Luba Roncari

Regulation of Planar Cell Polarity by Smurf Ubiquitin Ligases

Planar cell polarity (PCP) is critical for morphogenesis in metazoans. PCP in vertebrates regulates stereocilia alignment in neurosensory cells of the cochlea and closure of the neural tube through convergence and extension movements (CE). Noncanonical Wnt morphogens regulate PCP and CE in vertebrates, but the molecular mechanisms remain unclear. Smurfs are ubiquitin ligases that regulate signaling, cell polarity and motility through spatiotemporally restricted ubiquitination of diverse substrates. Here, we report an unexpected role for Smurfs in controlling PCP and CE. Mice mutant for Smurf1 and Smurf2 display PCP defects in the cochlea and CE defects that include a failure to close the neural tube. Further, we show that Smurfs engage in a noncanonical Wnt signaling pathway that targets the core PCP protein Prickle1 for ubiquitin-mediated degradation. Our work thus uncovers ubiquitin ligases in a mechanistic link between noncanonical Wnt signaling and PCP/CE.

Expression of activated epidermal growth factor receptors, Ras-guanosine triphosphate, and mitogen-activated protein kinase in human glioblastoma multiforme specimens.

OBJECTIVE Amplification of the epidermal growth factor receptor (EGFR) is a common event in the molecular pathogenesis of high-grade astrocytic tumors, occurring in 50% of glioblastoma multiforme (GBM) cases. A subset of GBMs also express a constitutively phosphorylated truncated receptor (EGFRvIII). Expression of transfected EGFRvIII in cells has been reported to activate the Ras-mitogen-activated protein kinase pathway and to provide a growth advantage. Novel therapeutic agents targeting signal transduction pathways are entering early clinical trials; determination of which GBMs express EGFRvIII might help identify patients who might benefit from these biological agents. METHODS A cohort of 15 flash-frozen surgical specimens (12 GBMs, 2 gliosarcomas, and 1 adult low-grade glioma) were evaluated for EGFR and EGFRvIII expression and for EGFR activation status using immunohistochemical (IHC) analysis, Western blotting, and reverse transcription-polymerase chain reaction assays. Levels of activated Ras-guanosine triphosphate were measured using a nonradioactive luciferase-based technique. Mitogen-activated protein kinase activation was determined using a myelin basic protein assay. IHC analysis was performed on paraffin-embedded, formalin-fixed, pathological specimens. Normal control samples included white matter specimens distal to tumors (n = 5), a sample obtained during a lobectomy for treatment of epilepsy (n = 1), and cultured fetal human astrocytes (n = 1). RESULTS We demonstrated higher levels of activated Ras and mitogen-activated protein kinase in GBM specimens, compared with normal brain tissue or the low-grade glioma. There was a very good correlation between results obtained using specialized molecular techniques and those obtained using routine IHC techniques. Screening for EGFRvIII expression may be of prognostic importance, because patients with EGFRvIII-positive tumors exhibited shorter life expectancies (mean survival time for patients with EGFRvIII-positive tumors, 4.5 +/- 0.6 mo; mean survival time for patients with EGFRvIII-negative tumors, 11.2 +/- 0.9 mo). CONCLUSION We demonstrated that routine IHC techniques using commercially available antibodies are capable of identifying which GBM specimens express EGFRvIII and whether the EGFRs are activated. Such a molecular classification of GBMs might allow us to determine which patients might benefit from biologically targeted therapies. In addition, characterization of specimens with respect to their EGFRvIII status seems to be of prognostic value.

A role for the TGFβ-Par6 polarity pathway in breast cancer progression

The role of polarity signaling in cancer metastasis is ill defined. Using two three-dimensional culture models of mammary epithelial cells and an orthotopic mouse model of breast cancer, we reveal that Par6 signaling, which is regulated directly by TGFβ, plays a role in breast cancer metastasis. Interference with Par6 signaling blocked TGFβ-dependent loss of polarity in acini-like structures formed by non-transformed mammary cells grown in three-dimensional structures and suppressed the protrusive morphology of mesenchymal-like invasive mammary tumor cells without rescuing E-cadherin expression. Moreover, blockade of Par6 signaling in an in vivo orthotopic model of metastatic breast cancer induced the formation of ZO-1-positive epithelium-like structures in the primary tumor and suppressed metastasis to the lungs. Analysis of the pathway in tissue microarrays of human breast tumors further revealed that Par6 activation correlated with markers of the basal carcinoma subtype in BRCA1-associated tumors. These studies thus reveal a key role for polarity signaling and the control of morphologic transformation in breast cancer metastasis.

Expression and hypoxic regulation of angiopoietins in human astrocytomas

Vascular endothelial growth factor (VEGF) is a major inducer of tumor angiogenesis and edema in human astrocytomas by its interaction with cognate endothelial-specific receptors (VEGFR1/R2). Tie1 and Tie2/Tek are more recently identified endothelial-specific receptors, with angiopoietins being ligands for the latter. These angiogenic factors and receptors are crucial for the maturation of the vascular system, but their role in tumor angiogenesis, particularly in astrocytomas, is unknown. In this study, we demonstrate that the angiopoietin family member Ang1 is expressed by some of the astrocytoma cell lines. In contrast to VEGF, Ang1 is down regulated by hypoxia. Ang2 was not overexpressed. Expression profiles of low-grade astrocytoma specimens were similar to those of normal brain, with low levels of Ang1, Ang2, and VEGF expression. Glioblastoma multiforme expressed higher levels of Angl, but not to the same degree as pseudopalisading astrocytoma cells around necrotic and hypoxic zones expressed VEGF, as shown in previous studies. Ang2 expression in the highly proliferative tumor vascular endothelium was also increased, as was phosphorylated Tie2/Tek. The expression profile of these angiogenic factors and their endothelial cell receptors in human glioblastomas multiforme was similar to that in a transgenic mouse model of glioblastoma multiforme. These data suggest that both VEGF and angiopoietins are involved in regulating tumor angiogenesis in human astrocytomas.

Expression of vascular endothelial growth factor by reactive astrocytes and associated neoangiogenesis

Injury to the central nervous system (CNS) invokes a reparative response known as astrogliosis, characterized largely by hypertrophy, proliferation and increased expression of glial fibrillary acidic protein (GFAP), resulting in reactive astrocytosis. Based on our prior observation that peritumoral reactive astrocytes express Vascular Endothelial Growth Factor (VEGF), a highly potent and specific angiogenic growth factor, we have hypothesized that reactive astrocytosis also contributes to the neovascularization associated with astrogliosis. To evaluate this hypothesis we evaluated human surgical/autopsy specimens from a variety of CNS disorders that induce astrogliosis and an experimental CNS needle injury model in wild type and GFAP:Green Fluorescent Protein (GFP) transgenic mice. Using computer image semi-quantitative analysis we evaluated the number of GFAP-positive reactive astrocytes, degree of VEGF expression by these astrocytes, associated Factor VIII-positive microvascular density (MVD) and Ki-67 proliferating endothelial cells. The degree of reactive astrocytosis correlated to levels of VEGF immunoreactivity and MVD in the neuropathological specimens. The mouse-needle-stick brain injury model demonstrated this correlation was temporally and spatially related and maximal after 1 week. These results, involving both human pathology specimens augmented by experimental animal data, supports our hypothesis that the neoangiogenesis associated with reactive astrogliosis is correlated to increased reactive astrocytosis and associated VEGF expression.