Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodelling genes

Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.

Up-regulation of the endothelial cell adhesion molecule intercellular adhesion molecule-1 (ICAM-1) by autoantibodies in autoimmune vasculitis

Autoimmune vasculitis is characterized by the presence of autoantibodies, particularly anti-neutrophil cytoplasmic antibodies (ANCA) and anti-nuclear antibodies (ANA), in patient sera. These autoantibodies have an incompletely understood role in development of vascular injury. The expression or up-regulation of cell adhesion molecules is an early phase in the development of an inflammatory vascular lesion. Autoantibody-positive sera from patients with vasculitis were assessed for their ability to modulate adhesion molecule expression by human umbilical vein endothelial cells (HUVEC). Autoantibody-positive serum samples from 11 out of 21 patients with primary vasculitis produced substantial up-regulation of ICAM-1 on HUVEC. Autoantibody-negative samples did not produce adhesion molecule up-regulation. Up-regulation of adhesion molecules on HUVEC was observed with samples positive for ANA, a phenomenon not previously reported. Preincubation of the sera with purified antigens recognized by ANCA failed to block this activation. In addition, MoAbs to ANCA antigens were ineffective at inducing ICAM-1 up-regulation, suggesting that activation is independent of the molecular specificity of the antibody. This capacity of ANCA- and ANA-positive sera to up-regulate adhesion molecules on endothelial cells may be a factor in the vessel wall inflammation seen in ANCA-associated vasculitis.

Overexpression of neurone glial-related cell adhesion molecule is an independent predictor of poor prognosis in advanced colorectal cancer

A downstream target of the Wnt pathway, neurone glial-related cell adhesion molecule (Nr-CAM) has recently been implicated in human cancer development. However, its role in colorectal cancer (CRC) pathobiology and clinical relevance remains unknown. In this study, we examined the clinical significance of Nr-CAM protein expression in a retrospective series of 428 CRCs using immunohistochemistry and tissue microarrays. Cox proportional hazards regression was used to calculate hazard ratios (HR) of mortality according to various clinicopathological features and molecular markers. All CRC samples were immunoreactive for Nr-CAM protein expression, compared to 10 / 245 (4%) matched normal tissue (P <0.0001). Of 428 CRC samples, 97 (23%) showed Nr-CAM overexpression, which was significantly associated with nodal (P = 0.012) and distant (P = 0.039) metastasis, but not with extent of local invasion or tumor size. Additionally, Nr-CAM overexpression was associated with vascular invasion (P = 0.0029), p53 expression (P = 0.036), and peritoneal metastasis at diagnosis (P = 0.013). In a multivariate model adjusted for other clinicopathological predictors of survival, Nr-CAM overexpression correlated with a significant increase in disease-specific (HR 1.66; 95% confidence interval 1.11-2.47; P = 0.014) and overall mortality (HR 1.57; 95% confidence interval 1.07-2.30; P = 0.023) in advanced but not early stage disease. Notably, 5-fluorouracil-based chemotherapy conferred significant survival benefit to patients with tumors negative for Nr-CAM overexpression but not to those with Nr-CAM overexpressed tumors. In conclusion, Nr-CAM protein expression is upregulated in CRC tissues. Nr-CAM overexpression is an independent marker of poor prognosis among advanced CRC patients, and is a possible predictive marker for non-beneficence to 5-fluorouracil- based chemotherapy.

p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase

p130(Cas) (crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130(Cas) suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130(Cas) is localized to focal adhesions. We demonstrate that p130(Cas) associates both in vitro and in vivo with pp125(FAK) (focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130(Cas) also associates with pp41/43(FRNK) (pp125(FAK)-related, non-kinase), an autonomously expressed form of pp125(FAK) composed of only the C-terminal noncatalytic domain. We show that the association of p130(Cas) with pp125(Fak) and pp41/43(FRNK) is direct, and is mediated by the binding of the SH3 domain of p130(Cas) to a proline-rich sequence present in both the C terminus of pp125(FAK) and in pp41/43(FRNK). In agreement with recent studies we show that p130(Cas) is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130(Cas) with pp125(FAK), a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.

Controlling Surface Topology and Functionality of Electrospun Fibers on the Nanoscale using Amphiphilic Block Copolymers To Direct Mesenchymal Progenitor Cell Adhesion

Surface patterning in three dimensions is of great importance in biomaterials design for controlling cell behavior. A facile one-step functionalization of biodegradable PDLLA fibers using amphiphilic diblock copolymers is demonstrated here to systematically vary the fiber surface composition. The copolymers comprise a hydrophilic poly[oligo(ethylene glycol) methacrylate] (POEGMA), poly[(2-methacryloyloxy)ethyl phosphorylcholine] (PMPC), or poly[2-(dimethylamino)ethyl methacrylate)] (PDMAEMA) block and a hydrophobic poly(l-lactide) (PLA) block. The block copolymer-modified fibers have increased surface hydrophilicity compared to that of PDLLA fibers. Mixtures of PLAPMPC and PLAPOEGMA copolymers are utilized to exploit microphase separation of the incompatible hydrophilic PMPC and POEGMA blocks at the fiber surface. Conjugation of an RGD cell-adhesive peptide to one hydrophilic block (POEGMA) using thiol-ene chemistry produces fibers with domains of cell-adhesive (POEGMA) and cell-inert (PMPC) sites, mimicking the adhesive properties of the extracellular matrix (ECM). Human mesenchymal progenitor cells (hES-MPs) showed much better adhesion to the fibers with surface-adhesive heterogeneity compared to that to fibers with only adhesive or only inert surface chemistries.

HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

What is the role of the cell adhesion protein N-cadherin in the molecular and morphological asymmetries of the Hensen’s node

Tese de doutoramento, Ciências Biomédicas (Ciências Morfológicas), Universidade de Lisboa, Faculdade de Medicina, 2014

Exploring Rac GTPase regulation : the molecular mechanisms governing the DOCK180 and ELMO interaction and the role of this complex in Rac-mediated cell migration

Les protéines DOCK180 et ELMO coopèrent ensemble biochimiquement et génétiquement afin d’activer la GTPase Rac1 lors de plusieurs évènements biologiques. Toutefois, le rôle que jouent ces protéines dans la signalisation par Rac est encore mal compris. Nous émettons l’hypothèse que Dock180 agit comme activateur de Rac, alors que ELMO est requis pour l’intégration de la signalisation de Rac plutôt que son activation per se. Nous postulons que ELMO agit comme signal de localisation intracellulaire afin de restreindre de façon spatio-temporelle la signalisation de Rac en aval de Dock180, et/ou que ELMO agit comme protéine d’échafaudage entre Rac et ses effecteurs pour amplifier la migration cellulaire. Dans l’objectif nº 1, nous démontrons que le domaine PH atypique de ELMO1 est le site d’interaction principal entre cette protéine et DOCK180. De plus, nous démontrons que la liaison entre ELMO et DOCK180 n’est pas nécessaire pour l’activation de Rac, mais est plutôt essentielle pour faciliter la réorganisation du cytosquelette induite par l’activation de Rac en aval de Dock180. Ces résultats impliquent que ELMO pourrait jouer des rôles additionnels dans la signalisation par Rac. Dans l’objectif nº 2, nous avons découvert l’existence d’une homologie structurelle entre ELMO et un module d’autorégulation de la formine Dia1, et avons identifié trois nouveaux domaines dans la protéine ELMO : les domaines RBD, EID et EAD. De façon analogue à Dia1, nous avons découvert que ELMO à l’état basal est autoinhibé grâce à des intéractions intramoléculaires. Nous proposons que l’état d’activation des protéines ELMO est régulé de façon similaire aux formines de la famille Dia, c’est-à-dire grâce à des interactions avec d’autres protéines. Dans l’objectif nº 3, nous identifions un domaine RBD polyvalent chez ELMO. Ce domaine possède une double spécificité pour les GTPases de la famille Rho et Arf. Nous avons découvert que Arl4A agit comme signal de recrutement membranaire pour le module ELMO/DOCK180/Rac. Nos résultats nous permettent de supposer que d’autres GTPases pourraient être impliquées dans l’activation et la localisation de cette voie de signalisation. Nous concluons qu’à l’état basal, ELMO et DOCK180 forment un complexe dans lequel ELMO est dans sa conformation autoinhibée. Bien que le mécanisme d’activation de ELMO ne soit pas encore bien compris, nous avons découvert que, lorsqu’il y a stimulation cellulaire, certaines GTPases liées au GTP peuvent intéragir avec le domaine RBD de ELMO pour relâcher les contacts intramoléculaires et/ou localiser le complexe à la membrane. Ainsi, les GTPases peuvent servir d’ancrage au complexe ELMO/DOCK180 pour assurer une regulation spatiotemporelle adequate de l’activation et de la signalisation de Rac.

Functionalised amyloid fibrils for roles in cell adhesion

We describe experiments designed to explore the possibility of using amyloid fibrils as new nanoscale biomaterials for promoting and exploiting cell adhesion, migration and differentiation in vitro. We created peptides that add the biological cell adhesion sequence (RGD) or a control sequence (RAD) to the C-terminus of an 11-residue peptide corresponding to residues 105-115 of the amyloidogenic protein transthyretin. These peptides readily self-assemble in aqueous solution to form amyloid fibrils, and X-ray fibre diffraction shows that they possess the same strand and sheet spacing in the characteristic cross-beta structure as do fibrils formed by the parent peptide. We report that the fibrils containing the RGD sequence are bioactive and that these fibrils interact specifically with cells via the RGD group displayed on the fibril surface. As the design of such functionalized fibrils can be systematically altered, these findings suggest that it will be possible to generate nanomaterials based on amyloid fibrils that are tailored to promote interactions with a wide variety of cell types. (c) 2007 Elsevier Ltd. All rights reserved.