OTX2 is critical for the maintenance and progression of Shh-independent medulloblastomas - PubMed (original) (raw)

OTX2 is critical for the maintenance and progression of Shh-independent medulloblastomas

David C Adamson et al. Cancer Res. 2010.

Abstract

OTX2 is a developmentally regulated transcription factor involved in early morphogenesis of the central nervous system. This gene is amplified and overexpressed in medulloblastoma cell lines, but the nature and extent of its genetic alterations in primary tumors have not been evaluated. Analysis of a large cohort of primary medulloblastomas revealed frequent focal copy number gain of a region minimally containing OTX2 as a single gene. OTX2 copy number gain was restricted to tumor subtypes that did not express a molecular signature of Wnt or Shh pathway activation. FISH analysis revealed copy number gain in a subset of cells within medulloblastoma samples, suggesting a late event in tumor progression. Gain of OTX2 copy number was associated with the presence of anaplastic histologic features and shorter survival in medulloblastoma patients. In support of a functional role, ectopic OTX2 expression enhanced proliferation and tumorigenicity of immortalized primary cells, whereas OTX2 knockdown in medulloblastoma cells prolonged the survival of animals bearing xenograft tumors. Mechanistic investigations revealed upregulation of MYC as a potential mechanism whereby OTX2 promotes tumor progression. Our findings define OTX2 as an important oncogenic driver in medulloblastoma.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Frequent gain and overexpression of OTX2 in medulloblastomas

(A) OTX2 copy number status in 212 medulloblastomas (201 primary MBs and 11 MB cell lines) was inferred using Affymetrix SNP arrays (100K and 500K). Each red line shown in the ideogram represents copy number gain for a single sample in the region shown. (B) Affymetrix exon arrays were used for gene expression profiling of 9 fetal cerebella, 5 adult cerebella, 103 primary medulloblastomas, and 7 medulloblastoma cell lines. OTX2 expression levels are shown as a heatmap. (C) Box and whisker plot representing distribution of OTX2 expression level within normal and MB sample groups.

Figure 2

Figure 2. OTX2 gain and overexpression are specific to medulloblastoma subgroups

A subset of the primary medulloblastomas (n=81) analyzed in Figure 1 were classified into four molecular subgroups based upon gene expression signatures obtained from exon arrays. (A) Focal gain of OTX2 (designated in red) is limited to Group C and D medulloblastomas. The minimal region of gain (~10.5 kb) is boxed. (B) OTX2 mRNA overexpression is highest in Group C and D tumors, as well as those expressing a signature of Wnt pathway activation.

Figure 3

Figure 3. Interphase FISH of OTX2 in primary medulloblastoma showing that OTX2 copy number gain is associated with tumor anaplasia and poor patient survival

(A) Increased OTX2 copy number is seen in primary medulloblastoma tumors with anaplastic features. The Y axis is the ratio of the mean copy number of OTX2 to the control chromosome 14 probe in each cell of each sample. (B) Kaplan-Meier survival analysis of patients stratified by mean OTX2 copy number. (C) Kaplan-Meier survival analysis of patients stratified by age.

Figure 4

Figure 4. OTX2 promotes cell proliferation in vitro and is required for tumor maintenance in vivo

(A) RK3E cells were transfected with pEGFP-N1 or pEGFP-OTX2 and colony formation potential was determined. Tumors derived from intracranial xenografts of stably transfected RK3E cells were stained for Ki67 and OTX2, and representative samples are pictured. Green arrows identify intra-tumor blood vessels. (B) MHH-1 cells were transfected with pEGFP-N1, pEGFP-OTX2, or pEGFP-OTX2 (K). After three doubling times, proliferation was determined with an MTT assay. (C) D425MED cells derived from OTX2 shRNA knockdown or scrambled shRNA were injected into the right cerebral hemisphere of mice. Representative H & E-stained tumor sections from each group are pictured.

Figure 5

Figure 5. MYC is a target gene of OTX2 in medulloblastoma cell lines

(A) Expression of endogenous OTX2 and MYC in medulloblastoma cell lines demonstrated by Western blotting. (B) Medulloblastoma cell lines were treated with OTX2 siRNA (+) or scrambled siRNA (−), and protein expression of OTX2, MYC, and GAPDH was detected by Western blotting. (C) Cells were treated with MYC siRNA or scrambled siRNA and after three doubling times assessed for viability with the MTT assay. (D) Medulloblastoma cell lines were treated with MYC siRNA (+) or scrambled siRNA (−), and protein expression of OTX2, MYC, and GAPDH was detected by Western blotting.

Figure 6

Figure 6. The OTX2 homeodomain is required for OTX2-mediated MYC transcription

(A) MHH-1 cells were cotransfected with the MYC promoter reporter construct (pBV-Del-3-Luc) and either pEGFP-N1, pEGFP-OTX2 or pEGFP-OTX2 (K), and then the relative luciferase reporter activity was measured. (B) OTX2 ChIP and PCR analyses were used to validate binding of endogenous OTX2 to the promoter region of MYC (primers were designed to span the region 375 bp directly upstream of the MYC TSS) in OTX2-expressing cell lines. D425MED and D384MED are OTX2-expressing medulloblastoma cell lines, whereas MHH1 does not express OTX2. i, input of total DNA from cell lysates; +, OTX2 antibody immunoaffinity-enriched DNA; −, IgG control antibody immunoaffinity-enriched DNA.

Similar articles

Cited by

References

    1. Kleihues P, Louis DN, Scheithauer BW, et al. The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol. 2002;61:215–225. discussion 26–9. - PubMed
    1. Pietsch T, Taylor MD, Rutka JT. Molecular pathogenesis of childhood brain tumors. J Neurooncol. 2004;70:203–215. - PubMed
    1. Zakhary R, Keles GE, Aldape K, Berger MS. Medulloblastoma and primitive neuroectodermal tumors. London: Churchill Livingstone; 2001.
    1. Packer RJ, Reddy A. New Treatments in Pediatric Brain Tumors. Curr Treat Options Neurol. 2004;6:377–389. - PubMed
    1. Gajjar A, Hernan R, Kocak M, et al. Clinical, histopathologic, and molecular markers of prognosis: toward a new disease risk stratification system for medulloblastoma. J Clin Oncol. 2004;22:984–993. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources