Beta-catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse.

During heart development endocardial cells within the atrio-ventricular (AV) region undergo TGFbeta-dependent epithelial-mesenchymal transformation (EMT) and invade the underlying cardiac jelly. This process gives rise to the endocardial cushions from which AV valves and part of the septum originate. In this paper we show that in mouse embryos and in AV explants TGFbeta induction of endocardial EMT is strongly inhibited in mice deficient for endothelial beta-catenin, leading to a lack of heart cushion formation. Using a Wnt-signaling reporter mouse strain, we demonstrated in vivo and ex vivo that EMT in heart cushion is accompanied by activation of beta-catenin/TCF/Lef transcriptional activity. In cultured endothelial cells, TGFbeta2 induces alpha-smooth muscle actin (alphaSMA) expression. This process was strongly reduced in beta-catenin null cells, although TGFbeta2 induced smad phosphorylation was unchanged. These data demonstrate an involvement of beta-catenin/TCF/Lef transcriptional activity in heart cushion formation, and suggest an interaction between TGFbeta and Wnt-signaling pathways in the induction of endothelial-mesenchymal transformation.

Fluorescent in situ hybridization as a screening test for HER2 amplification in G2 and G3 breast cancers of lobular and ductal histotype and metastases.

The aim of the present study was to evaluate the effectiveness of fluorescence in situ hybridisation (FISH), as a screening test, in moderately- (G2) or poorly- (G3) differentiated breast cancers of the ductal (IDC) and lobular (ILC) histotypes and distant metastases. HER2 FISH was performed on 486 G2 and 477 G3 both of IDC and ILC histotypes and in 241 metastases. A significant difference in the HER2 amplification was observed between G2 (14.8%) and G3 (31.9%), with no difference according to the histotype. However, the rate of amplification increased to 36% in the G2/hormone receptor-negative cases as compared to 10.6% in the G2/receptor-positive cases (p<0.0001). HER2 was amplified in 17% of metastases with some differences depending on the location. These data suggest that the HER2 FISH analysis may be an effective screening test in breast cancer metastases and G3 tumors, irrespective of the hormone receptor status or presence of lymphovascular invasion.

Management of Italian patients with advanced non-small-cell lung cancer after second-line treatment: results of the longitudinal phase of the LIFE observational study.

INTRODUCTION/BACKGROUND: Patients with advanced NSCLC who experience disease progression after second-line therapy might receive further active treatment. LIFE was an Italian cohort multicenter observational study composed of a cross-sectional and a longitudinal phase. PATIENTS AND METHODS: In the longitudinal phase, described here, the primary aim was to determine the proportion of patients receiving third-line therapy among those who received second-line active treatment according to clinical practice. The proportion of patients receiving further treatment lines was also estimated. RESULTS: The longitudinal phase was conducted between January and August 2012. Of 464 patients who began second-line therapy outside of clinical trials within the baseline evaluation, 56 (12.1%) were still receiving second-line therapy at the end of the observation period and 17 (3.7%) withdrew during or after second-line therapy. Of the remaining 391 patients, 158 (40.4%) received third-line treatment outside of clinical trials: 93 received a third-line chemotherapy and 65 a targeted agent. The main reason for interrupting third-line treatment was disease progression or death. During the same observation period, 25 of 113 patients who completed a third-line therapy received a fourth line of treatment. From diagnosis of NSCLC to the end of observation, biomarkers were tested in 323 patients (59.7%): epidermal growth factor receptor mutations in 315 (58.2%), Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutations in 83 (15.3%) and Anaplastic lymphoma kinase (ALK) translocation in 84 (15.5%). CONCLUSION: In Italian clinical practice, the proportion of patients with advanced NSCLC receiving more than 2 treatment lines of therapy is not negligible.

Advanced non-small cell lung cancer management in patients progressing after first-line treatment: results of the cross-sectional phase of the Italian LIFE observational study.

PURPOSE: LIFE (non-small cell Lung cancer management In patients progressing after First-linE of treatment in the metastatic setting) is a multicentre Italian observational study, including a cross-sectional and a longitudinal phase, with the aim of describing the therapeutic approach in clinical practice for advanced non-small cell lung cancer (NSCLC) patients, progressing after first-line treatment. METHODS: In this paper, the cross-sectional phase is outlined, with the primary endpoint of describing the proportion of patients receiving second-line treatment among those progressed during or after first-line treatment according to clinical practice. RESULTS: From July 2011 to January 2012, 603 patients were enrolled and 541 (90 %) were evaluable. A total of 464 (86 %) patients received a second-line therapy outside clinical trials. Chemotherapy and targeted therapies were administered to 65 and 34 % of patients, respectively (1 % both). No tissue collection was required within the observational trial, and biomarkers analysis was performed at diagnosis or later in 314 patients (58 %). In details, activating epidermal growth factor receptor mutations were detected in 21 % of 311 evaluable patients, Kirsten rat sarcoma 2 viral oncogene homolog mutation in 22 % of the 77 evaluable patients and anaplastic lymphoma kinase translocations analysis was performed in 74 patients and resulted positive in 23 % of cases. These high proportions were probably due to enriched patient population tested. CONCLUSIONS: These results showed a pattern of care for NSCLC second-line therapy which reflects international guidelines recommendations and current expected clinical practice. Interestingly, biomarkers analyses were performed in a higher percentage than expected.

Docetaxel versus paclitaxel for antiangiogenesis.

Cytoskeleton-toxic chemotherapeuticals, such as vinblastine and paclitaxel, display antiangiogenic activity. This study was designed to compare paclitaxel to its analog docetaxel and assess their doses still antiangiogenic in vitro and in vivo. Human endothelial cell functions involved in angiogenesis, namely proliferation, chemotaxis, morphogenesis, and secretion of matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator (uPA) were studied in vitro upon exposure to docetaxel and paclitaxel, whereas their effect on angiogenesis was studied in vivo by using the chick embryo chorioallantoic membrane (CAM) model. Proliferation of mouse embryo fibroblasts and human Kaposi's sarcoma, breast and endometrial carcinoma, and lymphoid tumor cells was also studied. In vitro, 0.5, 0.75, and 1 nM docetaxel and 2, 3, and 4 nM paclitaxel, i.e., non-cytotoxic doses, impacted all endothelial cell functions, but not protease secretion, in a dose-dependent fashion, whereas they did not affect the proliferation of other cells, except those of Kaposi's sarcoma. No apoptosis was induced by 0.5 nM docetaxel and 2 nM paclitaxel, and moderate apoptosis was induced by 1 nM docetaxel and 4 nM paclitaxel. The antiangiogenic effect rapidly disappeared on drug suspension and was accompanied ultrastructurally by thin lesions of cytoskeleton in the form of slight and equally reversible depolymerization and accumulation of microfilaments. Massive endothelial cell apoptosis with evident cytotoxicity and irreversibility were associated with 2 nM docetaxel and 5 nM paclitaxel, although these higher doses were ineffective on other cells except Kaposi's sarcoma cells. In vivo, 1, 2, and 3 nM docetaxel and 4, 8, and 12 nM paclitaxel displayed a dose-dependent antiangiogenic activity. We suggest that very low docetaxel and paclitaxel doses selectively cause organic and functional damage of endothelial cells and that docetaxel is four times stronger. Their antiangiogenic activity could be applied to treat Kaposi's sarcoma and cancers.

The mouse aorta model: influence of genetic background and aging on bFGF- and VEGF-induced angiogenic sprouting.

Angiogenesis can be studied ex vivo by culturing rat or mouse aortic rings in collagen gels. Unlike rat aorta explants, unstimulated mouse aortic rings were unable to spontaneously produce an angiogenic response under serum-free conditions. They, however, responded to bFGF and VEGF, generating networks of branching neovessels. Aortic rings from GFP-Tie2-transgenic mice generated GFP-labeled neovessels that could be easily identified by their distinctly green fluorescence. Aortic rings from 1- to 2-month-old mice produced microvessels faster, more uniformly and in greater number than aortic rings from 6- to 10-month-old mice, particularly in VEGF-treated cultures. Aortic rings from 129/SVJ mice were capable of a much stronger and sustained angiogenic response to bFGF than those of C57BL/6 or BALB/c mice, which were in turn more angiogenic than aortic rings from FVB mice. The same strains of mice responded differently to VEGF, as C57BL/6 mouse aortic rings produced more microvessels than those of BALB/c, FVB, and 129/SVJ mice, which were capable of only a limited response. The significant impact that aging and genetic background have on mouse aortic angiogenesis should be taken into account when the aortic-ring assay is used to evaluate function of genes that have been deleted or overexpressed in genetically modified mice.

Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins.

Recent studies have implicated the Tie2 tyrosine-kinase receptor and its main ligands--angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2)--as crucial regulators of mural cell recruitment during angiogenesis. Angiopoietin-mediated activation of Tie2 promotes perivascular mural cell assembly, but the mechanisms regulating this process are poorly understood because differentiated mural cells do not have the Tie2 receptor, which is reportedly expressed only in endothelial cells. There is also no direct evidence that Tie2 activation results in production of mural cell chemoattractants by the endothelium. In the rat aorta model of angiogenesis, developing microvessels recruit mural cells from the intimal/subintimal layers of the aortic wall. Ang-1 and Ang-2 promote angiogenesis in this system, stimulating branching morphogenesis and mural cell assembly. Mural precursor cells (MPCs) isolated with a nonenzymatic method from the intimal aspect of the rat aorta were positive for smooth muscle cell markers (alpha-smooth muscle actin and calponin) and negative for endothelial markers (factor-VIII-related antigen and CD31). These cells responded chemotactically to Ang-1 and Ang-2, and secreted MMP-2 when treated with these factors. Western-blot analysis, immunocytochemistry and RT-PCR demonstrated that MPCs express the Tie2 receptor. Immunoprecipitation showed phosphorylation of MPC Tie2 on tyrosine residues upon stimulation with Ang-1 or Ang-2. Surface expression of Tie2 was further demonstrated by isolating Tie2+/alpha-smooth muscle actin+ MPCs from primary aortic outgrowths with anti-Tie2-IgG-coated magnetic beads. Immunostaining of the rat aorta confirmed expression of Tie2 not only in endothelial cells but also in nonendothelial mesenchymal cells located in the aortic intimal/subintimal layers, which are the source of MPCs. These data indicate that the aortic wall contains Tie2+ nonendothelial mesenchymal cells and suggest that Tie2-related recruitment of mural cells during angiogenesis may occur through angiopoietin-mediated direct stimulation of these cells.

VE-cadherin expression and clustering maintain low levels of survivin in endothelial cells.

Survivin is strongly expressed in embryonic organs and in tumor cells but is low or absent in differentiated normal tissues. Resting endothelium expresses low levels of survivin but can up-regulate its synthesis on activation to proliferate. The mechanisms responsible for survivin down-regulation in resting conditions are still unknown. We report here that confluence and vascular endothelial-cadherin (VE-cadherin) expression induce contact inhibition of cell growth and survivin down-regulation in the endothelium. Using beta-catenin null and positive isogenic endothelial cell lines we found that the effect requires beta-catenin expression and its association to VE-cadherin cytoplasmic tail. Furthermore, in allantois organ cultures, survivin expression is up-regulated in areas of growing vessels where VE-cadherin is partially dismantled from junctions or in VE-cadherin -/- specimens. Overall, these data indicate that VE-cadherin and beta-catenin may negatively regulate survivin synthesis in endothelial cells. Consistently, in epidermal and pancreatic cell lines or ovarian tumors, epithelial-cadherin (E-cadherin) and survivin expression is inversely related, suggesting a non-cell-specific role of cadherins in reducing survivin synthesis.