J Neurooncol DOI 10.1007/s11060-014-1621-0
LABORATORY INVESTIGATION
Wnt activation affects proliferation, invasiveness and radiosensitivity in medulloblastoma Roberta Salaroli • Alice Ronchi • Francesca Romana Buttarelli • Filippo Cortesi Valeria Marchese • Elena Della Bella • Cristiano Renna • Caterina Baldi • Felice Giangaspero • Giovanna Cenacchi
•
Received: 28 December 2013 / Accepted: 21 September 2014 Ó Springer Science+Business Media New York 2014
Abstract Medulloblastomas (MBs) associated with the Wnt activation represent a subgroup with a favorable prognosis, but it remains unclear whether Wnt activation confers a less aggressive phenotype and/or enhances radiosensitivity. To investigate this issue, we evaluated the biological behavior of an MB cell line, UW228-1, stably transfected with human b-catenin cDNA encoding a nondegradable form of b-catenin (UW-B) in standard culture conditions and after radiation treatment. We evaluated the expression, transcriptional activity, and localization of bcatenin in the stably transfected cells using immunofluorescence and WB. We performed morphological analysis using light and electron microscopy. We then analyzed changes in the invasiveness, growth, and mortality in standard culture conditions and after radiation. We demonstrated that (A) Wnt activation inhibited 97 % of the invasion capability of the cells, (B) the growth of the UWB cells was statistically significantly lower than that of all the other control cells (p \ 0.01), (C) the mortality of
R. Salaroli A. Ronchi F. Cortesi V. Marchese E. D. Bella C. Renna G. Cenacchi (&) Department of Biomedical and Neuromotor Sciences, ‘‘Alma Mater Studiorum’’ University of Bologna, Via Massarenti 9, 40138 Bologna, Italy e-mail: [email protected] F. R. Buttarelli C. Baldi Department of Neurology and Psychiatry, Sapienza University of Rome, 00161 Rome, Italy F. Giangaspero Department of Radiology, Oncology and Anatomo-Pathology, Sapienza University of Rome, 00161 Rome, Italy F. Giangaspero Neuromed IRCCS, 86077 Pozzilli, Italy
irradiated UW-B cells was statistically significantly higher than that of the controls and their nonirradiated counterparts (p \ 0.05), and (D) morphological features of neuronal differentiation were observed in the Wnt-activated cells. In tissue samples, the Ki-67 labeling index (LI) was lower in b-catenin-positive samples compared to non-bcatenin positive ones. The Ki-67 LI median (LI = 40) of the nuclear b-catenin-positive tumor samples was lower than that of non-nuclear b-catenin-positive samples (LI = 50), but the difference was not statistically significant. Overall, our data suggest that activation of the Wnt pathway reduces the proliferation and invasion of MBs and increases the tumor’s radiosensitivity. Keywords Medulloblastoma Beta-catenin Wnt pathway Radiosensitivity
Introduction A medulloblastoma (MB) is a highly invasive embryonal tumor of the cerebellum and one of the leading causes of mortality in pediatric patients [1–3]. Metastatic disease at diagnosis remains the most important negative prognostic clinical marker of treatment failure in MB patients, despite the use of cranio-spinal irradiation and intensive chemotherapy [1, 4]. Postoperative radiotherapy is an effective treatment, but survivors have severe long-term side effects, and the response is sometimes limited by intrinsic radioresistance [3, 5]. Gene expression profiling studies [1–3] have suggested four molecular subtypes of MB, each of which is characterized by a distinct genetic profile, the activation of different oncogenic pathways, and diverse clinical outcomes. Subgroup A and B are characterized by Wnt and Hh signaling, respectively, and subgroups C and
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D are characterized by the expression of neuronal differentiation and photoreceptor genes, respectively [6–11]. Canonical Wnt signaling compromises the ability of a multiprotein complex containing axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3 b (GSK 3b) to target it for degradation and block its nuclear import, thereby inducing stabilization and nuclear localization of b-catenin. Nuclear b-catenin interacts with members of the LEF-TCF family of transcription factors, leading to the activation of various target genes, such as c-myc and cyclin D1 [4]. In sporadic MBs, the Wnt/b-catenin pathway is aberrantly activated in approximately 18–25 % of tumors [12– 14]. This tumor subgroup (subgroup A) is recognized as low risk, presenting in children between the ages of 6 and 12 years; it shows nuclear b-catenin immunopositivity, usually with a classic histological subtype and is associated with activating mutations in ctnnb1 (b-catenin gene) and monosomy of chromosome 6 [8, 9, 11, 15]. The frequency of metastatic disease at presentation is lower than in the other subgroups [4, 10]. Several clues point toward nuclear b-catenin having a positive role in the prognosis of MB, but the underlying molecular mechanisms are unknown [12, 15–17]. It remains unclear whether a favorable prognosis in patients harboring nuclear positivity for b-catenin is linked to reduced aggressiveness of the tumor or a different response to therapy [12]. Although there are numerous human xenograft mouse models of MB, no MB cell line with Wnt activation is available for conducting in vivo studies, which could help to resolve the role of nuclear b-catenin in the prognosis of MB. To overcome these limitations, we utilized the human MB cell line UW228-1, which is stably transfected with human b-catenin cDNA and encodes a nondegradable form of b-catenin. We evaluated changes in growth, mortality, and invasiveness in standard culture conditions and after ionizing radiation (IR) treatment. Moreover, in a series of formalin-fixed paraffin-embedded MB specimens, we compared the differences in the growth of Wnt-activated and nonactivated cases.
Modified Eagle’s Medium (high glucose) and Ham’s Nutrient Mixture F-12 (Euroclone, Milan, Italy), supplemented with 10 % heat-inactivated fetal bovine serum, 1 % penicillin–streptomycin, 1 % 2 mM L-glutamine, and 1 % nonessential amino acids (all produced by Euroclone, Milan, Italy). The cell cultures were expanded and maintained at 37 °C in 5 % CO2. Each experiment was started with cells at passage number eight. Plasmid construction, sequencing, and transfection The plasmid pCI-neo/b-catenin S33Y contains human bcatenin cDNA, which harbors an S33Y mutation downstream of the cytomegalovirus promoter in the NheI and SalI sites of the pCI-neo vector (Promega Corp, Madison, WI). S33Y amino acid substitution renders b-catenin refractory to phosphorylation by GSK 3b and therefore resistant to degradation [19]. Mutations in the human ctnnb1 gene encoding serine 33 substitutions occur in sporadic MBs [12, 14]. The vector also contains the neomycin resistance gene, allowing for the selection of transfected cells resistant to the antibiotic. The construct was kindly provided by Dr. Hans Clevers (Utrecht University, Utrecht, NL). Transfections were carried out using Lipofectamine 2000 reagent (Invitrogen, Paisley, UK) at a ratio of 10:1 (Lipofectamine/DNA, volume/mass), according to the manufacturer’s instructions. Cells were plated at a density of 100,000 per well in 6-well plates and collected 24 h after transfection. Stable transfected clones were derived from UW228-1 cells transfected with either pCI-neo/b-catenin S33Y (UW-B) or the pCIneo empty vector as a control line (UW-V) following treatment with the aminoglycoside antibiotic G418, which was used as a neomycin selection agent. After 48 h of culture growth, G418 was added at a concentration of 750 mg/mL. The cells were cultured in the selection medium for an additional 2 weeks. Clones were isolated by light microscopy following single-cell plating in 96-well plates. To avoid unique insertion events, four clones for UW-B and three clones for UW-V were derived from two different transfection experiments and characterized. Protein extraction and western blots
Materials and methods Cell culture We used a human MB cell line, UW228-1, without Wnt alterations, kindly provided by Dr. Charles G. Eberhart (John Hopkins University, Baltimore, MD), with the agreement of Dr. Mike Bobola (University of Washington, Seattle, WA) [18].This cell line was checked for mycoplasma (negative result) and then maintained in Dulbecco’s
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UW228-1, UW-V, and UW-B nuclear and total proteins were extracted and Western blotting was performed, as previously described [20]. The following primary antibodies were used: rabbit polyclonal anti-b-catenin (H-102, Santa Cruz Biotechnology, Santa Cruz, CA) 1:1,000, mouse monoclonal anticyclin D1 (A-12, Santa Cruz Biotechnology, Santa Cruz, CA) 1:100, mouse monoclonal anti-MYC (Ab-2, Calbiochem, Darmstadt, DE) 1:500, goat polyclonal anti-b-actin (I-19, Santa Cruz Biotechnology,
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Santa Cruz, CA) 1:1,000, and mouse monoclonal antilamin B1 (ZL-b, ABcam, Cambridge, UK) 1:400. The primary antibodies were detected with horseradish peroxidaselabeled secondary antibodies: antirabbit (NA934, GE Healthcare Europe GmbH, Freiburg, DE) 1:10,000, antimouse (NA93, GE Healthcare Europe GmbH; Freiburg, DE) 1:10,000, and antigoat (sc-2020, Santa Cruz Biotechnology, Santa Cruz, CA) 1:1,000. Total b-catenin c-myc, cyclin D1, and b-actin were detected in total protein extracts, and nuclear b-catenin and lamin B were detected in nuclear extracts. b-actin and lamin B were used to determine total and nuclear loading, respectively. The experiments were performed in triplicate. Immunofluorescence The UW-V and UW-B cells were plated at a density of 100,000 per well on glass coverslips in 6-well plates and fixed with 2 % paraformaldehyde (Merck, Darmstadt, DE) in PBS for immunofluorescence staining of b-catenin, as previously described [20]. For each sample, all cells on the coverslip were evaluated. The experiment was performed in triplicate. Light and transmission electron microscopy The cells were plated at a density of 200,000 viable cells per T25 flask on day 0 and were observed every 24 h for 5 days under a light microscope. Every day, ten nonoverlapping optical fields were photographed using a digital camera (Olympus C-5060, Olympus Italy, Segrate, Italy) at 109 magnification to obtain a semiquantitative evaluation of the cell-type composition of the cell population. The experiment was performed in triplicate. The relative amount of different cytotypes in the UW228-1, UW-V, and UW-B lines are reported in Fig. 4. For the ultrastructural analysis, the UV-B and UV-V cells were plated at a density of 76,800 per well in 6-well plates. At 24 and 96 h after seeding, the cells were fixed in 2.5 % of glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and postfixed in 1 % OsO4 in the same buffer. The samples were dehydrated in ethanol and embedded in araldite. All the cells in the thin sections were analyzed under a Philips CM10 TEM (Philips, Amsterdam, NL).
Cell growth analysis and cell mortality assay The number and viability/mortality of the cells were assessed by preparing a 1:1 dilution of the cell suspension in a trypan blue solution (Sigma-Aldrich, St. Louis, MO). The numbers of dead and total cells in every sample were counted in a Neubauer chamber. For growth curve building, the cells were plated at a density of 200,000 viable cells per T25 flask and irradiated on day 0 (24 h after seeding). The cell numbers were assessed every day for 5 days. Three replicate flasks were assayed at each time point in each of three independent experiments. For mortality analysis, 200,000 cells were seeded in T25 culture flasks and collected 48, 72, and 96 h after irradiation. Three replicate flasks were assayed at each time point in each of the three independent experiments. Invasion capability assay The UW228-1, UW-V, UW-B, UW228-1 IR, UW-V IR, and UW-B IR cells (50,000 cells in serum-free medium) were placed in the top chamber of a transwell migration Boyden chamber [21]. The lower chamber was filled with 35 ll of serum (10 %)-containing medium. The transwell insert contained an 8 lm pore membrane (NeuroProbe Inc., Gaithersburg, MD) with a thin layer of Matrigel (BD Bioscience, Bedford, MA). After incubation at 37 °C for 24 h, the cells that had not migrated to the lower chamber were removed from the upper surface of the transwell membrane with a cotton swab. Migrating cells on the lower membrane surface were fixed, stained with a May-Grunwald/Giemsa solution, and counted under a light microscope at 109 magnification. As negative controls, some cells were placed in a Boyden chamber in which the top and lower chambers were filled with serum-free medium. The experiment was performed in triplicate. Statistical analysis The data were analyzed with Dunnet’s test and Bonferroni’s multiple comparison test using the Graphpad software package for Windows (PRISM5). A value of p \ 0.05 was considered statistically significant. MB tissue samples
IR treatment The UW228-1, UW-V, and UW-B cells were plated 24 h prior to irradiation with an Irradiateur Biologique 437C (CIS-BIO; Cedex, France) c-ray machine at a dose of 2 Gy and a dose rate of approximately 2 Gy/min. Postirradiation, the cell growth, cell mortality rate, and invasiveness were evaluated at different time points.
We evaluated 187 MB samples from patients aged 0 through 16 years who were assessed at the Neuropathology Unit of Azienda Policlinico Umberto I, Sapienza University of Rome from 01/01/2009 to 31/12/2012. The cases were categorized according to the WHO classification [1]. All the samples were immunostained for b-catenin and Ki-67 with anti-b-catenin rabbit polyclonal antibody
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(Beckton Dickinson, Franklin Lakes, New Jersey; 1:600) and anti-Ki-67 (MIB-1) mouse monoclonal antibody (Dako, Glostrup, Denmark; 1:200). The immunostaining was performed in a Bond Max Leica autostainer with biotinylated secondary Ab, peroxidase complex, and DAB (all from Ultratech Sciteck, San Jose, CA). In each sample, the number of b-catenin-positive nuclei and staining for Ki-67 were evaluated using a Nikon Eclipse 50i microscope, which was equipped with a 209 objective lens and a Nikon DS-F1 camera, interfaced with a computer equipped with NIS Elements F software. In each slide, images were acquired from areas selected by a neuropathologist (FG) and then analyzed and processed using Image J software. Immunostaining was recorded on ten fields at 29 (1,000 neoplastic cells on average) as a percentage of positive tumor cells. b-catenin was recorded as positive when [10 % of the tumor cells showed nuclear staining. Samples containing a few scattered nuclear b-catenin-positive cells were not regarded as positive. Ki67 staining was recorded as a percentage of positive tumor nuclei (LI).
Results Transfection with the pCI-neo/b-catenin S33Y construct activated Wnt signaling in the UW228-1 cells
Fig. 1 Nuclear expression of b-catenin in the UW228-1, UW-V, and UW-B cells. a The graph shows the relative amount of b-catenin, and the column bars represent the mean of three independent experiments. Nuclear protein extracts were prepared from UW228-1, UW-V, and UW-B cells harvested 24, 28, 32, and 40 h after cell seeding. b The image shows one of three Western blots obtained: lane 1 UW228-1 at 24 h; lane 2 UW-V at 24 h; lane 3 UW-B at 24 h; lane 4 UW228-1 at 28 h; lane 5 UW-V at 28 h; lane 6 UW-B at 28 h; lane 7 UW228-1 at 32 h; lane 8 UW-V at 32 h; lane 9 UW-B at 32 h; lane 10 UW228-1 at 40 h; lane 11 UW-V at 40 h; lane 12 UW-B at 40 h. The expression of lamin B served as a nuclear loading control. Each sample was quantified densitometrically and normalized to the expression of lamin B
Western blot analysis showed an increase in nuclear b-catenin in the UW-B cell line compared to the control cell line, with the greatest elevation occurring 28 h after cell seeding. At this time point, nuclear b-catenin was increased 4.0-fold in the UW-B cells compared to the UW228-1 cells and 4.5-fold compared to the UW-V cells. After 32 h, nuclear b-catenin was increased 2.9-fold in the UW-B cells compared to the UW228-1 cells and 4.4-fold compared to the UW-V cells. Nuclear b-catenin levels did not essentially differ in the UW228-1 and UW-V cell lines at any time point (Fig. 1). The immunofluorescence analysis of the UW-B cells revealed strong b-catenin positivity at the nuclear level and a weak signal in both the cell cytoplasm and membrane in almost all the cells. b-catenin was localized at the perinuclear level in the UW-V cell line and at contact points among the cells (Fig. 2). Nuclear b-catenin in the UW-B cells showed transcriptional activity, as highlighted by a 1.8-fold increase in cyclin D1 and a 1.2-fold increase in c-myc protein levels (Fig. 3).
club/spindle-shaped (*30 %), and isolated giant cells. In comparison with the UW228-1 cell line, the UW-V line had a greater number of club/spindle-shaped cells (*50 %), with short unipolar or bipolar processes. In the UW-B cells, the ratio of polygonal/oval cells to and club/ spindle cells was altered, with the cell population containing a greater number of club/spindle cells (*70 %), with multipolar-directed processes. In addition, around 5 % of the cells had very long, thin cytoplasmic elongations. The remaining 25 % were polygonal/oval-shaped cells (Fig. 4). At the ultrastructural level, the phenotypes of both the transfected and nontransfected cell lines were undifferentiated. However, early neuronal differentiation, such as several intermediate filaments mixed with microtubules running parallel to cytoplasmic elongations, was observed in the club/spindle-shaped cells, which were particularly abundant in the UW-B line. In contrast, the polygonal/ovalshaped cells had abundant cytoplasm, with only a few microtubules and randomly distributed filaments (Fig. 5).
Wnt activation altered cell morphology and induced differentiation in the MB cell lines
Wnt activation altered the cell growth and enhanced the radiosensitivity of the MB cells
The UW228-1 parental cell line is characterized by three morphological cell types: polygonal/oval shaped (*70 %),
The growth of the nuclear b-catenin cell line decreased when cultured in standard conditions compared to the
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J Neurooncol Fig. 2 The expression and localization of b-catenin in the UW-V and UW-B cells. The FITC- secondary antibody was conjugated (green) with polyclonal swine antirabbit immunoglobulins; the nuclei are stained with DAPI (blue). We observed strong nucleopositivity for b-catenin only in the UW-B cells. Magnification of 9100
Fig. 3 Cyclin-D1 (a) and c-myc (b) expression levels in the UW-B cells compared to the UW-V cells 24, 28, 32, 40, and 48 h after cell seeding. Each sample was normalized with b-actin. The bar charts show the mean-fold increase in the UW-B cells compared to the UWV cells based on three independent experiments
vector control line. Statistical analysis of the number of UW-B and UW-V cells 120 h after seeding revealed a 12 % decrease (p \ 0.01, RM ANOVA). At 0, 24, 48, 72, and 96 h, there were 151 ± 4, 203 ± 3, 345 ± 16, 541 ± 79, and 1,096 ± 31 thousands of UW-B cells, respectively. In contrast, there were 148 ± 19, 261 ± 8, 423 ± 12, 809 ± 29, and 1,244 ± 57 thousands of UW-V cells at the same time points. As expected, the cell growth of the vector control line in response to IR treatment was slower than that of its nonirradiated counterpart. The growth of the irradiated UW-B cells was statistically significantly lower than that of all the other cell lines: 125 ± 6, 188 ± 10, 310 ± 28, 479 ± 23, and 693 ± 31 thousands of cells at 0, 24, 48, 72, and 96 h, respectively, and 136 ± 18, 214 ± 10, 396 ± 32, 523 ± 8, and 815 ± 23 thousands of cells for UW-V cells. Ninety-six hours after irradiation (120 h after cell seeding), the number of UW-B cells decreased 37 and 15 % compared to the UW-B cells (p \ 0.001) and UW-V cells, respectively, (p \ 0.01) (Fig. 6). Wnt activation increased the cell mortality rate
Fig. 4 Light microscopy of a UW228-1 polygonal/oval-shaped cells, b UW-V club/spindle-shaped cells, and c UW-B polygonal/ovalshaped cells and cells, with very long, thin cytoplasmic elongations. Magnification of 920. The relative amount of different cytotypes in the UW228-1, UW-V, and UW-B cells are shown in the table. ??? *70 %; ?? *50 %; ? *30 %
At all the time points (72, 96, and 120 h after cell seeding), the cell mortality rate of the UW-B cells was greater than that of the UW-V cells. The cell mortality rate of the UWB cells was 19.97 ± 0.90, 14.30 ± 3.74, and 13.58 ± 0.98 % at 72, 96, and 120 h, respectively, whereas the cell mortality rate of the UW-V cells was 9.80 ± 1.16, 8.53 ± 1.35, and 8.98 ± 0.95 % at the same time points. At the first time point (72 h after cell seeding), the difference in the cell mortality rate of the UW-V and UW-B cells was statistically significant (p \ 0.05). At 72, 96, and 120 h after seeding, the cell mortality rate of the UW-B cells exposed to IR was increased in comparison with that of the irradiated UW-V cells: 16.92 ± 2.90, 14.98 ± 1.66, and 20.19 ± 2.11 %, respectively, for the UW-B cells and 9.05 ± 2.01, 10.95 ± 1.37, and
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Fig. 5 Transmission electron microscopy of a UW228-1 round cells, with an irregular shaped nucleus and a hypertrophic nucleolus, bar = 5 lm; b Unipolar UW-V club/spindle cells; c Multipolar UWB club/spindle cells with very long, thin cytoplasmic elongations,
bar = 10 lm; d Detail of a long cytoplasmic elongation in a UW-B cell. Intermediate filaments mixed with microtubules, running parallel to cytoplasmic elongation, bar = 2 lm
B cells was statistically significantly higher than that of their UW-B nonirradiated counterparts and that of the irradiated UW-V cells, with a variation of 5.68 % (p \ 0.05) and 6.61 % (p \ 0.05), respectively (Fig. 7). Wnt activation inhibited the invasion capability of MB cells
Fig. 6 Cell growth of UW-V, UW-B, UW-V IR, and UW-B IR cells. The dotted line shows the IR-treated cells, and the continuous line denotes the untreated cells. The lines with no symbol are UW-V cells, and those with the symbol white square are UW-B lines. The cells were plated 24 h prior to irradiation. They were harvested and analyzed at the following time points: 0, 24, 48, 72, and 96 h after radiation. Each data point (±SEM; point) is the mean of three independent experiments. Vertical bars indicate the standard deviation; **p \ 0.01; ***p \ 0.001
14.51 ± 0.19 % for the UW-V cells. At 120 h after cell seeding, corresponding to 96 h after the IR treatment (24 h after seeding), the cell mortality rate of the irradiated UW-
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When the data were normalized against the invasiveness rate (100 ± 0.25 %) of the parental UW228-1 cell line, the mean invasiveness rate of the UW-V and UW-B cells was 102 ± 27 and 2.600 ± 0.003 %, respectively. In each sample, the number of migrating cells was counted. From this total, the number of migrating cells found in the corresponding negative controls was subtracted. Statistical analysis revealed a 97.7 % decrease in the number of migrating cells in the UW-B line compared to the UW2281 lines (p \ 0.0001). In response to IR, the invasiveness of the UW-B cells also decreased compared to the control cell line. When the data were normalized to those of the irradiated parental UW228-1 cell line (100 ± 0.003 %): the mean invasiveness of the UW-V cells was 97.6 ± 31 %, whereas that of the UW-B cells was 22.6 ± 6.5 %.
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Fig. 7 Mortality of UW-V and UW-B cells. Twenty-four hours after cell seeding, the UW-V and UW-B IR cells exposed to IR were irradiated, and all the samples were analyzed 48, 72 and 96 h after IR. Three replicate flasks were assayed at each time point in each experiment, and each sample was examined in three independent experiments. At each time point, the bar charts represent the mean of the percentages of dead and total cells (±SEM; bar). *p \ 0.05 Fig. 9 Proliferation rate of FFPE MB cases expressed as the Ki67 LI of nuclear b-catenin-positive cells vs. non-nuclear (cytoplasmic) bcatenin-positive cells
28, desmoplastic/nodular (D/N), and 3, MB with extensive nodularity (MBEN). The proliferation rate according to the Ki-67 LI ranged from 30 to 60 %. Nuclear b-catenin was detected in 20 cases (10 %) (14 classic, 2 D/N, 3 LC/A, and 1 MBEN). The median Ki-67 LI of the nuclear b-cateninpositive samples was 40 %, which was lower, although not statistically significant, than the value (50 %) of the nonnuclear beta-catenin samples (Fig. 9).
Fig. 8 Invasion capability of the UW228-1, UW-V, and UW-B cells and that of the UW228-1, UW-V, and UW-B cell lines exposed to IR 24 h after the experimental set up. a The bar charts represent the mean of migrating cells evaluated in three independent experiments. In each sample, the number of migrating cells was reduced compared to the number in corresponding negative controls. The number of migrating cells in the UW228-1 cell line are used as a reference (100 %); b The images represent the bottom surface of membranes containing migrating cells
Statistical analysis revealed a 77.3 % decrease in the number of UW-B invasive cells compared to UW228-1 cells (p \ 0.001). There was a 20.1 % increase in the number of UW-B cells exposed to IR compared to UW-B cells not exposed to IR, with no statistically significant difference (p [ 0.05) (Fig. 8). Proliferation index in nuclear b-catenin-positive tissue samples compared to non-nuclear b-catenin -positive samples The 187 FFPE MB samples were histologically classified as follows: 116, classic; 40, large cell/anaplastic (LC/A);
Discussion The activation of the Wnt pathway has been associated with a good outcome in a subgroup of MBs [2, 3]. However, the biological and molecular explanation for this association remains to be found. It is unclear whether the favorable prognosis in MB patients harboring nuclear positivity for b-catenin is linked to reduced aggressiveness or a different response to therapy [1, 2, 13, 15, 22]. In this study, an MB cell line was stably transfected with human b-catenin cDNA, which encodes a nondegradable form of b-catenin, to evaluate the biological effects in culture conditions and after IR treatment. Transfection with a pCIneo/b-catenin S33Y construct increased the expression of nuclear b-catenin and its target genes, c-myc and cyclin D1, in the UW-B clones. The data in the present study shed light on the activation of Wnt in a UW-B cell line, which is a useful cell system for understanding the biology of Wntactivated tumors. (1) We observed that Wnt activation altered the morphology of the cells and induced differentiation in the
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UW228-1 cells, changing the ratio of polygonal and spindle cells, with enrichment of multipolar spindles. We observed cells with very long, thin cytoplasmic elongations only in the UW-B line. At the ultrastructural level, in club/ spindle cells, which represent the prevalent cellular phenotype in UW-B, we noted features of early differentiation, such as intermediate filaments, toward a neuronal lineage. (2) The remarkable decrease in the growth of the UW-B cells when cultured in standard conditions suggests that Wnt activation reduces cell proliferation, in contrast with its role in other cancers [23–26]. It is well known that nuclear b-catenin may exert disparate effects on cells, potentially in a highly context- or cell-dependent fashion [12, 27–29]. In fact, nuclear b-catenin can promote proliferation and aggressive behavior in some cell systems and apoptosis and/ or growth arrest in others [11]. Recently, Cimmino et al. showed that norcantharidin suppresses Wnt signaling in MB cell lines and has potential therapeutic applications [14]. However, this cannot be ascribed unambiguously to the impairment of Wnt signaling: It may be due to the broadspectrum effect of norcantharidin [3]. In accordance with our data, the proliferation of protracted constitutively activated b-catenin dramatically increased in hGFAP-positive cells in another study [30]. The comparison of the Ki-67 Li of the nuclear b-catenin-positive and b-catenin-negative samples in the present study revealed that it was higher in the latter, with a lower proliferation rate in MB subgroup A in comparison to the other molecular subtypes of MB, which is in agreement with our in vitro data. (3) The marked increase in the cell mortality rate following Wnt activation in the MB cells suggests that mortality could be a cellular response to b-catenin accumulation. The further increase in the cell mortality rate of nuclear b-catenin-positive cells after the radiation treatment compared to their nonirradiated counterparts and the irradiated control cells (UW-V) indicates that b-catenin signaling is associated with enhanced radiosensitivity in MB cells. (4) One of the most striking effects of Wnt signaling activation in our cellular system is that it inhibited the invasion capability of MB cells. The transfected cells had 97 % less invasion capability in comparison to the control cells. Our observation, although limited to an in vitro study of only one MB cell line, may explain the low frequency of disseminated metastatic disease at presentation in patients with Wnt-activated MBs. To verify that our observations are general phenotypes of Wnt activation, we transfected other MB cell lines (DAOY, D283Med, and ONS76). However, UW228-1 was the only cell line with acceptable transfection efficiency. This undoubtedly prevents us from generalizing our results. However, as no commercial human cell lines with nuclear b-catenin accumulation are available, we proceeded with
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this single cell line. The findings of the study can be strengthened by confirming these data in other experimental models. While we are waiting for other in vitro models, our human MB cell line with a nondegradable form of b-catenin seems to be a simple and practical way to study the effect of Wnt activation. Few MB animal models are available, and irradiation of animals is not easy. C-myc and b-catenin cooperate with the loss of p53 to generate multiple members of the primitive neuroectodermal tumor family in mice, but these tumors are histologically similar to LC/A MBs, which are not a common MB variant [27]. On the other hand, orthotopic xenograft mouse models of each molecular subgroup of MB, molecularly faithful to the original patients’ tumors, have been developed [28]. In the near future, these models should facilitate preclinical drug screening for MB, but no specific study on the biological aggressiveness or radiosensitivity of subgroup A has been carried out on tumor models to date [27, 28]. In conclusion, this study, even with its intrinsic limitations, revealed a biological explanation for the decreased aggressiveness of MBs with Wnt activation and the better response of these tumors to radiotherapy compared to other types of cancer. It will be very important to perform a similar study in other in vitro and in vivo models to generalize the important conclusions of this study. Acknowledgments We thank Dr. Mike Bobola and Dr. Charles G. Eberhart for providing the human MB cell line UW228-1 and Dr. Hans Clevers for providing the plasmid construct. Conflict of interest of interest.
The authors declare that they have no conflict
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ZJ, Brun S, Lee Y, Zindy F, Lindsey JC, Taketo MM, Boop FA, Sanford RA, Gajjar A, Clifford SC, Roussel MF, McKinnon PJ, Gutmann DH, Ellison DW, Wechsler-Reya R, Gilbertson RJ (2010) Subtypes of medulloblastoma have distinct developmental origins. Nature 468:1095–1099 Ellison DW, Dalton J, Kocak M, Nicholson SL, Fraga C, Neale G, Kenney AM, Brat DJ, Perry A, Yong WH, Taylor RE, Bailey S, Clifford SC, Gilbertson RJ (2011) Medulloblastoma: clinicopathological correlates of SHH, WNT, and non-SHH/WNT molecular subgroups. Acta Neuropathol 121:381–396 Kool M, Korshunov A, Remke M, Jones DT, Schlanstein M, Northcott PA, Cho YJ, Koster J, Schouten-van Meeteren A, van Vuurden D, Clifford SC, Pietsch T, von Bueren AO, Rutkowski S, McCabe M, Collins VP, Ba¨cklund ML, Haberler C, Bourdeaut F, Delattre O, Doz F, Ellison DW, Gilbertson RJ, Pomeroy SL, Taylor MD, Lichter P, Pfister SM (2012) Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas. Acta Neuropathol 123:473–484 Ellison DW, Onilude OE, Lindsey JC, Lusher ME, Weston CL, Taylor RE, Pearson AD, Clifford SC, United Kingdom Children’s Cancer Study Group Brain Tumour Committee (2005) Betacatenin status predicts a favorable outcome in childhood medulloblastoma: the United Kingdom Children’s Cancer Study Group Brain Tumor Committee. J Clin Oncol 23:7951–7957 Fattet S, Haberler C, Legoix P, Varlet P, Lellouch-Tubiana A, Lair S, Manie E, Raquin MA, Bours D, Carpentier S, Barillot E, Grill J, Doz F, Puget S, Janoueix-Lerosey I, Delattre O (2009) Beta-catenin status in pediatric medulloblastomas: correlation of immunohistochemical expression with mutational status, genetic profiles, and clinical characteristics. J Pathol 218:86–94 Thompson MC, Fuller C, Hogg TL, Dalton J, Finkelstein D, Lau CC, Chintagumpala M, Adesina A, Ashley DM, Kellie SJ, Taylor MD, Curran T, Gajjar A, Gilbertson RJ (2006) Genomics identifies medulloblastoma subgroups that are enriched for specific genetic alterations. J Clin Oncol 24:1924–1931 Cimmino F, Scoppettuolo MN, Carotenuto M, De Antonellis P, Dato VD, De Vita G, Zollo M (2012) Norcantharidin impairs medulloblastoma growth by inhibition of Wnt/b-catenin signaling. J Neurooncol 106:59–70 Clifford SC, Lusher ME, Lindsey JC, Langdon JA, Gilbertson RJ, Straughton D, Ellison DW (2006) Wnt/Wingless pathway activation and chromosome 6 loss characterize a distinct molecular sub-group of medulloblastomas associated with a favorable prognosis. Cell Cycle 5:2666–2670 Kool M, Koster J, Bunt J, Hasselt NE, Lakeman A, van Sluis P, Troost D, Meeteren NS, Caron HN, Cloos J, Mrsic´ A, Ylstra B, Grajkowska W, Hartmann W, Pietsch T, Ellison D, Clifford SC, Versteeg R (2008) Integrated genomics identifies five medulloblastoma subtypes with distinct genetic profiles, pathway signatures and clinicopathological features. PLoS One 3:e3088 Rogers HA, Miller S, Lowe J, Brundler MA, Coyle B, Grundy RG (2009) An investigation of WNT pathway activation and association with survival in central nervous system primitive
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LABORATORY INVESTIGATION
Wnt activation affects proliferation, invasiveness and radiosensitivity in medulloblastoma Roberta Salaroli • Alice Ronchi • Francesca Romana Buttarelli • Filippo Cortesi Valeria Marchese • Elena Della Bella • Cristiano Renna • Caterina Baldi • Felice Giangaspero • Giovanna Cenacchi
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Received: 28 December 2013 / Accepted: 21 September 2014 Ó Springer Science+Business Media New York 2014
Abstract Medulloblastomas (MBs) associated with the Wnt activation represent a subgroup with a favorable prognosis, but it remains unclear whether Wnt activation confers a less aggressive phenotype and/or enhances radiosensitivity. To investigate this issue, we evaluated the biological behavior of an MB cell line, UW228-1, stably transfected with human b-catenin cDNA encoding a nondegradable form of b-catenin (UW-B) in standard culture conditions and after radiation treatment. We evaluated the expression, transcriptional activity, and localization of bcatenin in the stably transfected cells using immunofluorescence and WB. We performed morphological analysis using light and electron microscopy. We then analyzed changes in the invasiveness, growth, and mortality in standard culture conditions and after radiation. We demonstrated that (A) Wnt activation inhibited 97 % of the invasion capability of the cells, (B) the growth of the UWB cells was statistically significantly lower than that of all the other control cells (p \ 0.01), (C) the mortality of
R. Salaroli A. Ronchi F. Cortesi V. Marchese E. D. Bella C. Renna G. Cenacchi (&) Department of Biomedical and Neuromotor Sciences, ‘‘Alma Mater Studiorum’’ University of Bologna, Via Massarenti 9, 40138 Bologna, Italy e-mail: [email protected] F. R. Buttarelli C. Baldi Department of Neurology and Psychiatry, Sapienza University of Rome, 00161 Rome, Italy F. Giangaspero Department of Radiology, Oncology and Anatomo-Pathology, Sapienza University of Rome, 00161 Rome, Italy F. Giangaspero Neuromed IRCCS, 86077 Pozzilli, Italy
irradiated UW-B cells was statistically significantly higher than that of the controls and their nonirradiated counterparts (p \ 0.05), and (D) morphological features of neuronal differentiation were observed in the Wnt-activated cells. In tissue samples, the Ki-67 labeling index (LI) was lower in b-catenin-positive samples compared to non-bcatenin positive ones. The Ki-67 LI median (LI = 40) of the nuclear b-catenin-positive tumor samples was lower than that of non-nuclear b-catenin-positive samples (LI = 50), but the difference was not statistically significant. Overall, our data suggest that activation of the Wnt pathway reduces the proliferation and invasion of MBs and increases the tumor’s radiosensitivity. Keywords Medulloblastoma Beta-catenin Wnt pathway Radiosensitivity
Introduction A medulloblastoma (MB) is a highly invasive embryonal tumor of the cerebellum and one of the leading causes of mortality in pediatric patients [1–3]. Metastatic disease at diagnosis remains the most important negative prognostic clinical marker of treatment failure in MB patients, despite the use of cranio-spinal irradiation and intensive chemotherapy [1, 4]. Postoperative radiotherapy is an effective treatment, but survivors have severe long-term side effects, and the response is sometimes limited by intrinsic radioresistance [3, 5]. Gene expression profiling studies [1–3] have suggested four molecular subtypes of MB, each of which is characterized by a distinct genetic profile, the activation of different oncogenic pathways, and diverse clinical outcomes. Subgroup A and B are characterized by Wnt and Hh signaling, respectively, and subgroups C and
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D are characterized by the expression of neuronal differentiation and photoreceptor genes, respectively [6–11]. Canonical Wnt signaling compromises the ability of a multiprotein complex containing axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3 b (GSK 3b) to target it for degradation and block its nuclear import, thereby inducing stabilization and nuclear localization of b-catenin. Nuclear b-catenin interacts with members of the LEF-TCF family of transcription factors, leading to the activation of various target genes, such as c-myc and cyclin D1 [4]. In sporadic MBs, the Wnt/b-catenin pathway is aberrantly activated in approximately 18–25 % of tumors [12– 14]. This tumor subgroup (subgroup A) is recognized as low risk, presenting in children between the ages of 6 and 12 years; it shows nuclear b-catenin immunopositivity, usually with a classic histological subtype and is associated with activating mutations in ctnnb1 (b-catenin gene) and monosomy of chromosome 6 [8, 9, 11, 15]. The frequency of metastatic disease at presentation is lower than in the other subgroups [4, 10]. Several clues point toward nuclear b-catenin having a positive role in the prognosis of MB, but the underlying molecular mechanisms are unknown [12, 15–17]. It remains unclear whether a favorable prognosis in patients harboring nuclear positivity for b-catenin is linked to reduced aggressiveness of the tumor or a different response to therapy [12]. Although there are numerous human xenograft mouse models of MB, no MB cell line with Wnt activation is available for conducting in vivo studies, which could help to resolve the role of nuclear b-catenin in the prognosis of MB. To overcome these limitations, we utilized the human MB cell line UW228-1, which is stably transfected with human b-catenin cDNA and encodes a nondegradable form of b-catenin. We evaluated changes in growth, mortality, and invasiveness in standard culture conditions and after ionizing radiation (IR) treatment. Moreover, in a series of formalin-fixed paraffin-embedded MB specimens, we compared the differences in the growth of Wnt-activated and nonactivated cases.
Modified Eagle’s Medium (high glucose) and Ham’s Nutrient Mixture F-12 (Euroclone, Milan, Italy), supplemented with 10 % heat-inactivated fetal bovine serum, 1 % penicillin–streptomycin, 1 % 2 mM L-glutamine, and 1 % nonessential amino acids (all produced by Euroclone, Milan, Italy). The cell cultures were expanded and maintained at 37 °C in 5 % CO2. Each experiment was started with cells at passage number eight. Plasmid construction, sequencing, and transfection The plasmid pCI-neo/b-catenin S33Y contains human bcatenin cDNA, which harbors an S33Y mutation downstream of the cytomegalovirus promoter in the NheI and SalI sites of the pCI-neo vector (Promega Corp, Madison, WI). S33Y amino acid substitution renders b-catenin refractory to phosphorylation by GSK 3b and therefore resistant to degradation [19]. Mutations in the human ctnnb1 gene encoding serine 33 substitutions occur in sporadic MBs [12, 14]. The vector also contains the neomycin resistance gene, allowing for the selection of transfected cells resistant to the antibiotic. The construct was kindly provided by Dr. Hans Clevers (Utrecht University, Utrecht, NL). Transfections were carried out using Lipofectamine 2000 reagent (Invitrogen, Paisley, UK) at a ratio of 10:1 (Lipofectamine/DNA, volume/mass), according to the manufacturer’s instructions. Cells were plated at a density of 100,000 per well in 6-well plates and collected 24 h after transfection. Stable transfected clones were derived from UW228-1 cells transfected with either pCI-neo/b-catenin S33Y (UW-B) or the pCIneo empty vector as a control line (UW-V) following treatment with the aminoglycoside antibiotic G418, which was used as a neomycin selection agent. After 48 h of culture growth, G418 was added at a concentration of 750 mg/mL. The cells were cultured in the selection medium for an additional 2 weeks. Clones were isolated by light microscopy following single-cell plating in 96-well plates. To avoid unique insertion events, four clones for UW-B and three clones for UW-V were derived from two different transfection experiments and characterized. Protein extraction and western blots
Materials and methods Cell culture We used a human MB cell line, UW228-1, without Wnt alterations, kindly provided by Dr. Charles G. Eberhart (John Hopkins University, Baltimore, MD), with the agreement of Dr. Mike Bobola (University of Washington, Seattle, WA) [18].This cell line was checked for mycoplasma (negative result) and then maintained in Dulbecco’s
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UW228-1, UW-V, and UW-B nuclear and total proteins were extracted and Western blotting was performed, as previously described [20]. The following primary antibodies were used: rabbit polyclonal anti-b-catenin (H-102, Santa Cruz Biotechnology, Santa Cruz, CA) 1:1,000, mouse monoclonal anticyclin D1 (A-12, Santa Cruz Biotechnology, Santa Cruz, CA) 1:100, mouse monoclonal anti-MYC (Ab-2, Calbiochem, Darmstadt, DE) 1:500, goat polyclonal anti-b-actin (I-19, Santa Cruz Biotechnology,
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Santa Cruz, CA) 1:1,000, and mouse monoclonal antilamin B1 (ZL-b, ABcam, Cambridge, UK) 1:400. The primary antibodies were detected with horseradish peroxidaselabeled secondary antibodies: antirabbit (NA934, GE Healthcare Europe GmbH, Freiburg, DE) 1:10,000, antimouse (NA93, GE Healthcare Europe GmbH; Freiburg, DE) 1:10,000, and antigoat (sc-2020, Santa Cruz Biotechnology, Santa Cruz, CA) 1:1,000. Total b-catenin c-myc, cyclin D1, and b-actin were detected in total protein extracts, and nuclear b-catenin and lamin B were detected in nuclear extracts. b-actin and lamin B were used to determine total and nuclear loading, respectively. The experiments were performed in triplicate. Immunofluorescence The UW-V and UW-B cells were plated at a density of 100,000 per well on glass coverslips in 6-well plates and fixed with 2 % paraformaldehyde (Merck, Darmstadt, DE) in PBS for immunofluorescence staining of b-catenin, as previously described [20]. For each sample, all cells on the coverslip were evaluated. The experiment was performed in triplicate. Light and transmission electron microscopy The cells were plated at a density of 200,000 viable cells per T25 flask on day 0 and were observed every 24 h for 5 days under a light microscope. Every day, ten nonoverlapping optical fields were photographed using a digital camera (Olympus C-5060, Olympus Italy, Segrate, Italy) at 109 magnification to obtain a semiquantitative evaluation of the cell-type composition of the cell population. The experiment was performed in triplicate. The relative amount of different cytotypes in the UW228-1, UW-V, and UW-B lines are reported in Fig. 4. For the ultrastructural analysis, the UV-B and UV-V cells were plated at a density of 76,800 per well in 6-well plates. At 24 and 96 h after seeding, the cells were fixed in 2.5 % of glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and postfixed in 1 % OsO4 in the same buffer. The samples were dehydrated in ethanol and embedded in araldite. All the cells in the thin sections were analyzed under a Philips CM10 TEM (Philips, Amsterdam, NL).
Cell growth analysis and cell mortality assay The number and viability/mortality of the cells were assessed by preparing a 1:1 dilution of the cell suspension in a trypan blue solution (Sigma-Aldrich, St. Louis, MO). The numbers of dead and total cells in every sample were counted in a Neubauer chamber. For growth curve building, the cells were plated at a density of 200,000 viable cells per T25 flask and irradiated on day 0 (24 h after seeding). The cell numbers were assessed every day for 5 days. Three replicate flasks were assayed at each time point in each of three independent experiments. For mortality analysis, 200,000 cells were seeded in T25 culture flasks and collected 48, 72, and 96 h after irradiation. Three replicate flasks were assayed at each time point in each of the three independent experiments. Invasion capability assay The UW228-1, UW-V, UW-B, UW228-1 IR, UW-V IR, and UW-B IR cells (50,000 cells in serum-free medium) were placed in the top chamber of a transwell migration Boyden chamber [21]. The lower chamber was filled with 35 ll of serum (10 %)-containing medium. The transwell insert contained an 8 lm pore membrane (NeuroProbe Inc., Gaithersburg, MD) with a thin layer of Matrigel (BD Bioscience, Bedford, MA). After incubation at 37 °C for 24 h, the cells that had not migrated to the lower chamber were removed from the upper surface of the transwell membrane with a cotton swab. Migrating cells on the lower membrane surface were fixed, stained with a May-Grunwald/Giemsa solution, and counted under a light microscope at 109 magnification. As negative controls, some cells were placed in a Boyden chamber in which the top and lower chambers were filled with serum-free medium. The experiment was performed in triplicate. Statistical analysis The data were analyzed with Dunnet’s test and Bonferroni’s multiple comparison test using the Graphpad software package for Windows (PRISM5). A value of p \ 0.05 was considered statistically significant. MB tissue samples
IR treatment The UW228-1, UW-V, and UW-B cells were plated 24 h prior to irradiation with an Irradiateur Biologique 437C (CIS-BIO; Cedex, France) c-ray machine at a dose of 2 Gy and a dose rate of approximately 2 Gy/min. Postirradiation, the cell growth, cell mortality rate, and invasiveness were evaluated at different time points.
We evaluated 187 MB samples from patients aged 0 through 16 years who were assessed at the Neuropathology Unit of Azienda Policlinico Umberto I, Sapienza University of Rome from 01/01/2009 to 31/12/2012. The cases were categorized according to the WHO classification [1]. All the samples were immunostained for b-catenin and Ki-67 with anti-b-catenin rabbit polyclonal antibody
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(Beckton Dickinson, Franklin Lakes, New Jersey; 1:600) and anti-Ki-67 (MIB-1) mouse monoclonal antibody (Dako, Glostrup, Denmark; 1:200). The immunostaining was performed in a Bond Max Leica autostainer with biotinylated secondary Ab, peroxidase complex, and DAB (all from Ultratech Sciteck, San Jose, CA). In each sample, the number of b-catenin-positive nuclei and staining for Ki-67 were evaluated using a Nikon Eclipse 50i microscope, which was equipped with a 209 objective lens and a Nikon DS-F1 camera, interfaced with a computer equipped with NIS Elements F software. In each slide, images were acquired from areas selected by a neuropathologist (FG) and then analyzed and processed using Image J software. Immunostaining was recorded on ten fields at 29 (1,000 neoplastic cells on average) as a percentage of positive tumor cells. b-catenin was recorded as positive when [10 % of the tumor cells showed nuclear staining. Samples containing a few scattered nuclear b-catenin-positive cells were not regarded as positive. Ki67 staining was recorded as a percentage of positive tumor nuclei (LI).
Results Transfection with the pCI-neo/b-catenin S33Y construct activated Wnt signaling in the UW228-1 cells
Fig. 1 Nuclear expression of b-catenin in the UW228-1, UW-V, and UW-B cells. a The graph shows the relative amount of b-catenin, and the column bars represent the mean of three independent experiments. Nuclear protein extracts were prepared from UW228-1, UW-V, and UW-B cells harvested 24, 28, 32, and 40 h after cell seeding. b The image shows one of three Western blots obtained: lane 1 UW228-1 at 24 h; lane 2 UW-V at 24 h; lane 3 UW-B at 24 h; lane 4 UW228-1 at 28 h; lane 5 UW-V at 28 h; lane 6 UW-B at 28 h; lane 7 UW228-1 at 32 h; lane 8 UW-V at 32 h; lane 9 UW-B at 32 h; lane 10 UW228-1 at 40 h; lane 11 UW-V at 40 h; lane 12 UW-B at 40 h. The expression of lamin B served as a nuclear loading control. Each sample was quantified densitometrically and normalized to the expression of lamin B
Western blot analysis showed an increase in nuclear b-catenin in the UW-B cell line compared to the control cell line, with the greatest elevation occurring 28 h after cell seeding. At this time point, nuclear b-catenin was increased 4.0-fold in the UW-B cells compared to the UW228-1 cells and 4.5-fold compared to the UW-V cells. After 32 h, nuclear b-catenin was increased 2.9-fold in the UW-B cells compared to the UW228-1 cells and 4.4-fold compared to the UW-V cells. Nuclear b-catenin levels did not essentially differ in the UW228-1 and UW-V cell lines at any time point (Fig. 1). The immunofluorescence analysis of the UW-B cells revealed strong b-catenin positivity at the nuclear level and a weak signal in both the cell cytoplasm and membrane in almost all the cells. b-catenin was localized at the perinuclear level in the UW-V cell line and at contact points among the cells (Fig. 2). Nuclear b-catenin in the UW-B cells showed transcriptional activity, as highlighted by a 1.8-fold increase in cyclin D1 and a 1.2-fold increase in c-myc protein levels (Fig. 3).
club/spindle-shaped (*30 %), and isolated giant cells. In comparison with the UW228-1 cell line, the UW-V line had a greater number of club/spindle-shaped cells (*50 %), with short unipolar or bipolar processes. In the UW-B cells, the ratio of polygonal/oval cells to and club/ spindle cells was altered, with the cell population containing a greater number of club/spindle cells (*70 %), with multipolar-directed processes. In addition, around 5 % of the cells had very long, thin cytoplasmic elongations. The remaining 25 % were polygonal/oval-shaped cells (Fig. 4). At the ultrastructural level, the phenotypes of both the transfected and nontransfected cell lines were undifferentiated. However, early neuronal differentiation, such as several intermediate filaments mixed with microtubules running parallel to cytoplasmic elongations, was observed in the club/spindle-shaped cells, which were particularly abundant in the UW-B line. In contrast, the polygonal/ovalshaped cells had abundant cytoplasm, with only a few microtubules and randomly distributed filaments (Fig. 5).
Wnt activation altered cell morphology and induced differentiation in the MB cell lines
Wnt activation altered the cell growth and enhanced the radiosensitivity of the MB cells
The UW228-1 parental cell line is characterized by three morphological cell types: polygonal/oval shaped (*70 %),
The growth of the nuclear b-catenin cell line decreased when cultured in standard conditions compared to the
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J Neurooncol Fig. 2 The expression and localization of b-catenin in the UW-V and UW-B cells. The FITC- secondary antibody was conjugated (green) with polyclonal swine antirabbit immunoglobulins; the nuclei are stained with DAPI (blue). We observed strong nucleopositivity for b-catenin only in the UW-B cells. Magnification of 9100
Fig. 3 Cyclin-D1 (a) and c-myc (b) expression levels in the UW-B cells compared to the UW-V cells 24, 28, 32, 40, and 48 h after cell seeding. Each sample was normalized with b-actin. The bar charts show the mean-fold increase in the UW-B cells compared to the UWV cells based on three independent experiments
vector control line. Statistical analysis of the number of UW-B and UW-V cells 120 h after seeding revealed a 12 % decrease (p \ 0.01, RM ANOVA). At 0, 24, 48, 72, and 96 h, there were 151 ± 4, 203 ± 3, 345 ± 16, 541 ± 79, and 1,096 ± 31 thousands of UW-B cells, respectively. In contrast, there were 148 ± 19, 261 ± 8, 423 ± 12, 809 ± 29, and 1,244 ± 57 thousands of UW-V cells at the same time points. As expected, the cell growth of the vector control line in response to IR treatment was slower than that of its nonirradiated counterpart. The growth of the irradiated UW-B cells was statistically significantly lower than that of all the other cell lines: 125 ± 6, 188 ± 10, 310 ± 28, 479 ± 23, and 693 ± 31 thousands of cells at 0, 24, 48, 72, and 96 h, respectively, and 136 ± 18, 214 ± 10, 396 ± 32, 523 ± 8, and 815 ± 23 thousands of cells for UW-V cells. Ninety-six hours after irradiation (120 h after cell seeding), the number of UW-B cells decreased 37 and 15 % compared to the UW-B cells (p \ 0.001) and UW-V cells, respectively, (p \ 0.01) (Fig. 6). Wnt activation increased the cell mortality rate
Fig. 4 Light microscopy of a UW228-1 polygonal/oval-shaped cells, b UW-V club/spindle-shaped cells, and c UW-B polygonal/ovalshaped cells and cells, with very long, thin cytoplasmic elongations. Magnification of 920. The relative amount of different cytotypes in the UW228-1, UW-V, and UW-B cells are shown in the table. ??? *70 %; ?? *50 %; ? *30 %
At all the time points (72, 96, and 120 h after cell seeding), the cell mortality rate of the UW-B cells was greater than that of the UW-V cells. The cell mortality rate of the UWB cells was 19.97 ± 0.90, 14.30 ± 3.74, and 13.58 ± 0.98 % at 72, 96, and 120 h, respectively, whereas the cell mortality rate of the UW-V cells was 9.80 ± 1.16, 8.53 ± 1.35, and 8.98 ± 0.95 % at the same time points. At the first time point (72 h after cell seeding), the difference in the cell mortality rate of the UW-V and UW-B cells was statistically significant (p \ 0.05). At 72, 96, and 120 h after seeding, the cell mortality rate of the UW-B cells exposed to IR was increased in comparison with that of the irradiated UW-V cells: 16.92 ± 2.90, 14.98 ± 1.66, and 20.19 ± 2.11 %, respectively, for the UW-B cells and 9.05 ± 2.01, 10.95 ± 1.37, and
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Fig. 5 Transmission electron microscopy of a UW228-1 round cells, with an irregular shaped nucleus and a hypertrophic nucleolus, bar = 5 lm; b Unipolar UW-V club/spindle cells; c Multipolar UWB club/spindle cells with very long, thin cytoplasmic elongations,
bar = 10 lm; d Detail of a long cytoplasmic elongation in a UW-B cell. Intermediate filaments mixed with microtubules, running parallel to cytoplasmic elongation, bar = 2 lm
B cells was statistically significantly higher than that of their UW-B nonirradiated counterparts and that of the irradiated UW-V cells, with a variation of 5.68 % (p \ 0.05) and 6.61 % (p \ 0.05), respectively (Fig. 7). Wnt activation inhibited the invasion capability of MB cells
Fig. 6 Cell growth of UW-V, UW-B, UW-V IR, and UW-B IR cells. The dotted line shows the IR-treated cells, and the continuous line denotes the untreated cells. The lines with no symbol are UW-V cells, and those with the symbol white square are UW-B lines. The cells were plated 24 h prior to irradiation. They were harvested and analyzed at the following time points: 0, 24, 48, 72, and 96 h after radiation. Each data point (±SEM; point) is the mean of three independent experiments. Vertical bars indicate the standard deviation; **p \ 0.01; ***p \ 0.001
14.51 ± 0.19 % for the UW-V cells. At 120 h after cell seeding, corresponding to 96 h after the IR treatment (24 h after seeding), the cell mortality rate of the irradiated UW-
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When the data were normalized against the invasiveness rate (100 ± 0.25 %) of the parental UW228-1 cell line, the mean invasiveness rate of the UW-V and UW-B cells was 102 ± 27 and 2.600 ± 0.003 %, respectively. In each sample, the number of migrating cells was counted. From this total, the number of migrating cells found in the corresponding negative controls was subtracted. Statistical analysis revealed a 97.7 % decrease in the number of migrating cells in the UW-B line compared to the UW2281 lines (p \ 0.0001). In response to IR, the invasiveness of the UW-B cells also decreased compared to the control cell line. When the data were normalized to those of the irradiated parental UW228-1 cell line (100 ± 0.003 %): the mean invasiveness of the UW-V cells was 97.6 ± 31 %, whereas that of the UW-B cells was 22.6 ± 6.5 %.
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Fig. 7 Mortality of UW-V and UW-B cells. Twenty-four hours after cell seeding, the UW-V and UW-B IR cells exposed to IR were irradiated, and all the samples were analyzed 48, 72 and 96 h after IR. Three replicate flasks were assayed at each time point in each experiment, and each sample was examined in three independent experiments. At each time point, the bar charts represent the mean of the percentages of dead and total cells (±SEM; bar). *p \ 0.05 Fig. 9 Proliferation rate of FFPE MB cases expressed as the Ki67 LI of nuclear b-catenin-positive cells vs. non-nuclear (cytoplasmic) bcatenin-positive cells
28, desmoplastic/nodular (D/N), and 3, MB with extensive nodularity (MBEN). The proliferation rate according to the Ki-67 LI ranged from 30 to 60 %. Nuclear b-catenin was detected in 20 cases (10 %) (14 classic, 2 D/N, 3 LC/A, and 1 MBEN). The median Ki-67 LI of the nuclear b-cateninpositive samples was 40 %, which was lower, although not statistically significant, than the value (50 %) of the nonnuclear beta-catenin samples (Fig. 9).
Fig. 8 Invasion capability of the UW228-1, UW-V, and UW-B cells and that of the UW228-1, UW-V, and UW-B cell lines exposed to IR 24 h after the experimental set up. a The bar charts represent the mean of migrating cells evaluated in three independent experiments. In each sample, the number of migrating cells was reduced compared to the number in corresponding negative controls. The number of migrating cells in the UW228-1 cell line are used as a reference (100 %); b The images represent the bottom surface of membranes containing migrating cells
Statistical analysis revealed a 77.3 % decrease in the number of UW-B invasive cells compared to UW228-1 cells (p \ 0.001). There was a 20.1 % increase in the number of UW-B cells exposed to IR compared to UW-B cells not exposed to IR, with no statistically significant difference (p [ 0.05) (Fig. 8). Proliferation index in nuclear b-catenin-positive tissue samples compared to non-nuclear b-catenin -positive samples The 187 FFPE MB samples were histologically classified as follows: 116, classic; 40, large cell/anaplastic (LC/A);
Discussion The activation of the Wnt pathway has been associated with a good outcome in a subgroup of MBs [2, 3]. However, the biological and molecular explanation for this association remains to be found. It is unclear whether the favorable prognosis in MB patients harboring nuclear positivity for b-catenin is linked to reduced aggressiveness or a different response to therapy [1, 2, 13, 15, 22]. In this study, an MB cell line was stably transfected with human b-catenin cDNA, which encodes a nondegradable form of b-catenin, to evaluate the biological effects in culture conditions and after IR treatment. Transfection with a pCIneo/b-catenin S33Y construct increased the expression of nuclear b-catenin and its target genes, c-myc and cyclin D1, in the UW-B clones. The data in the present study shed light on the activation of Wnt in a UW-B cell line, which is a useful cell system for understanding the biology of Wntactivated tumors. (1) We observed that Wnt activation altered the morphology of the cells and induced differentiation in the
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UW228-1 cells, changing the ratio of polygonal and spindle cells, with enrichment of multipolar spindles. We observed cells with very long, thin cytoplasmic elongations only in the UW-B line. At the ultrastructural level, in club/ spindle cells, which represent the prevalent cellular phenotype in UW-B, we noted features of early differentiation, such as intermediate filaments, toward a neuronal lineage. (2) The remarkable decrease in the growth of the UW-B cells when cultured in standard conditions suggests that Wnt activation reduces cell proliferation, in contrast with its role in other cancers [23–26]. It is well known that nuclear b-catenin may exert disparate effects on cells, potentially in a highly context- or cell-dependent fashion [12, 27–29]. In fact, nuclear b-catenin can promote proliferation and aggressive behavior in some cell systems and apoptosis and/ or growth arrest in others [11]. Recently, Cimmino et al. showed that norcantharidin suppresses Wnt signaling in MB cell lines and has potential therapeutic applications [14]. However, this cannot be ascribed unambiguously to the impairment of Wnt signaling: It may be due to the broadspectrum effect of norcantharidin [3]. In accordance with our data, the proliferation of protracted constitutively activated b-catenin dramatically increased in hGFAP-positive cells in another study [30]. The comparison of the Ki-67 Li of the nuclear b-catenin-positive and b-catenin-negative samples in the present study revealed that it was higher in the latter, with a lower proliferation rate in MB subgroup A in comparison to the other molecular subtypes of MB, which is in agreement with our in vitro data. (3) The marked increase in the cell mortality rate following Wnt activation in the MB cells suggests that mortality could be a cellular response to b-catenin accumulation. The further increase in the cell mortality rate of nuclear b-catenin-positive cells after the radiation treatment compared to their nonirradiated counterparts and the irradiated control cells (UW-V) indicates that b-catenin signaling is associated with enhanced radiosensitivity in MB cells. (4) One of the most striking effects of Wnt signaling activation in our cellular system is that it inhibited the invasion capability of MB cells. The transfected cells had 97 % less invasion capability in comparison to the control cells. Our observation, although limited to an in vitro study of only one MB cell line, may explain the low frequency of disseminated metastatic disease at presentation in patients with Wnt-activated MBs. To verify that our observations are general phenotypes of Wnt activation, we transfected other MB cell lines (DAOY, D283Med, and ONS76). However, UW228-1 was the only cell line with acceptable transfection efficiency. This undoubtedly prevents us from generalizing our results. However, as no commercial human cell lines with nuclear b-catenin accumulation are available, we proceeded with
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this single cell line. The findings of the study can be strengthened by confirming these data in other experimental models. While we are waiting for other in vitro models, our human MB cell line with a nondegradable form of b-catenin seems to be a simple and practical way to study the effect of Wnt activation. Few MB animal models are available, and irradiation of animals is not easy. C-myc and b-catenin cooperate with the loss of p53 to generate multiple members of the primitive neuroectodermal tumor family in mice, but these tumors are histologically similar to LC/A MBs, which are not a common MB variant [27]. On the other hand, orthotopic xenograft mouse models of each molecular subgroup of MB, molecularly faithful to the original patients’ tumors, have been developed [28]. In the near future, these models should facilitate preclinical drug screening for MB, but no specific study on the biological aggressiveness or radiosensitivity of subgroup A has been carried out on tumor models to date [27, 28]. In conclusion, this study, even with its intrinsic limitations, revealed a biological explanation for the decreased aggressiveness of MBs with Wnt activation and the better response of these tumors to radiotherapy compared to other types of cancer. It will be very important to perform a similar study in other in vitro and in vivo models to generalize the important conclusions of this study. Acknowledgments We thank Dr. Mike Bobola and Dr. Charles G. Eberhart for providing the human MB cell line UW228-1 and Dr. Hans Clevers for providing the plasmid construct. Conflict of interest of interest.
The authors declare that they have no conflict
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