The role of the membrane cytoskeleton cross-linker ezrin in medulloblastoma cells

Abstract

Medulloblastoma is a highly malignant brain tumor that occurs predominantly in children. The molecular pathogenesis of medulloblastoma is under investigation. Previously, we used complementary DNA microarray analysis to compare patterns of gene expression in medulloblastoma samples versus normal cerebellum. The cytoskeletal protein ezrin was found to be overexpressed in medulloblastoma compared with normal cerebellum, an observation that was further validated by immunohistochemistry and real-time PCR analysis. To assess the role of ezrin in medulloblastoma, we studied ezrin's role in medulloblastoma migration, invasion, and adhesion. Western blotting and immunofluorescence showed high expression of ezrin in four medulloblastoma cell lines, and ezrin was primarily localized to filopodia. Ezrin-specific small interfering RNA suppressed the formation of filopodia and in vitro migration, invasion, and adhesion. We also used a stably transfected medulloblastoma cell line to study the effect of ezrin overexpression. We showed that high expression of ezrin promotes filopodia formation and in vitro invasion. Finally, athymic mice implanted with ezrin-overexpressing DAOY medullo-blastoma cell clones in the cerebellum showed shortened survival compared with controls. These findings suggest that, in addition to other cytoskeletal proteins, ezrin plays an important role in medulloblastoma adhesion, migration, and invasion.

Fig. 1.

Ezrin expression in medulloblastoma (MB) cell lines. (A) Top: Ezrin in normal cerebellum and MB cell lines revealed by Western blotting. β-actin was used as a loading control. Ezrin is overexpressed in all MB cell lines compared with normal cerebellum. Bottom: Quantification by densitometry of a representative experiment. (B) Ezrin gene expression analysis of primary patient MB samples (right) and MB cell lines (left) compared with normal human cerebellum using the Affymetrix Human Genome U133 Plus 2.0 Arrays. (C) Distribution of ezrin in DAOY, ONS76, UW228, and MED8A cells. Cells were immunolabeled for ezrin (green) and F-actin (red) to localize endogenous proteins by immunofluorescence. Yellow indicates colocalization of both proteins at filopodia and membrane extensions (arrows). Nuclei were stained using 4’,6-diamidino-2-phenylindole staining (blue). Scale bar, 25 μm.

Fig. 2.

Ezrin small interfering RNA (siRNA) depletes endogenous ezrin expression in medulloblastoma cells. (A) Top: Immunoblot analysis of ezrin protein expression in DAOY (left) and ONS76 (right) after ezrin knockdown by siRNA. Bottom: Quantification by densitometry of a representative experiment. Abbreviations: UN, untreated; NT, nontargeting siRNA; Ez1 and Ez2, two different ezrin knockdown sequences used. (B) Morphological change of DAOY and ONS76 cells after transfection with siRNA. After ezrin knockdown, cells were plated on cover slips for immunofluorescence. Cells were labeled with phalloidin for F-actin, and ezrin was labeled with antiezrin antibody by Alexa488-conjugated antimouse antibody. Arrows show filopodia bundles. Scale bar, 25 μm.

Fig. 3.

Effects of ezrin depletion on medulloblastoma (MB) cell migration, invasion, and adhesion. (A and B) MB cells transfected with ezrin small interfering RNA (siRNA) were seeded through a cell sedimentation manifold to establish a circular confluent monolayer on substratecoated wells (bovine serum albumin [BSA] or laminin [LN]). DAOY (A) and ONS76 (B) cells were incubated for 24 h, and images were used to measure migration. The average (AVG) migration rate was calculated as the change in the diameter of the circle circumscribing the cell population for a 24-h period. (C) Modified Boyden chamber invasion assay after transfection with ezrin siRNA in DAOY and ONS76 MB cell lines. After incubation for 24 h, the cells that migrated through the membrane were stained and representative fields were imaged. Abbreviations: NT, nontargeting siRNA; Ez1 and Ez2, two different ezrin knockdown sequences used. Magnification, ×200. (D) Invasion was quantified by counting cells. Ezrin siRNA significantly inhibited the invasion of MB cell lines. Columns represent the average (AVG) + SEM invasion rate (cells/fields). (E) Adhesion assay for MB cells tested on BSA and LN. Cells were seeded on 96-well plates coated with BSA or LN, incubated for 3 h, fixed with 4% paraformaldehyde, and then stained with crystal violet. The absorbance was measured at 595 nm (mean + SEM). *p < 0.05, **p < 0.01, versus control.

Fig. 4.

CD44 expression after transfection with ezrin small interfering RNA (siRNA). (A) Cells were seeded onto bovine serum albumin–coated or laminin-coated cover slips. After fixation, Texas red-X phalloidin and CD44 antibody were used to localize endogenous actin and CD44 by immunofluorescence. Images were acquired by serial focal planes to examine CD44 expression at the plasma membrane (PM, left) or a plane in the middle of the cell (Mid, right). Compared with control cells, ezrin knockdown reduced CD44 expression at filopodia and the plasma membrane in DAOY cells (arrows). Scale bars, 25 μm. (B) Top: Whole-cell lysates and Triton-soluble and -insoluble protein fractions were extracted from DAOY and ONS76 after transfection with ezrin siRNA. Protein (20 μg) was separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto an Immobilon-P polyvinyl difluoride membrane. CD44 protein was detected using an anti-CD44 antibody in each sample. Flotillin-2, a marker of the insoluble fraction, was used as a loading control. Bottom: Quantification by densitometry of a representative experiment. Abbreviations: NT, nontargeting siRNA; Ez3, ezrin siRNA.

Fig. 5.

Analysis of ezrin overexpression in medulloblastoma (MB) cell invasion. (A) DAOY cells stably expressing full-length FLAG–ezrin (Flag) or empty vector control (EV) were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to assess expression levels. The membranes were probed with anti-ezrin, anti-FLAG, and anti-β-actin and developed with chemiluminescence. Clones identified show high (EzH), intermediate (EzM), or low (EzL) expression of FLAG–ezrin. (B) Empty vector (EV) or FLAG–EzH stable cell lines were immunostained for ezrin (green) and phalloidin (red) and with 4’,6-diamidino-2-phenylindole (blue). The far right panels are enlargements shown by squares in the panels to the left. Scale bars, 25 μm. (C) DAOY cell clones described above were examined by Boyden chamber invasion assay. After a 22-h incubation, the cells that migrated through the membrane were stained and representative fields were imaged. Magnification, ×200. (D) Invasion was quantified by counting cells. Ezrin overexpression correlated with MB invasion rate and is statistically significant. Columns represent the average (AVG) + SEM invasion rate (cells/fields). **p < 0.01, versus control.

Fig. 6.

(A) Kaplan-Meier survival analysis for nude mice with intracerebellar human medulloblastoma (MB) cells transfected with either empty vector (EV) or a vector encoding full-length ezrin (EzH). The EV control mice lived longer than did the EzH mice (log-rank test, p < 0.05). The median survival of the EV group was 83 days, compared with 68.5 days in the EzH group. (B) Whole-mount photomicrographs from mouse brains depicting size and dissemination of MB cells. Mice were injected with empty vector (EV) or full-length ezrin (EzH), and the sections were stained with hematoxylin and eosin (a and b) and processed for immunohistochemistry with antiezrin (c and d). EzH-expressing MB cells showed greater leptomeningeal dissemination (b and c) than did EV controls. Original magnifications: ×1 for a and c; ×10 for b; ×40 for d. (C) Analysis of in vivo xenograft implant localization and invasion.