Cancer Gene Therapy (2009) 16, 351–361 r 2009 Nature Publishing Group All rights reserved 0929-1903/09 $32.00 www.nature.com/cgt 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 de- creased 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.