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. 2012 Jan;14(1):20-8.
doi: 10.1593/neo.111574.

Alternative splicing of CHEK2 and codeletion with NF2 promote chromosomal instability in meningioma

Hong Wei Yang  1 , Tae-Min Kim, Sydney S Song, Nihal Shrinath, Richard Park, Michel Kalamarides, Peter J Park, Peter M Black, Rona S Carroll, Mark D Johnson
Affiliations

Alternative splicing of CHEK2 and codeletion with NF2 promote chromosomal instability in meningioma

Hong Wei Yang et al. Neoplasia. 2012 Jan.

Abstract

Mutations of the NF2 gene on chromosome 22q are thought to initiate tumorigenesis in nearly 50% of meningiomas, and 22q deletion is the earliest and most frequent large-scale chromosomal abnormality observed in these tumors. In aggressive meningiomas, 22q deletions are generally accompanied by the presence of large-scale segmental abnormalities involving other chromosomes, but the reasons for this association are unknown. We find that large-scale chromosomal alterations accumulate during meningioma progression primarily in tumors harboring 22q deletions, suggesting 22q-associated chromosomal instability. Here we show frequent codeletion of the DNA repair and tumor suppressor gene, CHEK2, in combination with NF2 on chromosome 22q in a majority of aggressive meningiomas. In addition, tumor-specific splicing of CHEK2 in meningioma leads to decreased functional Chk2 protein expression. We show that enforced Chk2 knockdown in meningioma cells decreases DNA repair. Furthermore, Chk2 depletion increases centrosome amplification, thereby promoting chromosomal instability. Taken together, these data indicate that alternative splicing and frequent codeletion of CHEK2 and NF2 contribute to the genomic instability and associated development of aggressive biologic behavior in meningiomas.

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Figures

Figure 1
Figure 1
Chromosomal instability occurs in the context of 22q deletion during meningioma progression. (A) 500K SNP analyses for 12 initial (I) and recurrent (R) paired primary meningioma specimens illustrating accumulation of large-scale chromosomal changes at the time of recurrence. Note the association between 22q deletion and the presence of numerous segmental chromosomal abnormalities. Arrow identifies a pair of specimens in which the initial specimen lacked 22q deletion, whereas the recurrent tumor displayed 22q deletion and numerous additional large-scale chromosomal changes. (B) 500K SNP analyses of a portion of chromosome 22q in 47 human meningioma specimens. The location of the CHEK2 and NF2 genes is as indicated. Arrowheads point to two tumors with interstitial 22q deletions that involve NF2 and CHEK2. (C) Higher-resolution image of the SNP data shown in B illustrating frequent codeletion of NF2 and CHEK2 in meningioma.
Figure 2
Figure 2
Alternative splicing of CHEK2 in meningioma yields nonfunctional CHEK2 splice variants. (A) RT-PCR analysis of CHEK2 transcripts using total RNA extracted from 20 primary meningioma specimens (labeled A through T). Arrows indicate location of splice variants compared with the full-length CHEK2 mRNA (WT), M = marker. (B) Direct sequencing of CHEK2 clones illustrating the most commonly identified splice variants lacking the kinase domain. (C) Frequency of full-length (wt) CHEK2 clones versus various splice variants in 10 primary meningioma specimens. (D) Western blot illustrating Chk2 and NF2 protein expression in eight primary meningioma specimens. Note that in half of the specimens, alternate Chk2 isoforms were more abundant than full-length Chk2 (top band).
Figure 3
Figure 3
Impaired DNA repair capacity in meningioma cell lines with segmental 22q deletions. (A) 500K SNP analysis of genomic DNA obtained from four established human meningioma cell lines (CH157-MN, F5, IOMM-Lee, and Me3TSC). Dark blue represents copy number loss, whereas bright red represents gain. Each column contains data from a different cell line. Chromosome 22q is shown at higher magnification to the right. The approximate locations of the NF2 and CHEK2 genes are as indicated. Note that 22q deletions involving CHEK2 and NF2 occur in the CH157-MN and Me3TSC cell lines, but not the F5 and IOMM-Lee cell lines. (B) Cells from each of four human meningioma cell lines were exposed to ultraviolet irradiation (50 J/m2) for 5 minutes. The cells were then fixed and stained for γ-H2AX immunoreactivity (green) after 0, 1, 2, and 3 hours to detect DNA DSBs. Note the relative increase in nuclear γ-H2AX immunoreactivity in the CH157-MN and Me3TSC cell lines that harbor CHEK2 and NF2 deletions when compared with the cell lines that do not have such deletions (F5 and IOMM-Lee). (C) Quantitation of data shown in B. A significant increase in γ-H2AX immunoreactivity was noted after 3 hours in human meningioma cell lines harboring CHEK2 and NF2 deletions (CH157-MN and Me3TSC) when compared with meningioma cell lines that do not have such deletions (F5 and IOMM-Lee). Data shown are mean ± SEM. *P < .05, t test.
Figure 4
Figure 4
Chk2 depletion increases proliferation but decreases cell growth after DNA damage. (A) Western blot illustrating knockdown of Chk2 protein in CH157-MN cells stably expressing two different shRNAs directed against human Chk2. Control cells were transfected using an empty shRNA control vector. (B) MTT cell growth assay illustrating the effect of Chk2 depletion on the growth of human CH157-MN meningioma cells under control conditions. Data shown are mean ± SEM. *P < .05, **P < .0001, unpaired t test. (C) MTT cell growth assay illustrating effect of Chk2 depletion on the growth of CH157-MN cells after UV irradiation (50 J/m2 for 5 minutes). Data shown are mean ± SEM. *P < .05, t test. (D) MTT cell growth assay illustrating effect of Chk2 depletion on the growth of CH157-MN cells after overnight exposure to increasing concentrations of camptothecin. Data shown are mean ± SEM. *P < .05, t test. (E) Flow cytometry cell cycle analysis illustrating effect of Chk2 depletion on CH157-MN cell proliferation.
Figure 5
Figure 5
Chk2 depletion increases centrosome number and chromosomal instability in human meningioma cells. (A) Western blot illustrating correlation between Chk2 and Cdc25A expression using protein lysates derived from 13 primary human meningioma specimens. Arrows indicate samples with the lowest Chk2 expression. Densitometry measurements of the Chk2/Cdc25A ratio revealed a relative increase in Cdc25A expression in meningiomas with the lowest Chk2 levels. (B) Western blot illustrating effect of overexpressed Chk2 or Chk2 splice variants lacking the kinase domain on Cdc25A expression in 293T cells. Densitometry measurements revealed an increase in the p-Cdc25A/Cdc25A ratio and a decrease in the overall expression of Cdc25A after Chk2 overexpression. (C) Light micrographs illustrating effect of Chk2 depletion on centrosome number in IOMM-Lee meningioma cells. (D) Quantitative analysis of centrosome duplication induced by Chk2 depletion in CH157-MN meningioma cells. Cells with three or more centrosomes were considered abnormal. At least 680 cells were counted for each condition. P < .011, proportion test. (E) Array CGH analysis of DNA extracted from IOMM-Lee meningioma cells after Chk2 depletion. Cells were exposed to a sublethal dose of UV irradiation (50 J/m2 for 1 minute) and were then passaged 10 times before harvesting. Data for chromosomes 6, 7, and 8 are shown. Note the increase of chromosomal deletions in cells with shChk2 depletion.
Figure 6
Figure 6
A proposed model by which a monoallelic NF2 mutation prompts loss of the other NF2 allele through 22q deletion. Monoallelic mutation of NF2 leads to loss of the other NF2 allele through segmental 22q deletion. This results in frequent codeletion of CHEK2, which is located close to the NF2 gene. Concurrently, alternative splicing of CHEK2 generates nonfunctional and dominant negative forms of Chk2. The combined effect of these changes leads to decreased Chk2 function, increased centrosome amplification, and chromosomal instability.
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References

    1. Hansson CM, Buckley PG, Grigelioniene G, Piotrowski A, Hellstrom AR, Mantripragada K, Jarbo C, Mathiesen T, Dumanski JP. Comprehensive genetic and epigenetic analysis of sporadic meningioma for macro-mutations on 22q and micro-mutations within the NF2 locus. BMC Genomics. 2007;8:16. - PMC - PubMed
    1. Kalamarides M, Niwa-Kawakita M, Leblois H, Abramowski V, Perricaudet M, Janin A, Thomas G, Gutmann DH, Giovannini M. Nf2 gene inactivation in arachnoidal cells is rate-limiting for meningioma development in the mouse. Genes Dev. 2002;16:1060–1065. - PMC - PubMed
    1. Lekanne Deprez RH, Bianchi AB, Groen NA, Seizinger BR, Hagemeijer A, van Drunen E, Bootsma D, Koper JW, Avezaat CJ, Kley N, et al. Frequent NF2 gene transcript mutations in sporadic meningiomas and vestibular schwannomas. Am J Hum Genet. 1994;54:1022–1029. - PMC - PubMed
    1. Arslantas A, Artan S, Oner U, Durmaz R, Muslumanoglu H, Atasoy MA, Basaran N, Tel E. Comparative genomic hybridization analysis of genomic alterations in benign, atypical and anaplastic meningiomas. Acta Neurol Belg. 2002;102:53–62. - PubMed
    1. Goutagny S, Yang HW, Zucman-Rossi J, Chan J, Dreyfuss JM, Park PJ, Black PM, Giovannini M, Carroll RS, Kalamarides M. Genomic profiling reveals alternative genetic pathways of meningioma malignant progression dependent on the underlying NF2 status. Clin Cancer Res. 2010;16:4155–4164. - PubMed

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