signalling. We previously cloned and characterised alternatively spliced variants of ADAM15 that differ in their intracellular domains and demonstrated correlation of the expression of specific variants with breast cancer prognosis. In this study we have created isogenic cell panels (MDA-MB-231 and MCF-7) expressing five ADAM15 variants including wildtype and catalytically inactive forms. The expression of ADAM15 isoforms in MDA-MB-231 cells led to cell clustering to varying degree, without changes in EMT markers vimentin, slug and E-cadherin. Analysis of tight junction molecules revealed ADAM15 isoform specific, catalytic function dependent upregulation of Claudin-1. The expression of ADAM15A, and to a lesser degree of C and E isoforms led to an increase in Claudin-1 expression in MDA-MB-231 cells, while ADAM15B had no effect. In MCF-7 cells, ADAM15E was the principal variant inducing Claudin-1 expression. Sh-RNA mediated down-regulation of ADAM15 in ADAM15 over-expressing cells reduced Claudin-1 levels. Additionally, downregulation of endogenous ADAM15 expression in T47D cells by shRNA reduced endogenous Claudin-1 expression confirming a role for ADAM15 in regulating Claudin-1 expression. The PI3K/Akt/mTOR pathway was involved in regulating Claudin-1 expression downstream of ADAM15. Immunofluorescence analysis of MDA-MB-231 ADAM15A expressing cells showed Claudin-1 at cell-cell junctions, in the cytoplasm and nuclei. ADAM15 co-localised with Claudin-1 and ZO1 at cell-cell junctions. Immunoprecipitation analysis demonstrated complex formation between ADAM15 and ZO1/ZO2. These findings highlight the importance of ADAM15 Intra Cellular Domain-mediated interactions in regulating substrate selection and breast cancer cell phenotype

Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells

BACKGROUND: Astrocytomas are the most common type of primary central nervous system tumors. They are frequently associated with genetic mutations that deregulate cell cycle and render these tumors resistant to apoptosis. STAT3, signal transducer and activator of transcription 3, participates in several human cancers by inducing cell proliferation and inhibiting apoptosis and is frequently activated in astrocytomas. METHODS: RNA interference was used to knockdown STAT3 expression in human astrocytes and astrocytoma cell lines. The effect of STAT3 knockdown on apoptosis, cell proliferation, and gene expression was then assessed by standard methods. RESULTS: We have found that STAT3 is constitutively activated in several human astrocytoma cell lines. Knockdown of STAT3 expression by siRNA induces morphologic and biochemical changes consistent with apoptosis in several astrocytoma cell lines, but not in primary human astrocytes. Moreover, STAT3 is required for the expression of the antiapoptotic genes survivin and Bcl-xL in the A172 glioblastoma cell line. CONCLUSION: These results show that STAT3 is required for the survival of some astrocytomas. These studies suggest STAT3 siRNA could be a useful therapeutic agent for the treatment of astrocytomas

Identification and Classification of Conserved RNA Secondary Structures in the Human Genome

The discoveries of microRNAs and riboswitches, among others, have shown functional RNAs to be biologically more important and genomically more prevalent than previously anticipated. We have developed a general comparative genomics method based on phylogenetic stochastic context-free grammars for identifying functional RNAs encoded in the human genome and used it to survey an eight-way genome-wide alignment of the human, chimpanzee, mouse, rat, dog, chicken, zebra-fish, and puffer-fish genomes for deeply conserved functional RNAs. At a loose threshold for acceptance, this search resulted in a set of 48,479 candidate RNA structures. This screen finds a large number of known functional RNAs, including 195 miRNAs, 62 histone 3′UTR stem loops, and various types of known genetic recoding elements. Among the highest-scoring new predictions are 169 new miRNA candidates, as well as new candidate selenocysteine insertion sites, RNA editing hairpins, RNAs involved in transcript auto regulation, and many folds that form singletons or small functional RNA families of completely unknown function. While the rate of false positives in the overall set is difficult to estimate and is likely to be substantial, the results nevertheless provide evidence for many new human functional RNAs and present specific predictions to facilitate their further characterization

Peripheral T-lymphocytes express WNT7A and its restoration in leukemia-derived lymphoblasts inhibits cell proliferation

<p>Abstract</p> <p>Background</p> <p>WNT7a, a member of the Wnt ligand family implicated in several developmental processes, has also been reported to be dysregulated in some types of tumors; however, its function and implication in oncogenesis is poorly understood. Moreover, the expression of this gene and the role that it plays in the biology of blood cells remains unclear. In addition to determining the expression of the <it>WNT7A </it>gene in blood cells, in leukemia-derived cell lines, and in samples of patients with leukemia, the aim of this study was to seek the effect of this gene in proliferation.</p> <p>Methods</p> <p>We analyzed peripheral blood mononuclear cells, sorted CD3 and CD19 cells, four leukemia-derived cell lines, and blood samples from 14 patients with Acute lymphoblastic leukemia (ALL), and 19 clinically healthy subjects. Reverse transcription followed by quantitative Real-time Polymerase chain reaction (qRT-PCR) analysis were performed to determine relative <it>WNT7A </it>expression. Restoration of WNT7a was done employing a lentiviral system and by using a recombinant human protein. Cell proliferation was measured by addition of WST-1 to cell cultures.</p> <p>Results</p> <p>WNT7a is mainly produced by CD3 T-lymphocytes, its expression decreases upon activation, and it is severely reduced in leukemia-derived cell lines, as well as in the blood samples of patients with ALL when compared with healthy controls (<it>p </it>≤0.001). By restoring <it>WNT7A </it>expression in leukemia-derived cells, we were able to demonstrate that WNT7a inhibits cell growth. A similar effect was observed when a recombinant human WNT7a protein was used. Interestingly, restoration of <it>WNT7A </it>expression in Jurkat cells did not activate the canonical Wnt/β-catenin pathway.</p> <p>Conclusions</p> <p>To our knowledge, this is the first report evidencing quantitatively decreased <it>WNT7A </it>levels in leukemia-derived cells and that <it>WNT7A </it>restoration in T-lymphocytes inhibits cell proliferation. In addition, our results also support the possible function of <it>WNT7A </it>as a tumor suppressor gene as well as a therapeutic tool.</p

ADAM15 mediates upregulation of Claudin-1 expression in breast cancer cells

A Disintegrin and Metalloproteinase-15 (ADAM15) is a transmembrane protein involved in protein ectodomain shedding, cell adhesion and signalling. We previously cloned and characterised alternatively spliced variants of ADAM15 that differ in their intracellular domains and demonstrated correlation of the expression of specific variants with breast cancer prognosis. In this study we have created isogenic cell panels (MDA-MB-231 and MCF-7) expressing five ADAM15 variants including wild-type and catalytically inactive forms. The expression of ADAM15 isoforms in MDA-MB-231 cells led to cell clustering to varying degree, without changes in EMT markers vimentin, slug and E-cadherin. Analysis of tight junction molecules revealed ADAM15 isoform specific, catalytic function dependent upregulation of Claudin-1. The expression of ADAM15A, and to a lesser degree of C and E isoforms led to an increase in Claudin-1 expression in MDA-MB-231 cells, while ADAM15B had no effect. In MCF-7 cells, ADAM15E was the principal variant inducing Claudin-1 expression. Sh-RNA mediated down-regulation of ADAM15 in ADAM15 over-expressing cells reduced Claudin-1 levels. Additionally, downregulation of endogenous ADAM15 expression in T47D cells by shRNA reduced endogenous Claudin-1 expression confirming a role for ADAM15 in regulating Claudin-1 expression. The PI3K/Akt/mTOR pathway was involved in regulating Claudin-1 expression downstream of ADAM15. Immunofluorescence analysis of MDA-MB-231 ADAM15A expressing cells showed Claudin-1 at cell-cell junctions, in the cytoplasm and nuclei. ADAM15 co-localised with Claudin-1 and ZO1 at cell-cell junctions. Immunoprecipitation analysis demonstrated complex formation between ADAM15 and ZO1/ZO2. These findings highlight the importance of ADAM15 Intra Cellular Domain-mediated interactions in regulating substrate selection and breast cancer cell phenotype