Downregulation of Wnt2 and B-Catenin by Sirna

ORIGINAL ARTICLE Downregulation of Wnt2 and b-catenin by siRNA suppresses malignant glioma cell growth PPu1, Z Zhang1, C Kang1, R Jiang1, Z Jia1, G Wang1 and H Jiang2 1Department of Neurosurgery, Tianjin Medical University General Hospital; Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin, People’s Republic of China and 2Department of Neurology, Henry Ford Hospital, Detroit, MI, USA

Increasing evidence suggests that aberrant activation of Wnt signaling is involved in tumor development and progression. Our earlier study on gene expression profile in human gliomas by microarray found that some members of Wnt family were overexpressed. To further investigate the involvement of Wnt signaling in gliomas, the expression of core components of Wnt signaling cascade in 45 astrocytic glioma specimens with different tumor grades was examined by reverse transcription-PCR and immunohistochemistry. Wnt2, Wnt5a, frizzled2 and b-catenin were overexpressed in gliomas. Knockdown of Wnt2 and its key mediator b-catenin in the canonical Wnt pathway by siRNA in human U251 glioma cells inhibited cell proliferation and invasive ability, and induced apoptotic cell death. Furthermore, treating the nude mice carrying established subcutaneous U251 gliomas with siRNA targeting Wnt2 and b-catenin intratumorally also delayed the tumor growth. In both in vitro and in vivo studies, downregulation of Wnt2 and b-catenin was associated with the decrease of PI3K/p-AKT expression, indicating the interplay between Wnt/b-catenin and PI3K/AKT signaling cascades. In conclusion, the canonical Wnt pathway is of critical importance in the gliomagenesis and intervention of this pathway may provide a new therapeutic approach for malignant gliomas. Cancer Gene Therapy (2009) 16, 351–361; doi:10.1038/cgt.2008.78; published online 24 October 2008 Keywords: glioma; Wnt2; b-catenin; siRNA

Introduction pathways, the canonical Wnt/b-catenin pathway is most extensively studied and has been implicated in tumorigenesis. Wnts belong to a large family of cysteine-rich secreted Receptors for Wnt ligands contain at least 11 identified glycoproteins, consisting of at least 19 members in frizzled (Fz) transmembrane proteins. Most Wnt proteins 2,5,6 humans.1–3 Wnts play important roles in multiple cellular bind to multiple Fz and vice versa. However, recent processes during development, including cell differentia- studies show that one Wnt protein is able to signal in two 3,5,7 tion, migration, polarity and proliferation.1,3 Wnt signals distinct pathways depending on the receptor context. are transduced through at least three distinct signaling The complexity of specific ligand–receptor pairings 1–3,5,7,8 pathways, including canonical Wnt/b-catenin, Wnt/po- remains unclear. It is proposed that Wnt signaling larity and Wnt/calcium pathways. The major function of is initiated after Wnt ligand binding to its target Fz and the canonical Wnt/b-catenin signaling pathway is to co-receptor, low-density lipoprotein receptor-related pro- 3,4,8,9 regulate cell differentiation and proliferation during teins LRP5 or LPR6. In the absence of the Wnt development through the b-catenin/T-cell factor- signals, an intracellular multiprotein complex consisting mediated activation of Wnt target genes. The Wnt/ of adenomatous polyposis coli (APC), axin, glycogen polarity pathway regulates cell polarity and morpho- synthase kinase-3b (GSK-3b) and b-catenin can phos- genetic movements through the activation of c-Jun N- phorylate b-catenin, leading to its subsequent ubiquitina- terminal kinase. The Wnt/calcium pathway regulates cell tion and degradation. On activation of Wnt, the Wnt adhesion and motility through the activation of phos- ligand binds to its Fz receptor and transduces the signal to pholipase C, protein kinase C and Ca2 þ -calmodulin- a cytoplasmic protein Dishevelled (Ds), which in turn dependent kinase II.1,4,5 Among these three signaling inhibits the serine/threonine kinase GSK-3b. The decreased activity of GSK-3b leads to inactivation and dissociation of the multiprotein degradation complex, Correspondence: Professor P Pu, Department of Neurosurgery, Tianjin Medical University General Hospital, 154 Anshan Road, resulting in the accumulation and nuclear translocation of Tianjin 300052, Heping District, People’s Republic of China. cytosolic b-catenin. The b-catenin is the key mediator of E-mail: [email protected] canonical Wnt signaling and nuclear b-catenin is the 1,2,8 Received 29 February 2008; revised 3 August 2008; accepted 12 hallmark of an active Wnt pathway. In the nucleus, September 2008; published online 24 October 2008 b-catenin interacts with T-cell factor/LEF transcription Wnt2/b-catenin in glioma PPuet al 352 factors and activates downstream target genes, including tion of expression with the degree of malignancy of c-Myc, cyclin D1, c-Jun, c-fos, fra-1,and members of AP- gliomas was determined. 1 family, that are mainly involved in the regulation of cell fate and proliferation.2,8,10 RT-PCR analysis In addition to their roles during the development and For RT-PCR analysis, total RNA was extracted from adult tissues, the alteration of components of the Wnt tissue samples using Trizol reagent (Invitrogen, Carlsbad, pathway has also been implicated in oncogenesis. Most CA), reverse-transcribed to cDNA and amplified by PCR. notably, up to 85% of all sporadic colorectal cancers The following PCR conditions for amplification of Wnts (CRCs) have mutations in APC, and b-catenin mutations and its receptor Fz2, Fz5 and b-catenin were used: initial are present in approximately 10% of the remaining denaturation at 94 1C for 2 min, then 94 1C for 30 s, 52 1C CRCs.8 There are many ways to activate Wnt signaling; for 30 s and 72 1C for 30 s for 30 cycles; after the final although APC mutation is frequently found in the cycle, the reaction was held at 72 1C for 5 min. A portion majority of CRCs, it rarely occurs in cancers originating of b-actin gene was also amplified under the same outside of the gastrointestinal tract.11 The mutations of b- conditions as a control. PCR products were electrophor- catenin have been identified in a variety of tumors. esed on 2% agarose gel. The gel was stained with However, the alteration of Wnt per se is less studied in ethidium bromide, digitally photographed and scanned tumorigenesis. In our earlier study, the gene expression with UVI Gel Analyzing System (UVI Tech, Cambridge, profiles of 63 samples of different pathological types of England). Gene expression was calculated as the ratio of human gliomas and 5 samples of human normal brain mean band density of RT-PCR products to that of the tissues were analyzed using Atlas Human Cancer Array internal b-actin control. The pairs of primers used to 1.2, and overexpression of some members of the Wnt amplify each type of cDNA were as follows: pathway were observed in gliomas.12,13 To further evaluate the exact role of Wnt in gliomagenesis, we Target Primers Length analyzed the expression of Wnts in more fresh samples of genes (bp) primary astrocytic gliomas in the current study. We confirmed that Wnt2 and Wnt5a were significantly Wnt1 Upper: 50-GGCTGGGTTTCTGCTACGCT-30 336 overexpressed in gliomas. As it is generally accepted that Lower: 50-GCCTCGGTTGACGATCTTGC-30 Wnt5a activates the non-canonical Wnt pathway, which is Wnt2 Upper: 50-TCCAGGGTGATGTGCGATAAT-30 210 b-catenin independent,14 only the effects of downregula- Lower: 50-GGCAGATCCCGACTACTT-30 Wnt3 Upper: 50-GCTGACTTCCCGACTACTT-30 248 tion of Wnt2 and b-catenin in the canonical Wnt pathway 0 0 by siRNA were investigated in cultured malignant glioma Lower: 5 -ATCTCCGAGGCGCTGTCATA-3 Wnt4 Upper: 50-TCTGACAACATCGCCTACGG-30 337 cells and established subcutaneous gliomas in nude mice. Lower: 50-TCGACTATGGCTACCGCTTTG-30 Wnt5a Upper: 50-TCGACTATGGCTACCGCTTTG-30 331 Lower: 50-TGTTGGTGGGCGAGTTGAA-30 Wnt10b Upper: 50-GAATGCGAATCCACAACAAC-30 289 Materials and methods Lower: 50-GGGTCTCGCTCACAGAAGTCA-30 Tissue samples Wnt13 Upper: 50-CCCGGACTGATCTTGTCTAC-30 180 In total, 45 freshly resected astrocytic glioma samples Lower: 50-ACTGGGTAACACGGGTGACT-30 were collected at the Department of Neurosurgery at frizzled2 Upper: 50-CACCATCGTCATCGCTTGCTA-30 252 Lower: 50-TTGGTGAGGCGAGTGTAGAAC-30 Tianjin Medical University General Hospital during 2004 0 0 and classified according to WHO (World Health Organi- frizzled5 Upper: 5 -GCGGCACCAAGACGGACAAGC-3 296 Lower: 50-GGTGAAACGCCGCCACGACTC-30 zation) categories (2000). Tissues and clinical information b-Catenin Upper: 50-TGCCAAGTGGGTGGTATAGAG-30 332 are obtained as part of an approved study at the Lower: 50-CGCTGGGTATCCTGATGTGC-30 University. There are 19 cases of WHO II grade tumors, b-Actin Upper: 50-GCCGGGACCTGACTGACTA-30 327 including 17 cases of protoplasmic and fibrillary astro- Lower: 50-TGCGGATGTCCACGTCACACT-30 cytomas and 2 cases of oligodendroastrocytomas, 13 cases of anaplastic astrocytomas (WHO III grade) and 13 cases of glioblastomas (WHO IV grade). Four normal brain Immunohistochemical staining tissue specimens were obtained from internal decompres- For immunohistochemical staining, formalin-fixed tissue sion of patients with cerebral injury and temporal lobe samples were prepared as paraffin-embedded sections and resection for epilepsy. A portion of each tissue sample was stained with hematoxylin and eosin for histopathological snap frozen in the liquid nitrogen following resection and diagnosis. Unstained sections were deparaffinized and stored at À70 1C for isolation of RNA, and the remaining incubated overnight at 4 1C with primary antibodies portion was fixed with 10% formalin for histopathologi- against the proteins described above (Santa Cruz, San cal and immunohistochemical examination. All the tumor Diego, CA; in 1:100 dilution), then with biotinylated or normal brain tissues were diagnosed by two indepen- secondary antibody in a dilution of 1:200 at room dent neuropathologists. The expression of core compo- temperature for 1 h, followed by incubation with ABC nents of the Wnr/b-catenin signaling pathway in these peroxidase and diaminobenzedine. The sections were samples was examined by reverse transcription (RT)-PCR counterstained with hematoxylin. The expression levels analysis and immunohistochemical staining. The correla- of Wnt2, Fz2 and b-catenin in each specimen were scored

Cancer Gene Therapy Wnt2/b-catenin in glioma PPuet al 353 according to the percentage of the positive staining cells 72 h after plating, 20 ml of MTT (5 mg mlÀ1) was added to counted in five randomly selected high magnification each well and incubated for additional 4 h at 37 1C. The fields: 0––no expression, 1––positive cell ratio o25%, 2–– reaction was stopped by lysing the cells with 200 mlof positive cell ratio between 26 and 50%, and 3––positive DMSO for 5 min. Optical density was measured at 570 nm cell ratio 450%. and data were expressed as the percentage of control.

Cell culture and transfection Flow cytometric analysis of cell cycle kinetics Human glioblastoma U251MG cell line was obtained Parental and transfected U251 cells in the log phase of from the Institute of Biochemistry and Cell Biology, growth were harvested and fixed with 75% ethanol Chinese Academy of Science. Cells were cultured in six- overnight at 4 1C, and then incubated with RNase at well plates in DMEM supplemented with 10% fetal calf 37 1C for 30 min. Cell nuclei were stained with propidium serum. At 24 h after plating, the complete medium was iodide for 30 min. A total of 10 000 nuclei were examined replaced with serum-free medium. Cells were then by a FACS Calibur flow cytometer and DNA histograms transfected with 20 ng/10 ml siRNA using lipofectamine were analyzed by Modifit software (Becton Dickinson, (Invitrogen). After 6 h, serum-free medium was replaced Franklin Lakes, NJ). again with complete medium and cultured at 37 1Cin5% CO2. The target siRNA sequences against Wnt2 and b- Detection of cell apoptosis by Annexin staining and catenin were 50-GAAGATGGGAAGCGCCAAG-30 and TUNEL method 50-CAGGGGGUUGUGGUUAAGCUCUU-30, respec- Annexin V-cy3-labeled Apoptosis Detection kit 1 (Ab- tively. A scramble siRNA sequence (50-TTCTCCGAAC cam, Cambridge, England) was used for detection of GTGTCACGT-30) was used as a control (Gima Biol apoptotic cells by flow cytometry as described earlier.15 Engineering Inc., Shanghai, China). The data were analyzed using Cell Quest software (Becton Dickinson). The extent of cell apoptosis in the tumor Western blot analysis specimens was evaluated by TUNEL (TdT-mediated At 48–72 h after transfection with Wnt2 or b-catenin dUTP nick end labeling) method using an in situ Cell siRNA, total proteins from control and transfected U251 Death kit (Roche, Basel, Switzerland). Cell nuclei were glioma cells were extracted and the protein concentration counterstained with Hoechst 33342, visualized by fluor- was determined by Lowry method. 40 mg of protein escent microscopy and analyzed by IPP5.1 (Olympus, lysates from each sample was subjected to SDS-PAGE on Tokyo, Japan). 10% SDS-polyacrylamide gel. Separated proteins were transferred to a PVDF membrane The membrane was Three-dimensional cell growth on matrigel incubated with primary antibodies against Wnt2, Fz2, p- Each well of a 24-well plate was precoated with 200 mlof GSK3b, b-catenin, PI3K, p-AKT, cyclin D1 and c-Myc undiluted phenol red-free matrigel (BD Biosciences, (Santa Cruz; 1:500 dilution), followed by HRP-conju- USA). At 6 h after transfection with siRNA, cells were gated secondary protein (Zymed, San Diego, CA; 1:1000 harvested, diluted to a density of 2 Â 104 in 200 mlof dilution). The specific protein was detected using a complete medium per well, mixed with 100 ml of undiluted SuperSignal protein detection kit (Pierce, USA). After ice-cold matrigel for a ratio of 2:1 (v/v) and laid over the washing, the membrane was reprobed with an antibody bottom layer. After gelling, complete culture medium was against b-actin (1:500 dilution). The band density of added and changed every 2–3 days. Cell morphology was specific proteins was quantified after normalization with assessed at day 14 by capturing images at Â 40 the density of b-actin. magnification.

Immunofluorescence staining Establishment of subcutaneous glioma model and At 48–72 h after transfection with siRNA, the transfected treatment with siRNA targeting Wnt2 and b-catenin and parental cells growing on the coverslips were fixed All the animal experiments were carried out according to with À20 1C methanol, blocked with 1% BSA-PBS for the regulations and internal biosafety and bioethics 30 min and incubated overnight at 4 1C with primary guidelines of Tianjin Medical University and Tianjin antibodies (1:100 dilution) against Wnt2, Fz2, p-GSK3b, Municipal Science and Technology Commission. Female b-catenin, PI3K, p-AKT, cyclin D1 and c-Myc, then immune-deficient nude mice (6 weeks old; BALB/C-nu) incubated with fluorescein isothiocyanate/tetraethyl rho- were purchased from the animal center at the Cancer damine isothiocyanate (TRITC)-labeled secondary anti- Institute of the Chinese Academy of Sciences, bred at the body (1:200 dilution) for 30 min at 37 1C, washed with facility of laboratory animals, Tianjin Medical University, PBS and mounted with 0.5 M Na2CO3–50% glycerol. The and housed in microisolator individually ventilated cages images were captured by a fluorescence microscope. with water and food. A subcutaneous U251 glioma xenograft model was established as described earlier.16 MTT assay Once the tumor size reached approximately 5 mm in The viability of control and siRNA-transfected U251 cells diameter, the mice were randomly divided into four was measured by MTT (3-(4,5-dimethylthiazol-2)-2,5- groups (eight mice per group): (1) control untreated diphenyltetrazolium bromide) assay. Briefly, 4 Â 103 cells group; (2) Wnt2 siRNA-treated group; (3) b-catenin were plated into each well of a 96-well plate. At 24, 48 and siRNA-treated group and (4) scramble siRNA

Cancer Gene Therapy Wnt2/b-catenin in glioma PPuet al 354 (scr-siRNA) treated group. siRNA/oligofectamine mix- was also elevated in gliomas and higher in malignant ture (25 ml) containing 400 pmol of Wnt2 or b-catenin gliomas than in low-grade gliomas (Figure 1c). Spearman siRNA was injected intratumorally. The same amount of rank correlation analysis showed that Wnt2 expression scramble siRNA was used for control treatment. Because was positively correlated with the expression of all these of the short effect of synthetic siRNA, the siRNAs were proteins. injected every 4 days until the end of experiment. During the 32-day observation period, the tumor volume was measured with a caliper every 3 days using the following 2 Western blot analysis and immunostaining of Wnt2, formula: volume ¼ length Â width /2. At the end of b observation, the mice were killed and the tumor speci- -catenin and related proteins in U251 cells transfected b mens were prepared as paraffin-embedded sections. The with siRNA targeting Wnt2 or -catenin Western blot analysis showed that the expression of expression levels of Wnt2, Fz2, p-GSK3b, b-catenin, Wnt2, Fz2, p-GSK3b, b-catenin, cyclin D1, PI3K and p- PI3K, p-AKT, cyclin D1 and c-Myc were examined by AKTwas decreased, whereas c-Myc expression was not immunohistochemical staining and cell apoptosis was altered in U251 cells transfected with siRNA targeting determined by the TUNEL method. Wnt2 or b-catenin (Figure 2a). The expression of all these Statistical analysis proteins was similarly changed in U251 cells transfected A commercially available software package SPSS10.0 was with Wnt2 or b-catenin siRNA as detected by immuno- employed for statistical analysis of the data obtained. histochemical staining (Figures 2b and c). One-way analysis of variance and Spearman rank correlation analysis were used to analyze the significance between groups. The LSD method of multiple compar- The effect of Wnt2 and b-catenin siRNA on the viability, isons with parental and control groups was used when the apoptosis and invasive ability of U251 cells probability for analysis of variance was statistically The viability of U251 cells transfected with siRNAs was significant. Statistical significance was determined at determined by MTT assay. The viability of the cells Po0.05 level. transfected with either Wnt2 or b-catenin siRNA was decreased starting 24 h after transfection and continued to reduce during the 3-day observation period, whereas the Results viability of the cells transfected with scramble siRNA was The expression of the major components of Wnt not suppressed as compared with the parental cells. There signaling pathway in gliomas was no significant difference in the decrease in cell The mRNA expression of the important members of Wnt viability using either Wnt2 or b-catenin siRNA ligands and receptors, as well as its mediator b-catenin, (Figure 3a). was determined by RT-PCR in 45 astrocytic glioma The cell cycle kinetics of the parental and transfected specimens and 4 normal brain samples. The expression of cells showed that the S-phase fraction was decreased and Wnt2, Wnt5a and Fz2 mRNA was significantly upregu- the cell cycle was arrested in G0/G1 phase in U251 cells lated in gliomas as compared with normal brain samples. transfected with Wnt2 siRNA and b-catenin siRNA as There was no mRNA differential expression of Wnt3, compared with control cells (Figure 3b). These results Wnt4, Wnt10b, Wnt13, and b-catenin between glioma indicate the delay in cell cycle progression and decrease in and normal brain tissues, whereas Wnt1 and Fz5 mRNA cell proliferation in U251 cells transfected with siRNA showed either trace or no expression in normal brain or targeting Wnt2 or b-catenin. glioma samples (Figures 1a and b). Annexin V staining showed that apoptotic cells in U251 On the basis of the RT-PCR results, the expression of cells transfected with Wnt2 or b-catenin siRNA were Wnt2, Fz2, b-catenin and the downstream effectors cyclin significantly increased as compared with those in the D1 and c-Myc, as well as PI3K and p-AKT, were parental cells and cells transfected with scramble siRNA examined by immunohistochemistry. Wnt2, Fz2 and b- (Figure 3c). These findings suggest that siRNA targeting catenin were overexpressed in astrocytic gliomas as Wnt2 and b-catenin is able to induce apoptosis in glioma cells. compared with the normal brain tissues, and their Invasive growth is an important biological character- expression levels were elevated with the ascending order istic of malignant glioma cells. The invasive ability of of tumor malignancy (Table 1). It is interesting to note U251 cells transfected with siRNA targeting Wnt2 or b- that no mRNA differential expression of b-catenin was catenin was assessed by in vitro three-dimensional cell observed between normal brain tissues and different growth in matrigel matrix. The parental U251 cells and grades of gliomas, whereas b-catenin protein expression cells transfected with scramble siRNA grew as aggregated was markedly higher in gliomas than that in normal brain colonies from which highly invasive cells spread to form tissues, and the expression was further enhanced with the dendritic extensions, whereas the cells transfected with increasing malignant degree of tumors. These results siRNA targeting Wnt2 or b-catenin formed colonies indicate that increased b-catenin protein expression may without or with only traces of extended branching not be the result of the increased transcription, but is (Figure 3d). These results suggest that knockdown of likely derived from the inhibition of its degradation. The Wnt2 and b-catenin expression reduces the aggressive expression of cyclin D1, c-Myc, PCNA, PI3K and p-AKT capability of malignant glioma cells.

Cancer Gene Therapy Wnt2/b-catenin in glioma PPuet al 355

Figure 1 The mRNA expression of core components of Wnt pathways. (a) The mRNA expression of Wnt1–4, Wnt5a, Wnt10b, Wnt13, Fz2, Fz5 and b-catenin in astrocytic gliomas with different grades and normal brain tissues detected by semiquantitative reverse transcription (RT)-PCR. Lanes 1–6: WHO (World Health Organization) IV gliomas; 7–12: WHO III gliomas; 13–18: WHO II gliomas; 19–22: normal brain tissues. (b) Wnt1 and Fz5 had trace or no expression in either gliomas or normal brain tissues, whereas Wnt3, Wnt4, Wnt10b, Wnt13 and b-catenin had no significant differential expression between gliomas and normal brain tissues. Wnt2, Wnt5a and Fz2 were overexpressed in astrocytic gliomas as compared with the normal brain tissues. (c) Expression of Wnt2, Fz2, b-catenin, cyclin D1, c-Myc, PCNA, PI3K and p-AKT in different grades of gliomas examined by immunohistochemistry ( Â 200). Wnt2, Fz2 and b-catenin were overexpressed in gliomas as compared with normal brain tissues and elevated with the ascending order of tumor malignancy. Wnt2 expression was positively correlated with the expression of the remaining above-mentioned proteins as indicated by Spearman rank correlation analyses (Po0.01, except PCNA, Po0.05). The anti-glioma effect of siRNA targeting Wnt2 and 20.35 mm3. During the first 2 weeks of the observation b-catenin in vivo period, the tumors in either control or treated groups The in vivo anti-glioma effect of siRNA targeting Wnt2 grew slowly and there was no marked difference in tumor and b-catenin was investigated using U251 subcutaneous size between them. However, starting at day 16 after the glioma xenograft models. The mean volume of tumors in first treatment, the tumors in the control and scr-siRNA- all experimental mice before treatment was 82.97± treated mice grew rapidly until the end of the observation

Cancer Gene Therapy Wnt2/b-catenin in glioma PPuet al 356 period on day 32, and one of the control mice with the Expression of Wnt2, b-catenin and related proteins in largest volume of tumor died of cachexia. The tumors tumors treated with siRNA targeting Wnt2 and b-catenin treated with Wnt2 or b-catenin siRNA maintained a Similar to the results obtained from the in vitro study, the slower growth rate (Figures 4a and b). There was expression of Wnt2, Fz2, p-GSK3b, b-catenin, cyclin significant difference in tumor volume between the D1, PI3K and p-AKTin tumor specimens from Wnt2- or control and Wnt2- or b-catenin siRNA-treated mice in b-catenin siRNA-treated mice was prominently down- the last half of the observation period (Po0.01), but no regulated, whereas the c-Myc expression was not affected difference in tumor volume between Wnt2- and b-catenin (Figures 4c and d). There was no significant difference siRNA-treated mice. between tumors treated with Wnt2 or b-catenin siRNA either. However, the downregulation of PI3K signaling in Wnt2/b-catenin siRNA-treated tumors further demonstrated that cross-talk might exist between the Wnt and Table 1 Wnt2, frizzled2 and b-catenin expression in different grades of gliomas PI3K pathways.

2 Target Grade n Positive Total w P Detection of cell apoptosis in tumor samples treated rate (%) with Wnt2 and b-catenin siRNA + ++ +++ À Cell apoptosis in tumor samples derived from control and Wnt2 NB 4 0 0 0 0 4 treated mice was examined by the TUNEL method, which II 9 8 2 0 52.6 19 22.042 0.009 showed that the number of apoptotic cells was dramati- III 4 5 3 1 69.2 13 cally increased in the tumors treated with Wnt2 or IV 1 3 5 4 92.3 13 b-catenin siRNA as compared with those in tumor specimens from control or scr-siRNA-treated mice Frizzled2 NB 4 0 0 0 0 4 (Figures 5a and b). There was no difference in cell II 10 7 2 0 47.4 19 21.699 0.010 apoptosis induced by either Wnt2 or b-catenin siRNA. III 3 5 4 1 76.9 13 IV 2 2 4 5 84.6 13

b-Catenin NB 2 1 1 0 50 4 Discussion II 5 9 5 0 73.7 19 21.083 0.012 III 1 5 7 0 92.3 13 Malignant glioma is a highly invasive and challenging IV 0 3 6 4 100 13 tumor of the central nervous system. Currently available

Figure 2 The expression of Wnt2, Fz2, p-GSK3b, b-catenin, cyclin D1, c-Myc, PI3K and p-AKT in U251 cells treated with Wnt2 siRNA and b- catenin siRNA. (a) As detected by western blot analysis, siRNA targeting Wnt2 or b-catenin in U251 cells downregulated Wnt2 and b-catenin as well as expression of Fz2, p-GSK3b, cyclin D1, PI3K and p-AKT, whereas c-Myc expression was not significantly changed. (b) Expression of Wnt2, Fz2, p-GSK3b and b-catenin in U251 cells transfected with siRNA targeting Wnt2 or b-catenin as detected by immunofluorescence staining ( Â 400). Transfection with either Wnt2 or b-catenin siRNA downregulated the expression of Wnt2, Fz2, p-GSK3b and b-catenin significantly. (c) Expression of cyclin D1, c-Myc, PI3K and p-AKT in U251 cells transfected with Wnt2 siRNA or b-catenin siRNA as visualized by fluorescent microscopy ( Â 400). After transfection with siRNA targeting Wnt2 or b-catenin, the expression of cyclin D1, PI3K and p-AKT was lowered, but c-Myc expression was not altered.

Cancer Gene Therapy PPu Wnt2/ tal et b ctnni glioma in -catenin

Figure 3 Glioma cell viability, invasion and apoptosis were affected by transfection with Wnt2 or b-catenin siRNA. (a) Cell viability of U251 cells transfected with scramble, Wnt2 or b- catenin siRNA was determined by MTT ((3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide)) assay. The viability of cells treated with siRNA targeting Wnt2 or b-catenin was

acrGn Therapy Gene Cancer decreased during the 72-h observation period. (b) Cell cycle kinetics of U251 cells transfected with siRNA targeting Wnt2 or b-catenin was determined by flow cytometry. S-phase fraction was lowered and more cells were arrested in G0/G1 phase. (c) Apoptotic Index (AI) of control and transfected U251 cells was examined by Annexin V staining. As compared with the control and cells transfected with scr-siRNA, the AI of cells transfected with siRNA targeting Wnt2 or b-catenin was increased significantly (Po0.01). (d) Matrigel three-dimensional (3D) growth in control and transfected U251 cells demonstrated that control cells and cells transfected with scr-siRNA possessing highly invasive ability formed colonies with stellate-like extensions, whereas the cells transfected with siRNA targeting Wnt2 or b-catenin formed colonies without or with only traces of extended branching. 357 Wnt2/b-catenin in glioma PPuet al 358

Figure 4 The antiglioma effect of siRNA targeting Wnt2 and b-catenin in vivo.(a) Growth curves of subcutaneous U251 gliomas in nude mice with different treatments revealed that tumor growth significantly slowed down in Wnt2- or b-catenin siRNA-treated mice. (b) The appearance of xenograft subcutaneous U251 gliomas in control and differently treated groups. It was shown that the xenograft tumors treated with siRNA targeting Wnt2 or b-catenin were much smaller than those in control or scr-siRNA-treated mice. (c) Expression of Wnt2, Fz2, p-GSK3b and b- catenin in subcutaneous U251 gliomas treated with siRNA targeting Wnt2 or b-catenin was suppressed, as examined by immunohistochemistry ( Â 200). (d) Expression of cyclin D1, PI3K and p-AKT in subcutaneous U251 tumors treated with Wnt2 or b-catenin siRNA was lowered, whereas c-Myc expression was not changed, as detected by immunohistochemistry ( Â 200).

combined therapies offer only limited benefits for patients activation of the Wnt pathway is broadly involved in with malignant gliomas. There is an urgent need to oncogenesis. A number of Wnt genes, including Wnt1, develop new therapeutic approaches by better under- Wnt2, Wnt3a, Wnt5a, Wnt7a, Wnt7b, Wnt10b and standing the molecular pathogenesis of gliomas. Wnt13, have been associated with cell transformation The Wnt signaling pathway is essential for development and some tumor development.4,5,11,14,17,18 However, the and increasing evidence indicates that inappropriate report on the activation of Wnts in human brain tumors is

Cancer Gene Therapy Wnt2/b-catenin in glioma PPuet al 359

Figure 5 Wnt2 and b-catenin siRNA induce tumor cell apoptosis. (a) Apoptotic cells were increased in subcutaneous U251 gliomas treated with siRNA targeting Wnt2 or b-catenin detected by the TUNEL (TdT-mediated dUTP nick end labeling) method ( Â 200). (b) Apoptotic Index in subcutaneous U251 gliomas treated with siRNA targeting Wnt2 or b-catenin as compared to control, *Po0.01. limited. Howng et al.17 used RT-PCR analysis to immunohistochemistry shows that b-catenin protein investigate the expression of four Wnt genes, Wnt1, expression is lower in normal brain as compared with Wnt5a, Wnt10b and Wnt13, and other related compo- glioma samples. This finding suggests that b-catenin nents of Wnt signaling in a total of 45 brain tumor overexpression in gliomas may not result from increased samples (16 astrocytic tumors, 15 meningiomas and 14 transcription, but is likely due to the reduced degradation pituitary adenomas). The study showed that Wnt5a (41/ and accumulation. Accumulation of b-catenin protein has 45, 91%), Wnt10b (37/45, 82%) and Wnt13 (35/45, 78%) been identified as a key oncogenic process in the were expressed in most brain tumors, but no differential development of cancers. Utsuki et al.23 examined the expression was observed among three histopathological expression of b-catenin together with N-cadherin in 45 types of tumors, whereas Wnt1 was less expressed (6/45, astrocytic gliomas of different grades by immunohisto- 13%). However, no normal brain tissues were used as chemistry and found that their expression levels were control in the study. In our present study, the expression elevated with the ascending order of the tumor grade. The of seven Wnts (Wnt1, Wnt2, Wnt3, Wnt4, Wnt5a, activation or mutation of its upstream components, such Wnt10b and Wnt13), two Fzs (Fz2 and Fz5) and b- as Wnt activation, APC and axin mutation and aberra- catenin, was examined in 45 astrocytic gliomas and 4 tion of some other related molecules, may lead to the normal brain tissues by RT-PCR analysis. It was stabilization and accumulation of b-catenin. In addition, observed that Wnt2, Wnt5a and Fz2 were not expressed mutation of the b-catenin (CTNNB1) gene may also in normal brain tissues, but overexpressed in gliomas, and prevent its phosphorylation and degradation. CTNNB1 their expression levels were correlated positively with the gene has been found to be mutated in a variety of tumor grades. Similar observations were demonstrated by tumors.2,8,24–28 Most of the CTNNB1 gene mutations immunohistochemical staining. Wnt2 has been reported to occur in or around exon 3 of the CTNNB1 gene, affecting be upregulated in gastric cancer, CRC, melanoma, non- the phosphorylation sites for GSK3b and rendering small cell lung cancer and mesothelioma.19–22 However, the b-catenin refractory to degradation. There are a few expression of Wnt2 in gliomas has not been reported reports on the mutation of b-catenin in brain tumors, earlier. On the basis of our results from the present study, it including medulloblastomas, pituitary adenomas, cranio- seems that Wnt2 is also overexpressed in gliomas. pharyngiomas and astrocytic gliomas. However, the It should be noted that b-catenin mRNA expression in frequency of missense mutation in exon 3 of the CTNNB1 glioma samples shows no significant difference with that gene is significantly lower than the accumulation of of normal brain tissues as detected by RT-PCR. However, b-catenin,28–31 which, taking into account these limited

Cancer Gene Therapy Wnt2/b-catenin in glioma PPuet al 360 data, suggests that mutation of the CTNNB1 gene may be D1 but not c-Myc expression. Thus, the result of our not a major molecular event that results in the accumula- present study supports the view that c-Myc might not be a tion of b-catenin in the aberration of the Wnt/b-catenin critical target of Wnt/b-catenin signaling-mediated onco- pathway in either astrocytic gliomas or other types of genic events. brain tumors. Knockdown of Wnt/b-catenin simultaneously sup- As the Wnt2 activation and b-catenin accumulation are presses the activation of the PI3K/AKTpathway, which the hallmark of an active canonical Wnt pathway, which is one of the most important downstream effectors of is strongly involved in tumorigenesis, the silencing effect EGFR, suggesting the cross-talk between Wnt/b-catenin of Wnt2 and b-catenin expression by RNA interference and PI3K/AKTpathways. Tan et al.37 found that the on the cell growth of malignant gliomas are studied both transgenic mice overexpressing b-catenin in liver showed in vitro and in vivo. increased EGFR expression. The direct interaction of The in vitro studies have shown that transfection with Wnt/b-catenin signaling and EGFR/erbB2 heterodimers Wnt2 or b-catenin siRNA reduces the cell viability and has also been identified in mammary tumors.38 In invasive ability, whereas the apoptosis is induced. addition, a cross-talk has been reported between the Transfection with Wnt2 or b-catenin siRNA down- Wnt and the Hedgehog (Hh) as well as the Notch regulates the expression of not only Wnt2, b-catenin signaling pathways in the tumorigenesis.39,40 Using and its downstream effector, cyclin D1, but also PI3K and microarray analysis, Huang et al.41 demonstrated that p-AKT. However, c-Myc overexpression is not affected some important signaling pathways, such as PTEN-PI3K- by the siRNA transfection. There is no apparent AKT, NF-kB and p53, were involved in the apoptosis of difference between the effect of Wnt2 and b-catenin HeLa cells induced by silencing of b-catenin expression. siRNA on the suppression of the malignant phenotype in Taken together, these data indicate that direct and U251 cells. As expected, the b-catenin expression indirect links of Wnt/b-catenin with other signaling decreased in U251 cells transfected with Wnt2 siRNA. pathways are intertwining and complex. Further studies However, when b-catenin siRNA was transfected to U251 are needed to explore the precise mechanism underlying cells, Wnt2 expression in the upstream of the signaling the Wnt/b-catenin pathway in oncogenesis. pathway was also downregulated, which seems unex- In summary, results of the present study demonstrate pected. We believe that this is not due to the off-target that knockdown of the canonical Wnt signaling pathway effect as b-catenin-specific sequence has been searched using Wnt2 or b-catenin siRNA effectively suppresses the through Blast to avoid the homology to other genes as malignant glioma cell growth. RNA interference against described by Bouguignon et al.32 The signaling cascades Wnt2 and b-catenin has similar effects in the inhibition of usually do not operate in isolation, but integrate stimuli in cell proliferation of malignant pleural mesothelioma and cross-talked pathways and may have reciprocal feedback pediatric hepatic tumors.19,42 Therefore, the canonical regulation. Similarly, a signaling cascade not only Wnt signaling pathway may be a potential new ther- transmits the effects to the downstream members, a apeutic target for various human cancers.43 possibility may also exist that a feedback mechanism transmits information upwards, as suggested by Ventura et al.33 This observation and its underlying mechanism Acknowledgements should be further investigated. in vivo The results of the studies further confirm that This work is supported by National Natural Science Wnt2 or b-catenin siRNA inhibits the cell growth of Foundation of China, Grant number: 30300365, Program xenograft subcutaneous gliomas. Meanwhile, decreased for New Century Excellent Talents in University (NCET- expression of Wnt2 and b-catenin is consistent with the 07-0615) and Tianjin Science and Technology Committee, downregulation of Fz2, p-GSK3b, cyclin D1, PI3K and Grant Number 06YFSZSF01100. p-AKTexpression in tumors treated with target siRNAs, whereas c-Myc expression level remains unchanged. The c-Myc is known to be one of the downstream targets of the Wnt pathway and there is synergistic action between References Wnt and c-Myc in promoting oncogenic transformation 34 through the inhibition of apoptosis. However, knock- 1 Miller JR. The Wnts. Genomic Biol 2001; 3: 1–8. down of Wnt/b-catenin signaling fails to inhibit c-Myc 2 Karim R, Tse Q, Putti T, Scolyer R, Lee S. The significance expression in U251 cells in the present study. Kolligs of the Wnt pathway in the pathology of human cancers. et al.35 reported that mutated b-catenin transforms Pathology 2004; 36: 120–128. 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