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Basic and Translational Investigations |
Department of Neurological Surgery and Brain Tumor Research Center (J.K.W., S.Z., K.R.L., M.S.B., M.R.W., D.A.H.-K.), Department of Radiation Oncology (N.J., D.A.H.-K.), Department of Pathology (T. Tihan, S.V.), and Cancer Research Institute (T. Tamgüney, R.B., D.S.), University of California, San Francisco, San Francisco, CA 94143; and Institute of Cancer Genetics (R.P.), Columbia University, New York, NY; USA
Address correspondence to John K. Wiencke, Laboratory for Neuro and Molecular Epidemiology, 1st and Arguello, Ambulatory Care Bldg. AC-34, San Francisco, CA 94143-0441, USA (John.Wiencke{at}ucsf.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: low-grade glioma • methylation • PKB/Akt • PTEN • secondary glioblastoma
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Introduction |
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Glioblastoma (GBM) is a highly malignant brain tumor that is uniformly fatal. Efforts to improve the surgical, radiotherapeutic, and chemotherapeutic approaches to glioma treatment have failed to substantially increase long-term disease control.9 Although standard antineoplastic therapies have produced little clinical progress, genetic analyses of gliomas have increased our understanding of the molecular pathogenesis of these tumors.10 GBMs can either arise from low-grade gliomas or present as de novo tumors. low-grade tumors can arise as astrocytomas or oligodendrogliomas, which may depend on the cell type that sustained the initiating oncogenic alterations, or a mixed population termed oligoastrocytoma (OA). These two presentations of GBM have distinct, albeit overlapping, genetic alterations. While EGFR gene amplification and PTEN mutations are common in de novo GBMs, both of these alterations are infrequent in secondary GBMs. Conversely, secondary GBMs commonly contain TP53 mutations and PDGFR gene amplifications, aberrations that are less frequent in de novo GBMs.11
The significant proportion of de novo GBMs harboring PTEN mutations, 14%-47%,12,13 attests to the importance of this mutation in glioma pathogenesis. Moreover, low PTEN RNA levels,14,15 low protein levels,16 and loss of heterozygosity (LOH)17 each portends decreased survival in GBM patients. PTEN exerts its effects by dephosphorylating the phospholipid second messenger phosphatidylinositol (3,4,5)trisphosphate (PtdIns[3,4,5]P3) that is generated by phosphoinositide 3-kinase (PI3K).18 PtdIns(3,4,5)P3 recruits proteins that contain pleckstrin homology domains, such as protein kinase B (PKB/Akt), to the plasma membrane, allowing its phosphorylation and activation by another protein kinase, 3'-phosphoinositide-dependent kinase-1 (PDK1).19 Activation of PKB/Akt and other targets of PtdIns(3,4,5)P3 promotes a number of biological events important in tumorigenesis, including proliferation, survival, migration, and angiogenesis.20
Although PTEN mutations are exceedingly rare in low-grade gliomas, these tumors exhibit lower PTEN protein levels than does normal brain. Consequently, low-grade gliomas also display elevated levels of PKB/Akt phosphorylation and activity.16 We therefore hypothesized that methylation of the PTEN promoter may underlie decreased PTEN expression in tumors without PTEN mutations. Results we present here show that low-grade gliomas exhibit frequent PTEN promoter methylation, with a significantly lower frequency observed in de novo GBM specimens. In contrast, the PTEN promoter is frequently methylated in secondary GBMs. No PTEN promoter methylation was seen in 13 samples of normal brain, indicating that PTEN promoter methylation may represent an epigenetic mechanism to reduce PTEN expression during tumorigenesis. PTEN methylation in low-grade gliomas was associated with increased PKB/Akt phosphorylation, consistent with the known role of PTEN in regulating the PI3K/PKB/Akt pathway. We discuss the implications of these findings for our understanding of the genetics of de novo and secondary GBMs, as well as for therapeutic alternatives for low-grade gliomas.
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Materials and Methods |
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DNA Preparation and Bisulfite Treatment
Genomic DNA was isolated from approximately 25 mg wet weight of each frozen tissue sample using QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions and eluted twice in a total of 100 µl of elution buffer. This procedure yielded 5-40 µg of DNA. Bisulfite modification of genomic DNA was performed as described previously.21 Briefly, 1 µg purified DNA was diluted in 36 µl H2O, 4 µl 3.0 M NaOH was added, and DNA was denatured at 37°C for 15 min. The samples were then treated with 416 µl 3.6 M sodium bisulfite solution (pH 5.0) and 24 µl of 10 mM hydroquinone. All solutions were prepared fresh for each analysis. Samples were incubated at 55°C for 16 h. Two drops of mineral oil were layered on top of the solution to prevent evaporation. Bisulfite-modified DNA was purified with the Wizard DNA Clean-up System and vacuum manifold (Promega, Madison, WI, USA) according to the manufacturer's manual. Freshly prepared NaOH solution was added to a final concentration of 0.3 M, and samples were incubated at 37°C for 15 min, followed by neutralization with ammonium acetate (pH 7.0; final concentration, 3.0 M) and ethanol precipitation. Normal human peripheral blood lymphocyte DNA samples, treated and untreated with DNA methylase (M. Sss I; New England BioLabs, Beverly, MA, USA), were also modified as positive and negative controls, respectively.
Methylation-Specific PCR
To examine whether the PTEN promoter is methylated in glioma specimens, we used methylation-specific primers that had previously been used to demonstrate methylation of the PTEN promoter in a subset of non-small-cell lung cancer samples.22 These primers amplify a 181-base pair region of the PTEN promoter that starts 2,477 nucleotides upstream from the translation start site—methylated primers: forward, 5'-GTTTGGGGATTTTTTTTTCGC-3'; reverse, 5'-AACCCTTCCTACGCCGCG-3'; unmethylated primers: forward, 5'-TATTAGTTTGGGGATTTTTTTTTTGT-3'; reverse, 5'-CCCAACCCTTCCTACACCACA-3'. It should be noted that these primers do not amplify the highly homologous PTEN pseudogene located on chromosome 9p21, as these sequences lie outside the region of similarity.
The 25 µl PCR reaction contained 25 ng bisulfite DNA, 2% dimethylsulfoxide, 1.5 mM MgCl2, and 1 U Ampli Taq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA). PCR reactions were cycled in a Gene Amp 2700 thermocycler (Applied Biosystems) under the following conditions: preheat at 95°C for 10 min, 95°C for 30 s, 62°C for 30 s, 72°C for 30 s, 38 cycles, and a final extension at 72°C for 7 min. Aliquots (12 µl) of methylation-specific PCR (MSP) products were analyzed on 3% agarose gels, stained with ethidium bromide, and visualized under UV illumination. Results were recorded with a digital imaging system. For each PCR experiment, DNA from peripheral blood of normal blood donors treated with and without CpG methylase and bisulfite was included as positive and negative controls, respectively. We repeated MSP assays on all samples and found no discordant results among replicates. The MSP assay is sensitive to approximately 5% methylated product. To confirm the efficiency of the bisulfite modification and the specificity of MSP, bisulfite sequencing of the PCR products was carried out using the procedure reported previously.23
PTEN Sequencing
Fifty nanograms of genomic DNA was used for 40 rounds of PCR using the PTEN exon primers listed in Table 1. Primers that span the exon/intron boundaries were designed using the algorithm Primer3 (frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).
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PCR products were then sequenced in both directions on an ABI3700 DNA analyzer (Applied Biosystems), using the same primers used for amplification.
Western Blot Analysis
An approximately 50-mg sample of frozen tissue was homogenized in a Dounce homogenizer using 500 µl lysis buffer (20 mM Tris HCl, pH 7.4, 150 mM NaCl, 25 mM NaF, 1% NP40, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 1 mM NaVO4, and one protease inhibitor pill [Roche Applied Scientific, Indianapolis, IN, USA]/10 ml), followed by a 5-s treatment with a sonic dismembrator (Fisher Scientific, Pittsburgh, PA, USA). Insoluble material was removed by centrifugation at 13,000 x g for 15 min at 4°C. Protein concentration was estimated by the method of Lowry, and equal amounts of total protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to polyvinyl difluoride membranes, and blocked in 10 ml 5% milk in Tris-buffered saline containing 1% Tween-20 (TBST). Both the PTEN antibody (ABM-2052; Cascade Bioscience, Winchester, MA, USA) and the ß-actin antibody (A-5441; Sigma-Aldrich, St. Louis, MO, USA) were used at 1:5,000 dilution in TBST. Proteins were visualized using enhanced chemiluminescence (Amersham, Biosciences, Piscataway, NJ, USA).
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections were digested with protease XXV (Lab Vision Corp., Fremont, CA, USA) at 37°C for 10 min, and peroxidase activity was quenched with 3% hydrogen peroxide in phosphate-buffered saline. Sections were incubated with an antibody recognizing the Ser-473 phosphorylated form of PKB/Akt (from Cell Signaling Technology, Inc., Danvers, MA, USA; used at 1:50 dilution) and PTEN (from Cell Signaling Technology, Inc.; used at 1:1,000 dilution) at 4°C overnight. Primary antibody incubations were followed by incubation with biotinylated secondary antibody (1:200; no. BA-2000; Vector Labs, Burlingame, CA, USA) and avidin-biotin complex (1:100; no. PK6100; Vector Labs Vectastain ABC Kit) for 30 min each. Staining was visualized using 3,3'-diaminobenzidine tetrahydrochloride, and slides were counterstained with hematoxylin. Sections from nontumor brain and a PTEN-mutant GBM were used as controls for both phospho-PKB/Akt and PTEN. Staining for PKB/Akt was recorded as negative or positive in tumor cells. Staining for PTEN was recorded as positive and negative in tumor cells and in comparison to both normal neuronal and vascular cells and the positive control slide.
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We next examined whether methylation at the PTEN promoter affected the expression levels of PTEN protein in grade II astrocytomas and OAs. We analyzed PTEN expression by both Western blot analysis and immunohistochemistry (IHC), using sections of nontumor brain and GBM as a positive and negative control, respectively. Although nontumor brain showed strong staining, and the GBM showed negative staining by both techniques, neither approach showed a correlation between PTEN methylation and PTEN levels. Western blotting indicated that PTEN expression was uniformly high in 20 grade II tumors examined, regardless of PTEN methylation (data not shown). We hypothesized that this could be due to the infiltrative nature of low-grade glioma resulting in the contamination of normal tissue in the tumor sections analyzed. We therefore also analyzed PTEN levels by IHC to determine if regions of lower PTEN staining correlated with the presence of tumor material. This analysis showed that the majority of low-grade tumors analyzed (17 of 21) actually showed reduced PTEN staining in the tumor cells, with only four tumors showing diffusely positive staining, but this did not correlate with PTEN methylation (see Table 3 for the tumors in which PKB/Akt phosphorylation was also analyzed).
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Given the high frequency of PTEN methylation in grade II gliomas, we hypothesized that upon progression to higher grade gliomas, grade III tumors and secondary GBMs would retain this epigenetic aberration and display frequent PTEN methylation, in contrast to de novo GBMs in which PTEN methylation would prove rare. We obtained 19 grade III gliomas (10 OAs and 9 anaplastic astrocytomas) and 11 secondary GBM samples from patients who had pathologically documented prior low-grade gliomas, and found strong methylation of the PTEN promoter in 12 of 19 grade III gliomas and 9 of 11 secondary GBMs (Table 2, Fig. 1B). These frequencies are significantly different compared to the frequency of PTEN methylation in de novo GBMs (p < 0.001), but not (with the small number of samples) compared to that seen in low-grade gliomas (Table 2).
Promoter methylation may therefore represent an alternative mechanism of PTEN inactivation. PTEN mutation and methylation are likely to be mutually exclusive in any particular tumor. To test this hypothesis, we analyzed 14 of the de novo GBMs and nine of the secondary GBMs (the tumors that had sufficient DNA) for PTEN mutations. The analyses of PTEN mutations in these tumors showed that none of the nine secondary GBMs harbored PTEN mutations. Eight of nine secondary GBMs displayed PTEN methylation. in contrast, of the 14 de novo GBMs, four harbored PTEN mutations, and these were mutually exclusive with the two tumors that displayed PTEN methylation. Three PTEN mutations were in exon 5, and one was in exon 6, which together compose the core phosphatase domain. Mutations in GBMs consisted of M134I, T131P, R173C, and W111stop. Although the small number of tumors expressing mutant PTEN in this analysis precluded a statistically significant conclusion, the data are consistent with the hypothesis that PTEN methylation and mutation represent two independent mechanisms of PTEN inactivation.
We also analyzed potential associations of PTEN methylation and PKB/Akt phosphorylation with progression-free survival in the low-grade glioma patients. No statistically significant associations with clinical outcome were found. For the methylation analysis, we had information on 18 patients, of whom 10 had progressed. The hazard ratio for the difference was 0.28 (p = 0.06; 95% confidence interval, 0.07-1.13) favoring patients whose tumors were methylated. For PKB/Akt phosphorylation, only 12 patients had the necessary information, and 7 of these 12 had progressed. The hazard ratio for this difference was 0.32 (p = 0.16; 95% confidence interval, 0.06-1.69) favoring those with phosphorylation.
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Discussion |
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PTEN methylation in grade II gliomas correlated with PKB/Akt activation but did not correlate with PTEN expression levels as measured by either Western blot or IHC. Although some studies have seen a correlation between PTEN promoter methylation and PTEN expression,32,35 the correlations are not perfect and, in some cases, require tissue microdissection.26 Other studies have also noted no correlation between PTEN methylation and PTEN expression.31 Western blot analysis suggested that PTEN levels were uniformly high in the tumor samples, perhaps reflecting the invasive nature of low-grade gliomas, resulting in extensive normal tissue contamination within tumor specimens. Whereas contaminating normal tissue likely masks small reductions in PTEN expression by Western blot, the sensitive methylation assay used herein can detect methylation in a single PTEN allele, in samples containing only small proportions of tumor cells. In addition, as mentioned above, PTEN methylation may decrease expression in only a single allele in low-grade gliomas, thereby confounding detection of small decreases in expression. In contrast, very low levels of PKB/Akt phosphorylation in normal brain tissue16 facilitate detection of even small increases in PKB/Akt phosphorylation in tumor specimens, which allows visualization of this functional consequence of PTEN methylation. Conversely, IHC analysis suggested that most of the tumor cells present had low levels of PTEN staining, with only 4 of 21 tumors analyzed scored as having diffusely positive immunoreactivity. Regardless of whether this reflects that the PTEN IHC staining is not reliable in our hands (although it was performed twice independently in two different laboratories) or that PTEN levels in the low-grade tumors are decreased for multiple reasons, of which PTEN methylation is only one, it is clear that PTEN levels did not correlate with PKB/Akt phosphorylation (Table 3). Therefore, whether there is a simple linear relationship among PTEN methylation, decreased PTEN expression, and increased PKB/Akt phosphorylation remains to be proven. Nevertheless, PTEN methylation appears to be a marker for low-grade tumors that show increased PKB/Akt phosphorylation.
These technical difficulties for measuring PTEN levels aside, the role of promoter methylation in decreasing gene expression during tumorigenesis remains controversial.40 There is no clear mechanism leading to de novo methylation of CpG islands in somatic glial cells and resultant selective decreased expression of certain tumor suppressor proteins such as PTEN. Alternatively, promoter methylation may reflect gene inactivity rather than represent a primary cause of transcriptional repression. Although such mechanistic questions remain to be answered, PTEN methylation in human malignancies raises the possibility of using agents designed to antagonize CpG hypermethylation, for example, 5-azacytidine and 5-aza-2'-deoxycytidine (also known as Vidaza and decitabine, respectively). Vidaza has been approved for myelodysplastic syndrome41 and is currently in clinical trials for non-small-cell lung cancer and prostate cancer.42 Potential side effects induced by global demethylation notwithstanding, if methylation is a consequence of silencing rather than a cause, then demethylation may not restore gene activity. Of note, however, are studies in which treatment of a prostate cancer xenograft,43 a lung cancer cell line,22 and two melanoma cancer cell lines37 with decitabine resulted in increased PTEN expression. These latter two studies documented PTEN methylation at the same region of the promoter as analyzed in our studies, adding credence to the potential treatment of low-grade gliomas with such demethylating agents.
Although the small sample size precluded statistically significant conclusions, it was unexpected that PTEN methylation and increased PKB/Akt phosphorylation were associated with better outcome in low-grade gliomas. While decreased levels of PTEN generally portend poor clinical outcome in GBM16 and other tumors,44 there are reports of PTEN mutations correlating with favorable outcome in endometrial and lung cancer.31,45,46 This observation has been interpreted as PTEN alterations being required for tumor-associated initiating lesions, with alternative pathways being more important for tumor progression. Such correlations between genetic alterations and favorable prognosis in gliomas have also been noted for TP53 mutations47 and loss of chromosome 1p/19q.48 Clearly, further work is required to dissect the significance of differing PTEN genetic alterations in the establishment and development of human gliomas.
In addition to demethylating agents, our results point to the potential utility of inhibitors of the mammalian target of rapamycin (mTOR) in the treatment of low-grade gliomas. Rapamycin and its esterified analogues RAD-001 and CCI-779 have favorable toxicity profiles and carry additional appeal since tumors that express activated PKB, as is evident in the low-grade gliomas and secondary GBMs described herein, appear particularly sensitive to mTOR inhibition.49,50 Finally, studies that describe detection of tumor-associated gene methylation in peripheral blood samples from cancer patients51 further increase enthusiasm for future patient selection for treatment with these and other therapies.
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Acknowledgments |
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Received for publication June 16, 2006. Accepted for publication October 2, 2006.
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PTEN). The black elongated rectangle represents the PTEN transcribed region, and the initiating methionine codon is indicated. The black line extending to the left of the rectangle represents the PTEN promoter. The hatched rectangle represents the PTEN pseudogene sequence on chromosome 9 showing the region of high homology to the PTEN transcript. Tumor-associated PTEN methylation has been reported in regions a,