Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and have been associated with sensitivity to radio- and chemotherapy as well as favourable prognosis. By using microarray-based expression profiling, we found that oligodendroglial tumours with 1p and 19q losses showed significantly lower expression of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene (CITED4) at 1p34.2 as compared to tumours without 1p and 19q losses. Mutational analysis showed no CITED4 mutations in gliomas. However, 1p and 19q losses as well as low expression of CITED4 transcripts were significantly associated with hypermethylation of the CITED4-associated CpG island. In line with the latter finding, treatment of CITED4 hypermethylated glioma cell lines with 5-aza-2′-deoxycytidine and trichostatine A resulted in a marked increase of the CITED4 transcript levels. Furthermore, CITED4 hypermethylation was significantly associated with longer recurrence-free and overall survival of patients with oligodendroglial tumours. Taken together, our results indicate that CITED4 is epigenetically silenced in the vast majority of oligodendroglial tumours with 1p and 19q deletions and suggest CITED4 hypermethylation as a novel prognostic marker in oligodendroglioma patients.
Oligodendroglial tumours are primary neoplasms of the central nervous system that frequently demonstrate allelic losses on chromosomal arms 1p and 19q (Reifenberger and Louis, 2003). Recent data indicate that these combined 1p and 19q losses are mediated by an unbalanced t(1;19)(q10;p10) translocation (Griffin et al., 2006; Jenkins et al., 2006). Importantly, 1p/19q deletion is associated with favourable response to radio- and chemotherapy as well as prolonged survival (Cairncross et al., 1998, 2006; van den Bent et al., 2006). Thus, molecular testing for these losses provides clinically valuable information beyond the conventional histologic classification.
To date, several genes located on 1p or 19q have been reported as putative oligodendroglioma-associated tumour suppressor genes. For example, the cyclin-dependent kinase inhibitor 2C (CDKN2C, 1p32) gene is mutated or homozygously deleted in rare cases of anaplastic oligodendroglioma (Reifenberger and Louis, 2003). Other genes showed reduced expression in 1p-deleted gliomas, sometimes associated with promoter hypermethylation but never with mutation. These include the calmodulin-binding transcription activator 1 gene (CAMTA1, 1p36), the DNA fragmentation factor subunit β gene (DFFB, 1p36), the SHREW1 gene (1p36.32), the tumour protein p73 gene (TP73, 1p36.3) and the RAD54 gene (1p32) (Dong et al., 2002; Barbashina et al., 2005; McDonald et al., 2005, 2006). The p190RhoGAP gene (19q13.3) was reported as a candidate tumour suppressor that inhibits PDGF-induced murine oligodendrogliomas (Wolf et al., 2003). However, its role in human oligodendrogliomas is unknown. More recently, the myelin-related epithelial membrane protein gene 3 (EMP3, 19q13.3) was found to be epigenetically silenced in malignant gliomas and neuroblastomas (Alaminos et al., 2005).
We applied microarray-based expression profiling to identify novel genes that are differentially expressed between gliomas with and without 1p/19q losses (Tews et al., 2006). In total, 35 gliomas were studied, including seven oligodendrogliomas WHO grade II, eight diffuse astrocytomas WHO grade II, 14 anaplastic oligodendrogliomas WHO grade III and six anaplastic oligoastrocytomas WHO grade III (Tews et al., 2006). Differentially expressed genes allowing for the discrimination between gliomas with and without 1p/19q losses were revealed by significance analysis of microarrays (SAM) followed by prediction analysis for microarrays (PAM) (Tusher et al., 2001; Tibshirani et al., 2002; Tews et al., 2006). One of the genes with significantly lower expression in 1p/19q-deleted gliomas was the CREB-binding protein (CBP)/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene (CITED4) at 1p34.2 (OMIM 606815), which has been linked to cancer-relevant cellular processes (Braganca et al., 2002; Fox et al., 2004). Here, we report on a detailed molecular analysis of CITED4 aberrations in human gliomas and provide evidence for an association of CITED4 hypermethylation with longer survival of oligodendroglial tumour patients.
In total, we investigated tumour samples from 78 patients (40 female and 38 male; mean age at operation: 48 years; range: 11–91 years) with gliomas (21 oligodendrogliomas WHO grade II, three oligoastrocytomas WHO grade II, eight diffuse astrocytomas WHO grade II, 29 anaplastic oligodendrogliomas WHO grade III and 17 anaplastic oligoastrocytomas WHO grade III). The tumours were collected at the Departments of Neuropathology, Heinrich-Heine-University, Düsseldorf, and Charité University Hospital, Berlin, and investigated in an anonymized manner as approved by the local institutional review boards. All tumours were classified according to the WHO classification of tumours of the nervous system (Reifenberger et al., 2000). A tumour cell content of at least 80% was histologically assured for each specimen used for molecular analysis. Extraction of DNA and RNA from frozen tumour samples as well as DNA from leukocytes was carried out as reported elsewhere (van den Boom et al., 2003).
In line with our microarray results (Tews et al., 2006), quantitative real-time reverse transcription–polymerase chain reaction (PCR) analysis of 41 gliomas revealed CITED4 transcript levels reduced by at least 50% relative to non-neoplastic brain tissue in 17 of 21 gliomas (81%) with 1p losses spanning the CITED4 locus (Figure 1a). Twelve of these 17 tumours showed transcript levels reduced by at least 75% relative to non-neoplastic brain tissue when ARF1 was used as the reference gene. When ACTG1 was used as reference, all 17 tumours showed CITED4 transcript levels reduced by more than 75% relative to non-neoplastic brain tissue. In contrast, only 5 of 20 gliomas (25%) with retention of both CITED4 alleles showed reductions in the CITED4 transcript levels by at least 50% relative to non-neoplastic brain tissue (Figure 2). Statistical analyses confirmed that CITED4 mRNA expression was significantly lower (Student's t-test, P<0.001) and more commonly reduced (Fisher's exact test, P<0.001) in 1p/19q-deleted gliomas.
Mutation screening of 45 gliomas, including 22 tumours with 1p/19q losses, detected 15 different CITED4 polymorphisms, mostly single nucleotide exchanges (Table 1). Four polymorphisms (c.95T>A: Leu32Gln; c.170G>C, Arg57Pro; c.173A>G, Gln58Arg; c.393C>T, Gly131Gly) were linked to each other, that is, occurred together in nine patients. A 36-base pair in-frame deletion polymorphism (c.327-362del, GenBank accession no. NM_133467.2) was found in two patients, with one patient being constitutionally homozygous for this polymorphism. However, none of the 45 tumours carried somatic mutations, indicating that structural alterations of CITED4 are absent or very rare in gliomas.
We next investigated the role of aberrant methylation of the CITED4-associated CpG island in 62 gliomas, including the 41 tumours studied for CITED4 expression. CITED4 hypermethylation was detected in 31 of 34 gliomas with 1p/19q losses (91%) but only in 2 of 28 gliomas without 1p/19q losses (7%) (Figures 1b and 2) (Fisher's exact test, P<0.001). CITED4 hypermethylation was absent in leucocyte DNA from 16 patients with CITED4 hypermethylated gliomas and in non-neo;plastic brain samples from two unrelated individuals (Figure 2).
Correlation of CITED4 expression and methylation revealed that CITED4 transcript levels were reduced by at least 50% relative to non-neoplastic brain tissue in 15 of 19 tumours (79%) with CITED4 hypermethylation but only in 7 of 22 tumours (32%) without CITED4 hypermethylation. Statistical analyses confirmed that CITED4 mRNA levels were significantly lower (Student's t-test, P<0.01) and more commonly reduced (Fisher's exact test, P<0.01) in CITED4-hypermethylated gliomas. In line with these findings, 5-aza-2′-deoxycytidine and trichostatin A treatment of three CITED4-hypermethylated glioma cell lines (A172, T98G, U138MG) resulted in markedly increased mRNA levels (Figure 1c–d).
To assess the relationship between CITED4 hypermethylation and patient survival, we investigated 45 patients with oligodendroglial tumours (19 WHO grade II, 26 WHO grade III) and available follow-up data (Felsberg et al., 2004). The median follow-up time after diagnosis was 122 months. Twenty-three patients died of their tumours while 22 patients were still alive at last follow-up. Median overall survival after diagnosis was 103 months, median recurrence-free survival was 42 months. All patients were treated by open resection. Twenty-four of the 26 patients with WHO grade III tumours received adjuvant therapy, comprising radiotherapy in 16 patients, alkylating chemotherapy in one patient, and combined radio- and chemotherapy in seven patients. Univariate statistical analysis revealed that CITED4 hypermethylation was significantly associated with longer recurrence-free and overall survival in the entire group of 45 patients and in the 26 patients with WHO grade III tumours (Figure 3a–d). Similarly, significant associations were found between the 1p/19q allelic status and survival (Figure 3e–h). Multivariate analysis identified CITED4 hypermethylation and 1p/19q deletions as significant prognostic factors adjusted for WHO grade and patient age (Table 2).
The CITED4 gene encodes a 184-amino-acid protein of 24.5 kDa that binds CBP and the tumour suppressor protein EP300 (E1A-binding protein, 300 kDa), functions as a co-activator of the transcription factor AP-2 (Braganca et al., 2002), blocks binding of hypoxia-inducible factor 1 alpha (HIF1α) to EP300 and inhibits HIF1α transactivation as well as hypoxia-mediated reporter gene activation (Fox et al., 2004). Loss of nuclear expression or cytoplasmic translocation of CITED4 has been implicated in breast cancer development (Fox et al., 2004). Here, we report that CITED4 mRNA expression in gliomas is significantly lower in 1p/19q-deleted versus non-deleted tumours. Furthermore, we found reduced expression closely linked to CITED4 hypermethylation. In fact, our data indicate biallelic CITED4 inactivation by loss of one allele and hypermethylation of the other allele in the vast majority of 1p/19q-deleted oligodendroglial tumours. The absence of CITED4 hypermethylation in DNA extracted from leucocytes and non-neoplastic brain tissue indicates that CITED4 is not imprinted. However, it remains unclear whether CITED4 hypermethylation is already present in the yet unknown cell of origin of oligodendrogliomas or represents an acquired aberration selected for during tumorigenesis.
The molecular mechanisms by which CITED4 inactivation might promote oligodendroglial tumour growth are as yet unknown. CITED4 downregulation could interfere with EP300 tumour suppressor activity (Iyer et al., 2004), which, among other functions, plays a crucial role in p53 regulation (Grossman, 2001). Interestingly, 1p/19q-deleted oligodendrogliomas usually lack TP53 mutations (Reifenberger and Louis, 2003). However, further studies need to substantiate a tumour-suppressive function of the CITED4 protein and to clarify the functional consequences of its inactivation in oligodendroglial tumours.
We found a similar prognostic significance of CITED4 hypermethylation in oligodendroglioma patients as obtained for 1p/19q deletion. Thus, diagnostic assessment of CITED4 hypermethylation might be an alternative approach to 1p/19q-testing. However, the frequency of CITED4 hypermethylation in other gliomas, in particular anaplastic astrocytomas and glioblastomas, remains to be determined. Furthermore, future studies need to address whether CITED4 protein expression analysis, for example, by immunohistochemistry, might substitute for methylation analysis and 1p/19q-testing. It also needs to be investigated whether CITED4 inactivation contributes to radio- and chemosensitivity or just represents a surrogate marker for other alterations in 1p/19q-deleted oligodendrogliomas. For example, 1p/19q-deletion has been linked to hypermethylation of the O6-methylguanine DNA methyltransferase (MGMT) gene (Möllemann et al., 2005), which predicts response to alkylating chemotherapy in malignant gliomas (Hegi et al., 2005). Taken together, our results suggest CITED4 as an interesting novel candidate gene in oligodendroglial tumours and should stimulate further research into its functional roles and clinical significance.
loss of heterozygosity
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This study was supported by grants from the Deutsche Krebshilfe to GR and MS (70-3088-Sa1) as well as to GR and AvD (70-3163-Wi3), and from the German Bundesministerium für Bildung und Forschung (BMBF) within the National Genome Research Network 2 (NGFN-2) to PL, MH, GR, CH and AvD (01GS0460, 01GS0462 and 01GS0463). BT was a scholar of the Studienstiftung des Deutschen Volkes.
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Tews, B., Roerig, P., Hartmann, C. et al. Hypermethylation and transcriptional downregulation of the CITED4 gene at 1p34.2 in oligodendroglial tumours with allelic losses on 1p and 19q. Oncogene 26, 5010–5016 (2007) doi:10.1038/sj.onc.1210297
- gene expression profiling
- CpG island
- tumour suppressor gene
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