Luigi Insabato

Large Oncosomes in Human Prostate Cancer Tissues and in the Circulation of Mice with Metastatic Disease

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.

Effect of Helicobacter pylori infection and its eradication on cell proliferation, DNA status, and oncogene expression in patients with chronic gastritis

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 effect 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 withH pylori negative chronic gastritis, 53 withH 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 pyloriinfection. Atrophy was present in three of 10 patients withH 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 withH 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 whomH pylori persisted, atrophy, metaplasia, dysplasia, and markers of genomic instability remained unchanged. CONCLUSIONS ChronicH pylori infection seems to be responsible for genomic instability in a subset of cases of H pylori positive chronic atrophic gastritis; eradication ofH pylori infection can reverse inflammation and the related atrophy, metaplasia, and genomic instability.

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins

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 well-characterized 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, beta-dystroglycan, alpha-dystroglycan, and alpha-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. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

Loss of the tumor suppressor gene PTEN marks the transition from intratubular germ cell neoplasias (ITGCN) to invasive germ cell tumors

PTEN/MMAC1/TEP1 (hereafter PTEN) is a tumor suppressor gene (located at 10q23) that is frequently mutated or deleted in sporadic human tumors. PTEN encodes a multifunctional phosphatase, which negatively regulates cell growth, migration and survival via the phosphatidylinositol 3′-kinase/AKT signalling pathway. Accordingly, Pten+/− mice develop various types of tumors including teratocarcinomas and teratomas. We have investigated PTEN expression in 60 bioptic specimens of germ cell tumors (32 seminomas, 22 embryonal carcinomas and six teratomas) and 22 intratubular germ cell neoplasias (ITGCN) adjacent to the tumors for PTEN protein and mRNA expression. In total, 10 testicular biopsies were used as controls. In the testis, PTEN was abundantly expressed in germ cells whereas it was virtually absent from 56% of seminomas as well as from 86% of embryonal carcinomas and virtually all teratomas. On the contrary, ITGCN intensely expressed PTEN, indicating that loss of PTEN expression is not an early event in testicular tumor development. The loss of PTEN expression occurs mainly at the RNA level as determined by in situ hybridization of cellular mRNA (17/22) but also it may involve some kind of post-transcriptional mechanisms in the remaining 25% of cases. Analysis of microsatellites D10S551, D10S541 and D10S1765 in GCTs (n=22) showed LOH at the PTEN locus at 10q23 in at least 36% of GCTs (three embryonal carcinoma, three seminoma, two teratoma); one seminoma and one embryonal (9%) carcinoma presented an inactivating mutation in the PTEN gene (2/22). Finally, we demonstrated that the phosphatidylinositol 3′-kinase/AKT pathway, which is regulated by the PTEN phosphatase, is crucial in regulating the proliferation of the NT2/D1 embryonal carcinoma cells, and that the cyclin-dependent kinase inhibitor p27kip1 is a key downstream target of this pathway.

Targeting Axl With an High-affinity Inhibitory Aptamer

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.

Patterns of gene amplification in gastrointestinal stromal tumors (GIST).

Gastrointestinal stromal tumors (GIST) are the most common primary mesenchymal tumors of the gastrointestinal tract (GIT). They represent a wide clinico-pathological spectrum of tumors. No single histological or clinical parameter can predict the prognosis while the response to therapy is related to the type of KIT or PDGFRA mutation. Cytogenetic and CGH studies have identified frequent gross chromosomal aberrations but the target genes of these changes are unknown. To determine whether known oncogenes take part in genomic rearrangements and to investigate the potential clinical significance of their amplifications, nine known oncogenes (CMYC, MDM2, GLI1, CDK4, HER2, EGFR1, CCND1, FGF3, EMS) were analyzed by fluorescent in situ hybridization (FISH) on a tissue microarray (TMA) containing 94 primary GIST. Clinical follow-up information was available for 57 of these patients. Amplification was found for CMYC in three of 90 (3.3%), for MDM2 in five of 94 (5.3%), for EGFR1 in five of 94 (5.3%), and for CCND1 in seven of 79 (8.9%) evaluable cases. No amplifications were seen for HER2, GLI1, CDK4, FGF3, and EMS. Amplifications of MDM2 and CCND1 were associated with clinical and histological malignancy. In conclusion, our data show that gene amplification does occur in a subset of GIST. Identification of MDM2/CCND1 amplification may represent another molecular feature that could help in the evaluation of the behavior of GISTs.

Primary monotypic epithelioid angiomyolipoma of bone.

Primary monotypic epithelioid angiomyolipoma of bone

Proteolysis of MOB1 by the ubiquitin ligase praja2 attenuates Hippo signalling and supports glioblastoma growth

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.

The Levels of the Endocannabinoid Receptor CB2 and Its Ligand 2-Arachidonoylglycerol Are Elevated in Endometrial Carcinoma

The endocannabinoid system plays protective roles against the growth and the spreading of several types of carcinomas. Because estrogens regulate this system both in physiological states and cancer, in this paper we evaluated its involvement in endometrial carcinoma, a well-known estrogen-dependent tumor. To test whether the endocannabinoid system is expressed in endometrial cancer, tissue samples were collected both from 18 patients undergoing surgical treatment for endometrial adenocarcinoma and 16 healthy age-matched controls, and treated for Western blot and immunohistochemical analysis. Moreover, tissues were dounce homogenized and submitted to endocannabinoid measurement by liquid chromatography-mass spectrometry. To evaluate the physiological role of the endocannabinoid system, a human endometrial cancer cell-line (AN3CA) was used and transiently transfected with a plasmid containing the cDNA for the endocannabinoid receptor CB(2). Cells were incubated for 48 h with an agonist (JWH133) (10 mum) or antagonist (SR144528) (1 mum) of CB(2) 24 h after transfection, and cell proliferation was measured by the 3-[4,5-dimethyltiazol-2yl]-2,5 diphenyltetrazolium bromide formazan assay. In human endometrial carcinoma biopsies the expression of CB(2) receptor and the levels of its ligand, 2-arachidonoylglycerol increased, whereas monoacylglycerol lipase, an enzyme responsible for 2-arachidonoylglycerol degradation, was down-regulated. Immunohistochemical analysis revealed that CB(2) was overexpressed only in malignant endometrial cells. CB(2)-overexpressing AN3CA cells showed a significant reduction in cell vitality compared with parental AN3CA cells: incubation with the selective CB(2) antagonist SR144128 restored the viability of CB(2)-overexpressing cells to that of untransfected cells. In conclusion, the endocannabinoid system seems to play an important role in human endometrial carcinoma, and modulation of CB(2) activity/expression may account for a tumor-suppressive effect.

Malignant Gastrointestinal Leiomyosarcoma and Gastrointestinal Stromal Tumor With Prominent Osteoclast-like Giant Cells

CONTEXT One case of leiomyosarcoma and one case of gastrointestinal stromal tumor with prominent osteoclast-like giant cells have so far been reported in the digestive tract. OBJECTIVE To ascertain the clinicopathologic features and biologic behavior of these tumors, we report 3 additional cases of leiomyosarcoma of the gastrointestinal tract and one malignant gastrointestinal stromal tumor. DESIGN Histologic and immunohistochemical examinations were performed. Clinical and follow-up data were recorded, and the literature was reviewed. RESULTS The age of the patients ranged from 50 to 68 years (mean, 62 years). One of the lesions arose in the stomach, one in the ileum, and 2 in the colon. Three tumors showed a strong positivity for muscle actin and desmin and were diagnosed as leiomyosarcomas, 2 of them showing spindle cells and 1 of them showing epithelioid cells. The fourth tumor reacted strongly positive for c-Kit (CD117) and vimentin, and it was diagnosed as an epithelioid malignant gastrointestinal stromal tumor. All tumors were characterized by numerous osteoclast-like giant cells that were unevenly distributed and that, using immunohistochemistry, reacted strongly with CD68. CONCLUSIONS Malignant stromal tumors with osteoclast-like giant cells of the gastrointestinal tract are rare entities, are more commonly of a myogenic origin such as leiomyosarcoma, and seem to have an aggressive behavior.