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Hypermethylation and Transcriptional Downregulation of the Carboxyl-Terminal Modulator Protein Gene in Glioblastomas

Christiane B. Knobbe, Julia Reifenberger, Britta Blaschke, Guido Reifenberger

Affiliations of authors: Departments of Neuropathology (CBK, BB, GR) and Dermatology (JR), Heinrich-Heine-University, Düsseldorf, Germany

Correspondence to: Guido Reifenberger, MD, PhD, Department of Neuropathology, Heinrich-Heine-University, Moorenstr. 5, D-40225 Düsseldorf, Germany (e-mail: reifenberger{at}med.uni-duesseldorf.de)


    ABSTRACT
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The carboxyl-terminal modulator protein (CTMP) has been identified as a negative regulator of protein kinase B/Akt. Aberrant Akt signaling is frequently observed in glioblastomas, the most common and most malignant glial brain tumors. Because loss of CTMP function and/or expression may remove the inhibitory effects on Akt and promote tumorigenesis, we studied 93 primary glioblastomas and nine glioblastoma cell lines for CTMP deletion, mutation, promoter hypermethylation, and mRNA expression. None of the tumors or cell lines had CTMP-homozygous deletions or coding sequence mutations. However, CTMP mRNA expression was lower by at least 50% relative to non-neoplastic brain tissue in 37 (40%) glioblastomas and six (67%) glioma cell lines. Reduced CTMP mRNA levels were closely associated with hypermethylation of the CTMP promoter. Furthermore, treatment of CTMP-hypermethylated A172 glioma cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in partial demethylation of the CTMP promoter and increased CTMP mRNA expression. Thus, epigenetic downregulation of CTMP transcription is a common aberration in glioblastomas.


Glioblastomas are the most common and most malignant glial tumors of the brain. Molecular studies (1,2) have identified a variety of genetic alterations in glioblastomas that result in the inactivation of cell cycle regulatory pathways and in the activation of intracellular signaling pathways such as those that involve the mitogen-activated protein kinase and the phosphatidylinositol 3-kinase (PI3K) cascades. In glioblastomas, activation of the PI3K signaling pathway is frequently accomplished by amplification, genetic rearrangement, and/or overexpression of growth factor receptor genes such as the epidermal growth factor receptor (EGFR) gene (2). Furthermore, approximately one-third of glioblastomas carry mutations in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene, whose gene product dephosphorylates phosphatidylinositol-(3,4,5)-triphosphate and thereby inhibits the PI3K-dependent activation of protein kinase B/Akt signaling (3). Akt activation has been shown to be important for the development of glio-blastomas in different model systems (4,5), and phosphorylated (i.e., activated) Akt proteins have been detected in the majority of human glioblastomas (4,6). Maira et al. (7) identified the novel protein carboxyl-terminal modulator protein (CTMP) that binds Akt, inhibits its phosphorylation at threonine 308 and serine 473, and induces a less malignant phenotype in v-Akt-transformed cells. Thus, CTMP functions as an inhibitor of Akt that acts downstream of PTEN. Consequently, loss of CTMP function and/or expression may remove the inhibitory effects on Akt and allow for Akt-mediated signal transduction.

We investigated CTMP gene alterations and mRNA expression in 93 primary glioblastomas and nine glioma cell lines (A172, Hs683, U118MG, U178MG, T98G, CCF-STTG1 [all from American Type Culture Collection, Manassas, VA], U138MG [from German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany], U251MG, and U373MG [both from Dr. V. P. Collins, Department of Pathology, University of Cambridge, Cambridge, U.K.]). Human brain and tumor tissue samples were selected from the archives of the Department of Neuropathology, Heinrich-Heine-University, and investigated as approved by the local institutional review board at Heinrich-Heine-University. DNA and RNA were extracted as described (8). All tumors and cell lines were analyzed for CTMP coding region mutations (GenBank accession No. NM_176853) by using single-strand conformation polymorphism analysis and DNA sequencing (8,9), and for homozygous deletion of CTMP by using duplex polymerase chain reaction (PCR) for the simultaneous amplification of fragments from CTMP and the reference gene adenine phosphoribosyltransferase (APRT; GenBank accession No. NM_000485) (8,10). None of the tumors or cell lines had somatic mutations in CTMP or had a homozygous deletion of CTMP. Peripheral blood samples were available from 57 of the 93 patients. These samples were subjected to loss of heterozygosity analysis at the microsatellite loci D1S2344 and D1S305, which map proximal and distal to CTMP at 1q21, respectively. None of the samples showed allelic loss.

We next assessed the CTMP mRNA levels in the 93 glioblastomas by real-time reverse transcription–PCR using primers binding to sequences shared by transcript variants 1 and 2 (GenBank accession Nos. NM_053055 and NM_176853). CTMP mRNA levels were normalized to the housekeeping gene ADP-ribosylation factor 1 (ARF1; GenBank accession No. NM_001658) and were compared with the normalized CTMP mRNA levels in non-neoplastic brain tissue samples obtained from an individual at autopsy or from patients undergoing surgery for chronic epilepsy or traumatic brain injury. Compared with those in non-neoplastic brain tissue, CTMP mRNA levels were reduced by at least 50% in 37 of 93 (40%) glioblastomas and in six of nine (67%) glioma cell lines (Figs. 1, A, and 2). To determine whether methylation differences in the CTMP promoter were responsible for the reduced mRNA expression, we selected 15 glioblastomas (10 tumors with CTMP mRNA levels reduced by >=50% and five tumors with CTMP mRNA levels of >=60% relative to non-neoplastic brain tissue), the nine cell lines, and non-neoplastic brain tissue samples from four different individuals for methylation analysis by sequencing the CTMP 5'-CpG island after sodium bisulfite modification of the DNA (10). For each sample, the genomic CTMP sequence from nucleotides -525 to 233 (GenBank accession No. AJ313515) was amplified in three fragments, each of which was cloned into pCR 2.1 vectors (Invitrogen, Carlsbad, CA). At least five clones of each fragment were sequenced to determine the methylation status. Hypermethylation (i.e., methylation of the majority of the 67 investigated CpG sites per sample) was observed in eight of 10 glioblastomas and in all cell lines with reduced CTMP mRNA levels (Figs. 1, B, and 2, A). By contrast, hypermethylation was observed neither in the non-neoplastic brain samples nor in the tumors and cell lines with normal CTMP mRNA levels (Fig. 2, A). To assess whether hypermethylation was responsible for the decreased mRNA expression, we treated the cell lines A172 and T98G, which were positive and negative for CTMP promoter hypermethylation, respectively, with the demethylating agent 5-aza-2'-deoxycytidine, the histone deacetylase inhibitor trichostatin A, or a combination of both. Relative to untreated A172 cells, CTMP mRNA expression increased in A172 cells treated with either drug (data not shown) or the combination (Fig. 1, D). Furthermore, the treatment of A172 cells with the combination of 5-aza-2'-deoxycytidine and trichostatin A resulted in a partial demethylation of the hypermethylated CTMP CpG island (Fig. 2, A). CTMP mRNA expression was unaltered in T98G cells after treatment with either drug alone (data not shown) or the combination (Fig. 1, D).



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Fig. 1. A) Reduced carboxyl-terminal modulator protein (CTMP) mRNA expression in glioblastoma GB10D shown by real-time reverse transcription–polymerase chain reaction (PCR). x-axis, cycle number; y-axis, relative amount of PCR product. Note identical curves for the housekeeping gene ADP-ribosylation factor 1 (ARF1) in GB10D and non-neoplastic brain tissue (NB), indicating equal expression (right panel). By contrast, the CTMP curve of GB10D is located to the right of the CTMP curve of NB, indicating lower CTMP expression in GB10D (left panel). The calculated CTMP mRNA level in GB10D was 20% of the level in NB. B) Sequence of the 5'-CpG island of CTMP after sodium bisulfite treatment of DNA revealed methylation of CpG sites in GB10D (arrows) but not in NB (shown is the reverse sequence from nucleotides 12 to 33). C) Assessment of CTMP methylation by PCR-based analysis of HpaII-digested (+) or undigested (–) DNA. Similar signals for HpaII-digested and undigested DNA indicate complete methylation of the HpaII site within each fragment (CTMP fragments 1, 3, or 5). No signal for HpaII-digested DNA indicates absent methylation, and a weak signal (e.g., fragment 5 in GB21D and GB110D) indicates partial methylation. D) Induction of CTMP mRNA expression in A172 but not T98G cells by 5-aza-2'-deoxycytidine (A172 = 1 µM for 72 hours; T98G = 0.5 µM for 48 hours) and trichostatin A (1 µM for 24 hours) treatment. Before treatment (–), A172 cells expressed very low levels of CTMP mRNA. After treatment (+), A172 cells expressed increased CTMP mRNA levels that are comparable with levels in NB. T98G cells expressed levels of CTMP mRNA comparable with levels in NB, independent of the treatment. bp = size of the respective PCR product in base pairs. E) Analysis of Akt expression and phosphorylation in tissue lysates from selected glioblastomas using western blot analysis with polyclonal antibodies specific for either total Akt protein or Akt phosphorylated at serine 473 (pAkt). The blots were additionally stained with a monoclonal antibody specific for {beta}-actin ({beta}-Act) to assess protein loading. Case numbers are given at the top of each lane. Signals for pAkt are markedly increased in all glioblastomas compared with signals in NB. kDa = molecular weight in kilodaltons.

 


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Fig. 2. A) Methylation patterns in the 5'-CpG island of the carboxyl-terminal modulator protein (CTMP) in nine glioma cell lines, 15 primary glioblastomas, and four non-neoplastic brain samples (NB1–NB4). Methylation at each of the 67 investigated CpG sites located between nucleotides -525 and 233 was determined by amplification of three overlapping fragments (top) from sodium bisulfite–treated DNA. The fragments were cloned, and at least five clones from each fragment were sequenced per original sample. The results are represented as quintiles of methylation in a gray-scale pattern indicating the percentage of clones with methylation at each CpG site (i.e., the percentage of methylated clones among the totally sequenced clones). CTMP expression (CTMP expr.) was determined by real-time reverse transcription–polymerase chain reaction (PCR). Hatched boxes = CTMP mRNA levels reduced by at least 50% relative to levels in non-neoplastic brain samples; white boxes = CTMP mRNA levels of 60% or more relative to levels in non-neoplastic brain. The location of exon 1 and the start codon (ATG) are indicated. B) Comparison between CTMP hypermethylation, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene mutations, and epidermal growth factor receptor (EGFR) amplification in 93 glioblastomas (case numbers listed to the left of each panel). CTMP methylation was assessed by PCR analysis of eight fragments from the CTMP 5'-CpG island that contained HpaII restriction sites (1–8). Black boxes = signal intensity for HpaII-digested template equal to that for undigested template (complete methylation); gray boxes = weaker signal intensity for HpaII-digested template than that for undigested template (partial methylation); white boxes = no signal for HpaII-digested template (no methylation). Tumors with PTEN mutation or EGFR amplification are indicated by horizontally and vertically striped boxes, respectively (data are from (11)]. Reduced CTMP transcript levels (hatched boxes) are closely associated with CTMP hypermethylation. PTEN mutation and EGFR amplification are not associated with CTMP hypermethylation.

 
To screen all 93 glioblastomas for CTMP hypermethylation, we digested tumor DNA with the methylation-sensitive restriction enzyme HpaII, and used PCR to amplify eight different HpaII site–containing fragments from the CTMP CpG island (Figs. 1, C, and 2, B). PCR conditions were optimized for each fragment by using DNA samples that had been analyzed by sequencing sodium bisulfite–modified DNA. HpaII restriction analysis revealed CTMP hypermethylation (i.e., methylation of at least four of eight HpaII site–containing fragments) in 29 of 37 (78%) glioblastomas with reduced CTMP mRNA levels and in two of 56 (4%) glioblastomas with normal CTMP mRNA levels (Fig. 2, B). The relationship between CTMP hypermethylation and reduced mRNA expression was statistically significant (P<.001, two-sided Fisher’s exact test).

We next compared CTMP hypermethylation and/or reduced mRNA expression with previously published data (11) on PTEN and EGFR gene alterations in the 93 glioblastomas (Fig. 2, B). PTEN mutations were present in similar fractions of glioblastomas with and without reduced CTMP mRNA expression (27% versus 30%; P = .599, chi-square test) and with and without CTMP hypermethylation (32% versus 27%; P = .749, chi-square test). Similarly, five of six cell lines with CTMP hypermethylation (A172, U138MG, U178MG, U251MG, and U373MG) and two of three cell lines without CTMP hypermethylation (T98G and U118MG) carry PTEN mutations (12,13). Thus, we conclude that there is no association between PTEN inactivation and CTMP aberrations in glioblastomas. We also detected no relationship between CTMP hypermethylation and EGFR gene amplification (Fig. 2, B; P = .505, chi-square test).

To investigate the Akt activation status in primary glioblastomas, we performed western blot analyses of 15 selected tumors, including samples with CTMP hypermethylation (GB103D), PTEN mutations (GB47D and GB96D), EGFR amplification (GB98D, GB131D, GB139D, and GB140D), and alteration in none (GB60D, GB105D, GB147D, GB181D, and GS11D) or in any two or three of these genes (GB101D, GB191D, and GS3D). Relative to Akt protein levels in non-neoplastic brain tissue, all tumors showed increased levels of serine 473–phosphorylated Akt protein (Fig. 1, E). In agreement with previous studies (4,6), these findings indicate that Akt is activated in the majority of glioblastomas, including those tumors with alterations in expression or function of PTEN, CTMP, and/or EGFR.

We found that CTMP mRNA levels were reduced by 50% or more compared with non-neoplastic brain tissue in 40% of primary glioblastomas and in 67% of glioma cell lines. Our findings suggest reduced CTMP expression as a novel molecular mechanism involved in the pathogenesis of glioblastomas. Reduced CTMP expression was closely associated with CTMP 5'-CpG island hypermethylation and could be restored by 5-aza-2'-deoxycytidine and trichostatin A treatment. These findings suggest a role for epigenetic DNA modification in the regulation of CTMP promoter activity. We found that CTMP gene promoter hypermethylation and reduced mRNA expression in glioblastomas are not associated with PTEN mutations and EGFR amplification. Whether our findings regarding CTMP expression and regulation are unique to glioblastomas or are also relevant to other tumors remains to be determined.


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Supported by grants SFB 503/B7 and GRK 320 from the Deutsche Forschungsgemeinschaft, grants 10-1639-Re3 and 70-3088-Sa1 from the Deutsche Krebshilfe, and grant 9772182 from the Medical Faculty of Heinrich-Heine-University, Düsseldorf, Germany (all to G. Reifenberger). J. Reifenberger is supported by the Lise Meitner Program of the Ministry of Science and Research of Northrhine-Westphalia.

All PCR primer sequences used in this study are available on request from G. Reifenberger.


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Manuscript received August 14, 2003; revised December 24, 2003; accepted January 12, 2004.


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