Misregulated E-Cadherin Expression Associated with an Aggressive Brain Tumor Phenotype

BACKGROUND: Cadherins are essential components of the adherens junction complexes that mediate cell-cell adhesion and regulate cell motility. During tissue morphogenesis, changes in cadherin expression (known as cadherin switching) are a common mechanism for altering cell fate. Cadherin switching is also common during epithelial tumor progression, where it is thought to promote tumor invasion and metastasis. E-cadherin is the predominant cadherin expressed in epithelial tissues, but its expression is very limited in normal brain. METHODOLOGY/PRINCIPAL FINDINGS: We identified E-cadherin expression in a retrospective series of glioblastomas exhibiting epithelial or pseudoepithelial differentiation. Unlike in epithelial tissues, E-cadherin expression in gliomas correlated with an unfavorable clinical outcome. Western blotting of two panels of human GBM cell lines propagated either as xenografts in nude mice or grown under conventional cell culture conditions confirmed that E-cadherin expression is rare. However, a small number of xenograft lines did express E-cadherin, its expression correlating with increased invasiveness when the cells were implanted orthotopically in mouse brain. In the conventionally cultured SF767 glioma cell line, E-cadherin expression was localized throughout the plasma membrane rather than being restricted to areas of cell-cell contact. ShRNA knockdown of E-cadherin in these cells resulted in decreased proliferation and migration in vitro. CONCLUSIONS/SIGNIFICANCE: Our data shows an unexpected correlation between the abnormal expression of E-cadherin in a subset of GBM tumor cells and the growth and migration of this aggressive brain tumor subtype

VEGF and Angiopoietin-1 Exert Opposing Effects on Cell Junctions by Regulating the Rho GEF Syx

Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser806, which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx−/− mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function

Human Glucocorticoid Receptor β Binds RU-486 and Is Transcriptionally Active

Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, α and β. In contrast to the canonical hGRα, hGRβ is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGRα. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRβ in COS-1 and U-2 OS cells. Surprisingly, yellow fluorescent protein (YFP)-hGRβ was predominantly located in the cytoplasm and translocated to the nucleus following application of the glucocorticoid antagonist RU-486. This effect of RU-486 was confirmed with transiently expressed wild-type hGRβ. Confocal microscopy of coexpressed YFP-hGRβ and cyan fluorescent protein-hGRα in COS-1 cells indicated that the receptors move into the nucleus independently. Using a ligand binding assay, we confirmed that hGRβ bound RU-486 but not the hGRα ligand dexamethasone. Examination of the cellular localization of YFP-hGRβ in response to a series of 57 related compounds indicated that RU-486 is thus far the only identified ligand that interacts with hGRβ. The selective interaction of RU-486 with hGRβ was also supported by molecular modeling and computational docking studies. Interestingly, microarray analysis indicates that hGRβ, expressed in the absence of hGRα, can regulate gene expression and furthermore that occupation of hGRβ with the antagonist RU-486 diminishes that capacity despite the lack of helix 12 in the ligand binding domain

Targeting Src family kinases inhibits bevacizumab-induced glioma cell invasion.

Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM). Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab's influence on glioma biology and block it's actions towards cell invasion. To explore the mechanism(s) of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion.We show that 1) activation of Src family kinases (SFKs) is common in GBM, 2) the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3) SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab.Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic

Dasatinib inhibits activation of SFKs and downstream targets in glioma.

<p><b>A.</b> Migration of SF767 cells in the absence or presence of 10 µM dasatinib or 10 µM PP2 was determined using a trans-well migration assay. Inhibition of SFKs with dasatinib or PP2 resulted in fewer SF767 cells migrating toward the chemoattractant. The differences vs. control (DMSO) treatment are statistically significant (n = 4; one-way ANOVA with Dunnett's Multiple Comparisons post test; *** indicates p<0.0001). <b>B.</b> Immunofluorescent staining of actin (white) in SF767 cells after control (DMSO) treatment or treatment with dasatinib (10 µM for 24 hours). For each treatment, the top image is the x-z orientation and bottom is the x-y orientation. Blue staining is DAPI; scale bar is 10 µm. <b>C.</b> Whole cell lysates of SF767 cells treated for 24 hours with 10 µM dasatinib (+) or DMSO (−) were western blotted for total Src, Y416-phosphorylated Src, total p120-catenin, Y228-phosphorylated p120, Y172-phosphorylated Vav2, actin (as a loading control), or active or total Rac1.</p

Activation of Src and p120 phosphorylation correlates with increased glioma invasiveness.

<p><b>A.</b> Seventeen glioma cell lines propagated as xenografts in mouse flank were examined by western blot for expression of active (Y416-phosphorylated) and total Src and Y228-phosphorylated and total p120 catenin proteins. Actin is a loading control. The positive control lysate is from MDA231 cells. Xenograft lines were previously classified based on their relative invasiveness as highly or moderately invasive (data boxed in gray) or minimally or non-invasive (not boxed). <b>B.</b> The level of Y228-phosphorylated p120 catenin expression (relative to actin expression) as determined by Western blot for each cell line was plotted vs. relative glioma invasiveness. The lines through the data indicate the median for each invasiveness category; n = 8 highly/moderately invasive lines and n = 9 minimally/non-invasive lines; **indicates a statistical difference (one-tailed, unpaired t test) between the two categories of invasiveness at p<0.005. <b>C.</b> Human prostate tumor, breast tumor, astrocytoma, and GBM samples were examined by immunohistochemistry for total p120 catenin expression (using the 15D2 antibody), and Y228-phosphorylated p120 catenin expression. Bar: 100 µm.</p

The invasive front of human GBM is a site of SFK activation.

<p>The spatial distribution of active SFK (as assessed by Y416-phosphorylated Src), Y228-phosphorylated p120 catenin, Y410-phosphorylated p130cas and Y172-phosphorylated Vav2 was assessed by immunohistochemistry of orthotopic GBM39 xenograft samples. Core indicates an image from the non-invading tumor core; rim indicates an image from the tumor's leading edge where the tumor interfaces with and can invade into the surrounding normal brain. Bar: 100 µm.</p

Dasatinib suppresses SFK signaling and bevacizumab-induced GBM cell invasion.

<p><b>A.</b> Orthotopically located GBM10 xenografts treated with or without dasatinib were stained immunohistochemically for Y416-phosphorylated Src, Y228-phosphorylated p120, and Y410-phosphorylated p130cas. Upper panels: Left panels are lower magnification; right panels are higher magnification of boxed inset. Bar: 100 µm. <b>B.</b> The number of single GBM10 cells invading into the surrounding normal brain parenchyma in placebo- (control), dasatinib-, bevacizumab-, and dasatinib+bevacizumab-treated samples was counted in 18 unique fields from at least 3 separate mice per treatment. Data are expressed as % control and represent the mean ± SD (<i>n</i> = 18; ** represents p<0.001, ANOVA with Dunnett's Multiple Comparisons post test). <b>C.</b> Orthotopically located GBM10 xenografts treated with bevacizumab with or without dasatinib were stained immunohistochemically for human Vimentin to show the localization and invasion pattern of the GBM10 tumor cells. Bar: 100 µm. <b>D.</b> Migration of GBM10 cells in the absence or presence of 10 µM dasatinib, 0.5 mg/ml bevacizumab, or dasatinib+bevacizumab was determined using a trans-well migration assay. Inhibition of SFKs with dasatinib alone or in combination with bevacizumab resulted in fewer GBM10 cells migrating toward the chemoattractant; bevacizumab treatment alone had no effect on GBM10 migration. * indicates a statistically significant difference vs. control treatment (n = 3; one-way ANOVA with Dunnett's Multiple Comparisons post test; * indicates p<0.05).</p

Correction: Folate receptor-α (FOLR1) expression and function in triple negative tumors.

Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense overexpression of FOLR1 in breast tumors and poor prognosis, yet there is limited examination of the distribution of FOLR1 across clinically relevant breast cancer subtypes. To explore this further, we used RNA-seq data from multiple patient cohorts to analyze the distribution of FOLR1 mRNA across breast cancer subtypes comprised of estrogen receptor positive (ER+), human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) tumors. FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors. However, subsets of high level FOLR1 expressing tumors were observed in all clinical subtypes. These observations were supported by immunohistochemical analysis of tissue microarrays, with the largest number of 3+ positive tumors and highest H-scores of any subtype represented by triple negatives, and lowest by ER+ tumors. FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status. To delineate the importance of FOLR1 overexpression in triple negative cancers, RNA-interference was used to deplete FOLR1 in overexpressing triple negative cell breast lines. Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions. Taken together, our data suggests patients with triple negative cancers expressing high FOLR1 expression represent an important population of patients that may benefit from targeted anti-FOLR1 therapy. This may prove particularly helpful for a large number of patients who would typically be classified as triple negative and who to this point have been left without any targeted treatment options