| Article Access Statistics|
| Viewed||2075 |
| Printed||59 |
| Emailed||0 |
| PDF Downloaded||56 |
| Comments ||[Add] |
| Cited by others ||2 |
Click on image for details.
|Year : 2014 | Volume
| Issue : 1 | Page : 57-61
The expression of disabled-2 is common reduced in meningiomas
Ziyin Zhang1, Yihua Chen2, Xuemei Xie2, JianJian Tang1
1 Department of Neurosurgery, Meishan City People's Hospital, Meishan 620010, China
2 Department of Pathology, People's Liberation Army General Hospital of Chengdu Military Region, Chengdu 610083, Sichuan, China
|Date of Submission||24-Oct-2013|
|Date of Decision||15-Jan-2014|
|Date of Acceptance||31-Jan-2014|
|Date of Web Publication||07-Mar-2014|
|Date of Print Publicaton||07-Mar-2014|
Department of Pathology, People's Liberation Army General Hospital of Chengdu Military Region, Chengdu 610083, Sichuan
Source of Support: It is supported by the national natural science foundation of China (81372497), Conflict of Interest: None
Aims: Disabled-2 (Dab2) is frequently down-regulated in several types of cancers. We examined the expression level of Dab2 in human meningiomas and meningioma cells, aimed to investigate its role in the oncogenesis and development of meningiomas. Materials and Methods: Western blot analysis was employed to detect Dab2 expression in 90 fresh tissues of meningiomas, 10 leptomeninges and two kinds of human malignant meningioma cell lines. Independent samples t-test, analysis of variance, Pearson Chi-square test and likelihood ratio test were used to analyze the expression level of Dab2 and its relations to clinic-pathological characteristics of meningiomas. Results: Dab2 was significantly down-regulated in classic meningiomas than the atypical or anaplastic meningiomas. The reduced or loss of expression of Dab2 were significantly correlated with the lower classification of meningiomas and negatively correlated with the invasive ability of adjacent tissues. Furthermore, it was reduced or lost in malignant meningioma cell lines (IOMM-Lee and KT21-MG1). The lower classification of meningiomas correlated with previous comorbidities; not with the gender, age of patients and smoking. Conclusions: Dab2 is expressed at variable level in meningiomas with different grade of malignancy and probably plays a pivotal role in the early stage of oncogenesis of malignant meningiomas.
Keywords: Classification, disabled-2, meningiomas, reduced, western blot
|How to cite this article:|
Zhang Z, Chen Y, Xie X, Tang J. The expression of disabled-2 is common reduced in meningiomas. Neurol India 2014;62:57-61
| » Introduction|| |
Meningiomas account for approximately 25% tumors of the central nervous system (CNS) and the pathogenesis of these tumors is unclear. Most of the meningiomas are non-invasive according to the classifications of World Health Organization (WHO).  However, the atypical or anaplastic meningiomas have a high risk of recurrence and they grow rapidly with the associated complications related to mass effect and elevated intracranial pressure, especially when meningiomas are in critical locations. , Recent studies have shown that reduced expression of disabled-2 (Dab2) can promote epithelial - to-mesenchymal transition and may promote the oncogenesis of mesenchymomas. , As a clear putative tumor suppressor gene, , aberrantly low expression of Dab2 has been reported in several human malignant cancers, including breast cancers,  urothelial carcinomas of the bladder,  lung cancers , and gastric carcinomas.  However, expression of Dab2 in CNS tumors have not yet been reported. In the present study, we first examined the expression level of Dab2 in fresh tissues of meningiomas and human meningioma cells by western blot. We also compared the expression level of Dab2 in different grades of meningiomas and investigated the relationships between Dab2 expression and clinic-pathological characteristics of meningiomas in order to explore the possible mechanisms of Dab2 in the oncogenesis and development of meningiomas.
| » Materials and Methods|| |
A total of 90 fresh tissue samples of meningiomas were collected from patients who underwent surgery for meningioma at the People's Liberation Army General Hospital of Chengdu Military Region between January 2009 and December 2012. Patients included 58 females and 32 males with an age range between 42 and 77 years (mean age 59 ± 6.5 years). Tumors were classified as grade I (classic meningiomas, n = 46), grade II (atypical meningiomas, n = 24) and grade III (anaplastic meningiomas, n = 20) according to WHO classification.  Six cases of non-tumor meningoceles were obtained from autopsies and four cases from patients with cerebral trauma, all non-tumor cases. Fresh tissues were stored at − 70°C liquid nitrogen. This study was approved by the committee on Human Research at General Hospital of Chengdu Military Region and strictly conducted following the regulations of the institutional review boards. Informed consent were obtained from the every patients.
Human meningeal cell (HMC) line and two human malignant meningioma cell lines (IOMM-Lee and KT21-MG1) were examined in this study (Sciencell, USA). Cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and streptomycin at 37°C in a 5% CO 2 humidified atmosphere.
Protein extraction and western blot analysis
Fresh tissues of meningiomas and leptomeninges were cut into very small pieces and made into cell suspension. Adherent cells were digested with trypsin. Samples were lysed in radioimmunoprecipitation assay lysis buffer and incubated on ice for 0.5 h, then supernatant was removed after centrifuging at a speed of 12,000 g for 15-20 min. The protein samples were quantified by bicinchoninic acid protein assay, as for the manufacturer's protocol (Beyotime Institute of Biotechnology, China). A total of 60 μg protein were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels, then transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were immediately blocked with 5% non-fat milk for 2.5 h and incubated for 16 h at 4°C with primary antibodies: Anti-Dab2 (rabbit anti-human polyclonal, dilution 1:800, Santa Cruz Biotechnology Inc., CA) and anti-β-tubulin (rabbit monoclonal 1:800, BD Transduction Laboratories, USA). Washed the membranes in tween-20 tris-buffered saline at least 10 min, then incubated with goat anti-rabbit secondary antibody at 37°C for 2.5 h (dilution 1:5000, Zhongshan jinqiao, Beijing, China). The protein bands were detected and analyzed by enhanced chemiluminescence system (ECL Plus, Bio Rad Biosciences, USA). The relative expression quantity of protein was scored as the ratio of Dab2 intensity to β-tubulin staining gray intensity.
SPSS 19.0 for windows software was used for TE analysis, independent samples t-test and analysis of variance (ANOVA) were used to evaluate Dab2 expression in the meningiomas and non-tumor leptomeninges. The Pearson Chi-square test, or likelihood ratio test were used to determine the relationship between Dab2 expression and clinicopathological characteristics of meningiomas. All the experiments were independently repeated 3 times. It was considered statistically significant when P < 0.05.
| » Results|| |
Expression level of Dab2 was significantly down-regulated in the meningioma tissues
Total protein was extracted from 90 fresh tissues of meningiomas and 10 cases of non-tumor leptomeninges and the expression of Dab2 was studied by western blot. The loss of expression of Dab2 was observed in 28 (31.1%) meningioma tissues and it was reduce in 62 (68.9%) meningioma tissues [Figure 1]a. The mean gray intensity of Dab2 was 1.77 ± 0.093 in meningioma tissues and 0.46 ± 0.032 in non-tumor tissues [Figure 1]b. There was a significant reduction in the Dab2 expression in meningiomas (t = 13.02, P < 0.001).
|Figure 1: Expression level of disabled-2 (Dab2) in the meningioma tissues and non-tumor leptomeninges by western blot (a) T: Tumor (meningioma) tissues, N: Non-tumor leptomeninges. We observed the expression loss of Dab2 in 54 cases (60%) of meningioma tissues, such as case 1 and case 4. Case 2 and case 3 represented the reduced expression of Dab2 in tumor tissues. βtubulin was the internal consults. (b) The bar chart of expression level of Dab2 in meningioma tumor tissues versus non-tumor leptomeninges, which revealed that Dab2 expression was signifi cantly reduced in the meningioma tissues|
Click here to view
Reduced expression of Dab2 was common in human malignant meningioma cell lines
Western blot analysis was also performed to detect the expression level of Dab2 in IOMM-Lee, KT21-MG1 and HMC. We observed a sharp reduction of Dab2 expression in KT21-MG1 cells [Figure 2]a and expression level of Dab2 in HMC were significantly higher in IOMM-Lee (t = 15.24, P < 0.001) and KT21-MG1 (t = 24.56, P = 0.000) [Figure 2]b.
|Figure 2: Expression level of disabled-2 (Dab2) in human malignant meningioma cell lines by western blot (a) Dab2 expressed in malignant meningioma cell lines (KT21-MG1 and KT21-MG1) and human meningeal cell (HMC). (b) A sharp reduction of Dab2 expression were observed in KT21-MG1 cells, and Dab2 expression in malignant meningioma cells were signifi cantly down-regulated than HMC (Bar, significantly difference, **P< 0.01)|
Click here to view
Reduced or loss expression of Dab2 were significantly correlated with the low classification of meningiomas
The expression level of Dab2 in classic meningiomas (grade I), atypical meningiomas (grade II) and anaplastic meningiomas (grade III) were 0.781 ± 0.138, 0.398 ± 0.116 and grade III 0.103 ± 0.081, respectively [Figure 3]a. Results of ANOVA showed that, it was significantly higher in grade I than the others (P < 0.001) [Table 1]. Expression loss of Dab2 was observed in 4 cases (14.3%) of grade I, 9 cases (32.1%) of grade II and 15 cases (53.6%) of grade III [Figure 3]b, Likelihood ratio test showed that expression loss of Dab2 was correlated with the classification of meningiomas (χ2 = 12.37, P = 0.002) [Table 2]. Results of Spearman's rank correlation analysis showed that, reduced expression of Dab2 was negatively correlated with previous comorbidities such as hypertension and diabetes mellitus and adjacent tissues invasion [Table 3]. However, there was no significant difference in the expression of Dab2 among all the subtypes of classic meningiomas (WHO I) (data not shown). Analogously, we found that the lower classification of meningiomas was correlated with complications, such as hypertension and diabetes mellitus; but not with the gender, age and smoking of patients by Pearson Chi-square test [Table 4].
|Figure 3: The expression level of disabled-2 (Dab2) in different grades of meningiomas (a) Expression loss of Dab2 were observed in 4 cases of grade I, 9 cases of grade II and 15 cases of grade III; reduced expression of Dab2 were observed in 42 cases of grade I, 15 cases of grade II and 5 cases of grade III. (b) The mean gray intesity of grade I was 0.781 ± 0.138, grade II 0.398 ± 0.116 and grade III 0.103 ± 0.081. Results of analysis of variance showed that, it was signifi cantly higher in grade I than the others (P < 0.001)|
Click here to view
|Table 1: Expression level of Dab2 in different classifi cations of meningiomas by ANOVA (Games-Howell test)|
Click here to view
|Table 2: Expression loss of Dab2 in a different classifi cation of meningiomas by likelihood ratio test|
Click here to view
|Table 3: Results of Spearman's rank correlation analysis between Dab2 expression and clinicopathological factors of meningiomas|
Click here to view
|Table 4: Correlations between classifi cation of meningiomas and clinicopathological factors by Pearson Chi-square test or|
likelihood ratio test
Click here to view
| » Discussion|| |
In the present study, we for the first time observed that Dab2 expression was significantly reduced in meningiomas when comparing with leptomeninges. Importantly, Dab2 was expressed to a variable level in meningiomas with different grade of malignancy, suggesting a pivotal role of Dab2 reduction in the early stage of cell immortalization and consequently promotes the oncogenesis of malignant meningiomas. Furthermore, the reduced expression of Dab2 was negatively correlated with the associated comorbidities, such as hypertension and diabetes mellitus and invasive ability in meningiomas. This data are consistent with our previous study, which reported that Dab2 was significantly decreased in lung cancer tissues by immunochemistry and western blot. ,
Meningioma is a kind of mesenchymal tumor and originates from the arachnoidal cells of leptomeninges.  We further detected that Dab2 was strongly positive in HMC, instead, a sharp reduction of Dab2 expression were observed in both KT21-MG1 and IOMM-Lee cells. This phenomenon is similar to Dab2 down-regulation in breast cancer cells,  urothelial carcinoma cells,  gastric cancers,  and lung cancer cells. , More interestingly, Dab2 is significantly increased in various CNS inflammations, including experimental cryo-injury and allergic encephalomyelitis in rats cords of rats with experimental autoimmune encephalomyelitis is significantly increased and lower expression of Dab2 results in diseases of CNS. ,,
A plurality of studies have shown that, Dab2 expression is up-regulated in the development of embryonic CNS in mouse and is able to be enhanced by retinoic acid. ,, It has been immunodetected in several macrophages and T cells and plays an important role in the repairing of experimental autoimmune encephalomyelitis of rats. ,, Furthermore, Dab2 causes the activation of Ras/mitogen-activated protein kinase signaling pathways and ultimately results in the constructing of an autocrine transforming growth factor-β (TGF-β) signaling loop. , Herein, we speculated that the reduced expression of Dab2 promoted the oncogenesis of meningiomas by TGF-β signaling ways. However, further studies are needed to be performed to elucidate the concrete explanations for the reducing of Dab2 in meningiomas.
| » Conclusion|| |
This is the first study that detected the reduced expression of Dab2 in meningioma tissues and meningioma cells, Dab2 were expressed at variable level in meningiomas with different grade of malignancy. Furthermore, the reduced or loss expression of Dab2 were significantly negatively correlated with complications in the present study, suggesting a pivotal role of Dab2 reduction in the oncogenesis and development of meningiomas. It would offer a new approach for the diagnostics or therapeutics for meningiomas.
| » References|| |
|1.||Louis OH, Wiestler OD, Cavenee WK. World Health Organization Classification of Tumors of the Central Nervous System. Lyon: IARC Press; 2007. |
|2.||Campbell BA, Jhamb A, Maguire JA, Toyota B, Ma R. Meningiomas in 2009: Controversies and future challenges. Am J Clin Oncol 2009;32:73-85. |
|3.||Lusis E, Gutmann DH. Meningioma: An update. Curr Opin Neurol 2004;17:687-92. |
|4.||Martin JC, Herbert BS, Hocevar BA. Disabled-2 downregulation promotes epithelial-to-mesenchymal transition. Br J Cancer 2010;103:1716-23. |
|5.||Xu J, Lamouille S, Derynck R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res 2009;19:156-72. |
|6.||Tong JH, Ng DC, Chau SL, So KK, Leung PP, Lee TL, et al. Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma. BMC Cancer 2010;10:253. |
|7.||Fulop V, Colitti CV, Genest D, Berkowitz RS, Yiu GK, Ng SW, et al. DOC-2/h Dab2, a candidate tumor suppressor gene involved in the development of gestational trophoblastic diseases. Oncogene 1998;17:419-24. |
|8.||Bagadi SA, Prasad CP, Srivastava A, Prashad R, Gupta SD, Ralhan R. Frequent loss of Dab2 protein and infrequent promoter hypermethylation in breast cancer. Breast Cancer Res Treat 2007;104:277-86. |
|9.||Kim JA, Bae SH, Choi YJ, Kim KH, Park SS. Feed-back regulation of disabled-2 (Dab2) p96 isoform for GATA-4 during differentiation of F9 cells. Biochem Biophys Res Commun 2012;421:591-8. |
|10.||Xu HT, Yang LH, Li QC, Liu SL, Liu D, Xie XM, et al. Disabled-2 and Axin are concurrently colocalized and underexpressed in lung cancers. Hum Pathol 2011;42:1491-8. |
|11.||Xie XM, Zhang ZY, Yang LH, Yang DL, Tang N, Zhao HY, et al. Aberrant hypermethylation and reduced expression of disabled-2 promote the development of lung cancers. Int J Oncol 2013;43:1636-42. |
|12.||Xu S, Zhou Y, Du WD, Chen G, Zhou FS, Schneider M, et al. Association of the variant rs2243421 of human DOC-2/DAB2 interactive protein gene (hDAB2IP) with gastric cancer in the Chinese Han population. Gene 2013;515:200-4. |
|13.||Xu HT, Xie XM, Li QC, Liu SL, Dai SD, Liu Y, et al. Atonal homolog 1 expression in lung cancer correlates with inhibitors of the Wnt pathway as well as the differentiation and primary tumor stage. APMIS 2013;121:111-9. |
|14.||Xu HT, Li QC, Dai SD, Xie XM, Liu DI, Wang EH. The expression patterns and correlations of chibby, β-catenin, and DNA methyltransferase-1 and their clinicopathological significance in lung cancers. APMIS 2011;119:750-8. |
|15.||Cautain B, Damoiseaux J, Bernard I, van Straaten H, van Breda Vriesman P, Boneu B, et al. Essential role of TGF-beta in the natural resistance to experimental allergic encephalomyelitis in rats. Eur J Immunol 2001;31:1132-40. |
|16.||Moon C, Lee J, Ahn M, Shin T. Involvement of Disabled-2 protein in the central nervous system inflammation following experimental cryoinjury of rat brains. Neurosci Lett 2005;378:88-91. |
|17.||Ahn M, Oh H, Lee W, Kim H, Moon C, Shin T. Immunohistochemical studies on disabled-2 protein in the spinal cords of rats with experimental autoimmune encephalomyelitis. Brain Res 2011;1416:51-60. |
|18.||Cheung KK, Mok SC, Rezaie P, Chan WY. Dynamic expression of Dab2 in the mouse embryonic central nervous system. BMC Dev Biol 2008;8:76. |
|19.||Smith ER, Smedberg JL, Rula ME, Hamilton TC, Xu XX. Disassociation of MAPK activation and c-Fos expression in F9 embryonic carcinoma cells following retinoic acid-induced endoderm differentiation. J Biol Chem 2001;276:32094-100. |
|20.||Jiang Y, He X, Howe PH. Disabled-2 (Dab2) inhibits Wnt/β-catenin signalling by binding LRP6 and promoting its internalization through clathrin. EMBO J 2012;31:2336-49. |
|21.||Sarrió D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J. Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res 2008;68:989-97. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]
|This article has been cited by|
||Neural progenitor fate decision defects, cortical hypoplasia and behavioral impairment in Celsr1-deficient mice
| ||C Boucherie,C Boutin,Y Jossin,O Schakman,A M Goffinet,L Ris,P Gailly,F Tissir |
| ||Molecular Psychiatry. 2018; 23(3): 723 |
|[Pubmed] | [DOI]|
||Aberrant Hypermethylation at Sites -86 to 226 of DAB2 Gene in Non-Small Cell Lung Cancer
| ||Chang Li,Jun Chen,Tengfei Chen,Zhenlei Xu,Chun Xu,Cheng Ding,Yuxuan Wang,Jun Zhao,Chang Li,Zhe Lei,Hong-Tao Zhang,Jun Zhao,Chang Li,Zhe Lei,Hong-Tao Zhang,Jun Zhao |
| ||The American Journal of the Medical Sciences. 2015; 349(5): 425 |
|[Pubmed] | [DOI]|