Oncosomes are tumor-derived microvesicles that transmit signaling complexes between cell and tissue compartments. Herein, we show that amoeboid tumor cells export large (1- to 10-μm diameter) vesicles, derived from bulky cellular protrusions, that contain metalloproteinases, RNA, caveolin-1, and the GTPase ADP-ribosylation factor 6, and are biologically active toward tumor cells, endothelial cells, and fibroblasts. We describe methods by which large oncosomes can be selectively sorted by flow cytometry and analyzed independently of vesicles <1 μm. Structures resembling large oncosomes were identified in the circulation of different mouse models of prostate cancer, and their abundance correlated with tumor progression. Similar large vesicles were also identified in human tumor tissues, but they were not detected in the benign compartment. They were more abundant in metastases. Our results suggest that tumor microvesicles substantially larger than exosome-sized particles can be visualized and quantified in tissues and in the circulation, and isolated and characterized using clinically adaptable methods. These findings also suggest a mechanism by which migrating tumor cells condition the tumor microenvironment and distant sites, thereby potentiating advanced disease.

No abstract

Axl is a tyrosine kinase receptor that was first identified as a transforming gene in human myeloid leukemia. Recent converging evidence suggests its implication in cancer progression and invasion for several solid tumors, including lung, breast, brain, thyroid, and pancreas. In the last decade, Axl has thus become an attractive target for therapeutic development of more aggressive cancers. An emerging class of therapeutic inhibitors is now represented by short nucleic acid aptamers. These molecules act as high affinity ligands with several advantages over conventional antibodies for their use in vivo, including their small size and negligible immunogenicity. Furthermore, these molecules can easily form conjugates able to drive the specific delivery of interfering RNAs, nanoparticles, or chemotherapeutics. We have thus generated and characterized a selective RNA-based aptamer, GL21.T that binds the extracellular domain of Axl at high affinity (12 nmol/l) and inhibits its catalytic activity. GL21.T blocked Axl-dependent transducing events in vitro, including Erk and Akt phosphorylation, cell migration and invasion, as well as in vivo lung tumor formation in mice xenografts. In this respect, the GL21.T aptamer represents a promising therapeutic molecule for Axl-dependent cancers whose importance is highlighted by the paucity of available Axl-specific inhibitory molecules.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the wellcharacterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, ␤-dystroglycan, ␣-dystroglycan, and ␣-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Duchenne muscular dystrophy (DMD) is one of the most prevalent and severe inherited diseases of childhood, characterized by progressive muscular wasting and weakness. The deficient gene product, dystrophin, 1 is a peripheral membrane protein of ϳ426 kd, which is expressed in muscle tissues and the brain. At the plasma membrane, dystrophin associates with a large multimeric complex, termed the dystrophin-glycoprotein complex (DGC). 2 The DGC is composed of two subcomplexes: the dystroglycan complex (␣ and ␤ subunits) and the sarcoglycan complex (␣, ␤, ␥, and ␦ subunits). The Nterminal region of dystrophin interacts directly with the cytoskeletal protein actin, while the dystrophin C-terminal domain binds to the plasma membrane through interactions with ␤-dystroglycan. As such, dystrophin is thought

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The solid-pseudopapillary tumor of the pancreas (SPTP) is an unusual low-grade malignant epithelial tumor affecting predominantly adolescent girls and young women. Although approximately 500 cases of SPTP have been described in the last 40 yr, its pathogenesis remains uncertain. However, the clinical features of this neoplasm are very characteristic and SPTP must be suspected in any young woman with a cystic or partially cystic pancreatic mass. In this report, we describe the cytologic features of seven cases of SPTP investigated by preoperative fine-needle aspirates. The analysis of the cytologic features in these cases and in 43 cases collected from the literature reveals that they are highly characteristic and quite distinct from those of other cystic or solid tumors of the pancreas. On this basis, a cytologic diagnosis of SPTP may be rendered with great confidence, not only in clinically typical examples, but also in unusual presentations, such as in older patients, in males, in ectopic locations, and in metastatic sites.

Human glioblastoma is the most frequent and aggressive form of brain tumour in the adult population. Proteolytic turnover of tumour suppressors by the ubiquitin–proteasome system is a mechanism that tumour cells can adopt to sustain their growth and invasiveness. However, the identity of ubiquitin–proteasome targets and regulators in glioblastoma are still unknown. Here we report that the RING ligase praja2 ubiquitylates and degrades Mob, a core component of NDR/LATS kinase and a positive regulator of the tumour-suppressor Hippo cascade. Degradation of Mob through the ubiquitin–proteasome system attenuates the Hippo cascade and sustains glioblastoma growth in vivo. Accordingly, accumulation of praja2 during the transition from low- to high-grade glioma is associated with significant downregulation of the Hippo pathway. These findings identify praja2 as a novel upstream regulator of the Hippo cascade, linking the ubiquitin proteasome system to deregulated glioblastoma growth.

Background-Helicobacter pylori, the main cause of chronic gastritis, is a class I gastric carcinogen. Chronic gastritis progresses to cancer through atrophy, metaplasia, and dysplasia. Precancerous phenotypic expression is generally associated with acquired genomic instability. Aim-To evaluate the eVect of H pylori infection and its eradication on gastric histology, cell proliferation, DNA status, and oncogene expression. Methods/Subjects-Morphometric and immunohistochemical techniques were used to examine gastric mucosal biopsy specimens from eight controls, 10 patients with H pylori negative chronic gastritis, 53 with H pylori positive chronic gastritis, and 11 with gastric cancer. Results-All patients with chronic gastritis were in a hyperproliferative state related to mucosal inflammation, regardless of H pylori infection. Atrophy was present in three of 10 patients with H pylori negative chronic gastritis and in 26 of 53 with H pylori positive chronic gastritis, associated in 18 with intestinal metaplasia. DNA content was abnormal in only 11 patients with atrophy and H pylori infection; eight of these also had c-Myc expression, associated in six cases with p53 expression. Fifty three patients with H pylori positive chronic gastritis were monitored for 12 months after antibiotic treatment: three dropped out; infection was eradicated in 45, in whom cell proliferation decreased in parallel with the reduction in gastritis activity; atrophy previously detected in 21/45 disappeared in five, regressed from moderate to mild in nine, and remained unchanged in seven; complete metaplasia disappeared in 4/14, and markers of genomic instability disappeared where previously present. In the five patients in whom H pylori persisted, atrophy, metaplasia, dysplasia, and markers of genomic instability remained unchanged. Conclusions-Chronic H pylori infection seems to be responsible for genomic instability in a subset of cases of H pylori positive chronic atrophic gastritis; eradication of H pylori infection can reverse inflammation and the related atrophy, metaplasia, and genomic instability. (Gut 1999;44:789-799)