Deltex-1 Activates Mitotic Signaling and Proliferation and Increases the Clonogenic and Invasive Potential of U373 and LN18 <em>Glioblastoma</em> Cells and Correlates with Patient Survival

<div><p><i>Glioblastoma</i> (GBM) is a highly malignant primary tumor of the central nervous system originating in glial cells. GBM results in more years of life lost than any other cancer type. Low levels of Notch receptor expression correlates with prolonged survival in various high grade gliomas independent of other markers. Different downstream pathways of Notch receptors have been identified. We tested if the Notch/Deltex pathway, which is distinct from the canonical, CSL-mediated pathway, has a role in GBM. We show that the alternative or non-canonical Notch pathway functioning through Deltex1 (DTX1) mediates key features of glioblastoma cell aggressiveness. For example, DTX1 activates the RTK/PI3K/PKB and the MAPK/ERK mitotic pathways and induces anti-apoptotic Mcl-1. The clonogenic and growth potential of established glioma cells correlated with DTX1 levels. Microarray gene expression analysis further identified a DTX1-specific, MAML1-independent transcriptional program - including <i>microRNA-21</i>- which is functionally linked to the changes in tumor cell aggressiveness. Over-expression of DTX1 increased cell migration and invasion correlating to ERK activation, miR-21 levels and endogenous Notch levels. In contrast to high and intermediate expressors, patients with low <i>DTX1</i> levels have a more favorable prognosis. The alternative Notch pathway via DTX1 appears to be an oncogenic factor in glioblastoma and these findings offer new potential therapeutic targets. </p> </div

Proliferation and mitotic signaling pathways are modified by DTX1 in established glioma cell lines.

<p>(A) Western blot analysis of signaling cascade proteins. Blots for total EGFR (t-EGFR), phosphorylated Akt/PKB (p-Akt/PKB), total Akt/PKB (t-Akt/PKB), phosphorylated Erk (p-Erk), total Erk (t-Erk), Mcl-1 and β-actin (actin) are shown. (B) Proliferation analysis by BrdU incorporation assay. Relative average values of 5 individual experiments are shown per genotype. (C) Proliferation analysis by cell counting. Equal numbers of cells were seeded, grown for 3d under standard conditions and counted afterwards. (D) Apoptosis in U373 cells after treatment with 100 µM temozolomide (TMZ) or DMSO control. Relative values of sub-G₁ cells measured by PI staining are shown normalized to control cells treated with vector control. Averages of at least three independent experiments are shown. Values are normalized to controls. Error bars: ±SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

DTX1 controls miR-21 expression in a p300 dependent manner.

<p>(A) Real time quantitative PCR analysis of miR-21 expression in U373 cells with modified DTX1 levels. (B) p300 and (C) miR-21 expression levels in U373-DTX1-myc cells transfected with siRNA targeting p300 (sip300, red) or control siRNA (siCTRL, blue) analyzed by qPCR normalized to U373-DTX1-myc. (D) miR-21 expression in U373-DTX1-myc cells treated with miR-21 inhibitor (α-miR-21, green) or an inhibitor control (α-CTRL, yellow). Average relative expression values of at least four independent experiments are shown. Error bars: ±SEM. * p<0.05, ** p<0.01, *** p<0.001. (E) Western blot analysis of signaling cascades in U373-DTX1-myc cells transfected with sip300 or siCTRL. Blots were probed for phosphorylated and total Akt/PKB, phosphorylated and total Erk, Mcl-1 and actin.</p

Migration and invasion potential of glioma cells is regulated by DTX1.

<p>(A) Boyden chamber trans-well migration and invasion assay with U373 and LN18 glioma cells on collagen coated membranes with 8 µm porosity. Counts were performed after 24h. (B) Scratch test wound healing assay. A scratch wound was inflicted and immediately imaged (time 0h). Follow up images were taken after 12, 24 and 48 hours. Wound closing was assessed using standard imaging software. (C) Western blot analysis of known pro-migratory factors in glioblastoma probing for Snail-1, Akt2/PKBβ, and beta-actin. (D) Boyden chamber trans-well migration assay with U373-DTX1-myc cells not treated (white), treated with a miR-21 inhibitor (α-miR-21, green) or an inhibitor control (α-CTRL , yellow). Average values are shown from at least three individual experiments. Error bars: ±SEM. * p<0.05, *** p<0.001.</p

Click-induced ABR thresholds of wild-type versus Akt knockout mice at 3–4 months of age.

<p>Normal hearing threshold was about 25 dB SPL. The ABR thresholds of single Akt1^-/- knockout and Akt2^-/-/Akt3^-/- double knockout mice were significantly higher than their corresponding wild-types (* p<0.05). Bars represent mean ± standard deviation. (Numbers of mice measured: n = 11 for wild-type, n = 5 for Akt1^-/-, n = 8 for Akt2^-/-, n = 10 for Akt3^-/-, n = 8 for Akt2^-/- Akt3^-/-).</p

Akt1, Akt2 and Akt3 mRNA relative expression in the organ of Corti (OC), the spiral ganglion (SG) and the stria vascularis (SV) of 5-day-old C57/B6 mice.

<p>Expression was measured by quantitative real-time PCR with GAPDH as an endogenous control. Akt1, Akt2 and Akt3 expression levels are presented relative to expression in the brain. Each bar represents mean ± standard deviation. Each experiment was repeated three times, with different biological replicates in triplicate. For each experiment mRNA of 20 ears were pooled.</p

A) Gentamicin-induced hair cell (HC) damage in cultured neonatal OC explants from Akt1, Akt2, Akt3 single and double knockout mice.

<p>Micrographs illustrate phalloidin-labeled organs of Corti (OCs). OCs were exposed to 0.5 mM gentamicin for 24h. Akt1^+/- and Akt1^-/- mice show no additional HC loss compared to wild-type littermates. Akt2^+/- and Akt3^+/- mice show additional HC loss compared to wild type mice. Akt2^-/- and Akt3^-/- knockout mice show additional increased HC loss, with only a small number of outer HC (OHC) remaining compared to their heterozygous littermates. The most pronounced HC loss is seen in Akt2^-/- Akt3^-/- knockout mice, where only a few OHCs survive and a large number of inner HCs (IHCs) are lost. The three OHC rows and the single inner HC row can be well recognized in OCs untreated with gentamicin. Genotypes are described as wild-type, heterozygous (+/-) and homozygous (-/-). Scale bar = 20 μm. <b>B)</b> Quantitative analysis of surviving hair cells (HCs) after gentamicin treatment <i>in vitro</i>. OCs were exposed to 0.5 mM gentamicin for 24h. Each bar represents mean ± standard deviation. <i>Asterisks</i> indicate a significant (p<0.05) difference between the indicated groups. (Numbers of OC explants analyzed: Wild-type n = 17, Akt1^-/- n = 1, Akt1^+/- n = 9, Akt2^-/- n = 6, Akt2^+/- n = 9, Akt3^-/- n = 5, Akt3^+/- n = 18, Akt2^-/- Akt3^-/-n = 12).</p

Light microscopy images of Akt single and double knockouts as well as wild-type (WT) mouse cochleas (sagittal section) stained with hematoxylin/eosin.

<p>No differences were observed between the morphology of the single and double knockout mice compared with WTs. Arrows and abbreviations indicate the location of the inner HC (IHC), the outer HC (OHC), tectrial membran (TM) SG neuron (SGN) and the stria vascularis (SV). Scale bar = 100 μm (A, E, I, M, Q); Scale bar = 100 μm (B, F, J, N, R); Scale bar = 100 μm (C, D, G, H, K, L, O, P, S, T).</p

<i>Ndr1/2</i>-double null mice are embryonic lethal, but a single <i>Ndr</i> allele is sufficient to sustain normal development.

<p>Genotype (GT) distribution of offspring from <i>Ndr-</i>single allele intercrosses (<i>Ndr1</i>^+/-;<i>Ndr2</i>^-/- x <i>Ndr1</i>^-/-;<i>Ndr2</i>^+/-) at weaning. Actual offspring numbers are indicated, together with the expected and obtained Mendelian ratios. No <i>Ndr1/2</i>-double null mice were recovered. All other genotypes were obtained at approximately the expected Mendelian ratios. A total of 415 offspring were analyzed (n = 415).</p><p><i>Ndr1/2</i>-double null mice are embryonic lethal, but a single <i>Ndr</i> allele is sufficient to sustain normal development.</p

Murine NDR kinases are essential for cardiac looping.

<p>(A) OPT 3D reconstruction of wild-type (Ai) and <i>Ndr1/2</i>-double null (Aii) embryos at E8.5. Blue: anatomy. Red: <i>Nkx2</i>.<i>5</i> whole mount <i>in situ</i> hybridization. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right. Two <i>Ndr1/2</i>-double null and two control embryos were examined. Using bright field microscopy ten <i>Ndr1/2</i>-double null and ten control embryos were analysed at E8.5 to confirm the observed phenotype (data not shown). (B, C) Bright field images of wild-type (Bi, Ci) and <i>Ndr1/2</i>-double null (Bii, Cii) developing hearts at E9.5. B: frontal view; C: lateral view. Scale bars = 100μm. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right, d: dorsal, v: ventral. Five <i>Ndr1/2</i>-double null and five control embryos were analyzed. (D, E) OPT virtual section of wild-type (Di, Ei) and <i>Ndr1/2</i>-double null (Dii, Eii) embryos shown in 6A. Panel D: coronal plane; panel E: transversal plane. Labels: a: anterior, p: posterior, d: dorsal, v: ventral. Arrows point to the heart. Two <i>Ndr1/2</i>-double null and two control embryos were analyzed for OPT as shown in 6D and 6E. (F, G, H) Hematoxylin and Eosin stained transversal sections of a wild-type (Fi, Gi, Hi) and <i>Ndr1/2</i>-double null (Fi, Gi, Hi) hearts at the 6-somite stage. The myocardium (MC) and headfolds (HF) are indicated in (Fi) and (Fii). Arrows in (Fii), (Gii) and (Hii) point to remaining cells in the cardiac jelly and lumen. Note the similar section plan between the embryos shown in (E) and (G). Three <i>Ndr1/2</i>-double null and three control embryos were analyzed. (J) Scheme showing the approximate level of the sections within the embryo. The distance between sections is about 30μm.</p