Integrative Genomic Analysis Identifies NDRG2 As a Candidate Tumor Suppressor Gene Frequently Inactivated in Clinically Aggressive Meningioma

Research Article

Integrative Genomic Analysis Identifies NDRG2 as a Candidate Tumor Suppressor Gene Frequently Inactivated in Clinically Aggressive Meningioma

Eriks A. Lusis,1 Mark A. Watson,2 Michael R. Chicoine,3 Meghan Lyman,4 Peter Roerig,5 Guido Reifenberger,5 David H. Gutmann,4 and Arie Perry1

1Division of Neuropathology, Departments of 2Pathology and Immunology, 3Neurosurgery, and 4Neurology, Washington University School of Medicine, St. Louis, Missouri; and 5Department of Neuropathology, Heinrich-Heine-University, Du¨sseldorf, Germany

Abstract and TSLC1 are also common (2, 4–6). However, there are few Although meningiomas are common central nervous system known genetic changes associated with the malignant progression tumors, little is known about the genetic events responsible of meningioma. Although losses of chromosomal arms 14q and 1p for malignant progression. In this study, we employed gene are common in anaplastic tumors (WHO grade III) and are expression profiling to identify transcripts whose expression generally associated with poor prognosis (7, 8), the relevant tumor was lost in anaplastic (WHO grade III) versus benign (WHO suppressor genes that map to these loci have not been identified. grade I) meningioma. Approximately 40% of genes down- Loss of heterozygosity studies have had limited success in regulated in anaplastic meningioma were localized to chro- identifying minimal regions of deletion, in part, due to the fact mosomes 1p and 14q. One specific gene located at 14q11.2, that the entire chromosome or chromosomal arm is lost in most NDRG2, was consistently down-regulated in grade III menin- examples. In the current study, we used a strategy combining expression profiling with known cytogenetic alterations, leading gioma, a finding which we validated at both the transcript and protein levels in independent sets of clinically and patholog- to the identification of NDRG2 as a potential meningioma- ically diverse meningiomas. Loss of NDRG2 expression was associated tumor suppressor gene on 14q11.2. We subsequently also seen in a subset of lower-grade meningiomas, including validated its role in meningioma tumor progression using atypical meningiomas (WHO grade II) with clinically aggres- multiple methods for measuring RNA and protein expression sive behavior. Furthermore, we found that the loss of NDRG2 within independent cohorts of clinically well-characterized expression was significantly associated with hypermethylation meningiomas. Lastly, hypermethylation of the CpG island within of the NDRG2 promoter. Collectively, these data identify the NDRG2 promoter region was shown to be the likely NDRG2 as the first specific candidate tumor suppressor gene mechanism of inactivation in the majority of meningiomas with on chromosome 14q that is inactivated during meningioma loss of NDRG2 expression. progression. In addition, these findings highlight the utility of combining genomic, epigenetic, and expression data to Materials and Methods identify clinically significant tumor biomarkers, and suggest Tissue specimens. Tissue specimens were collected by the Siteman that NDRG2 expression will be a useful and functionally Cancer Center Tissue Procurement Facility and the Department of relevant biomarker to predict aggressive behavior in patients Neuropathology, Heinrich-Heine-University, under approved protocols with meningioma. (Cancer Res 2005; 65(16): 7121-6) from the Institutions’ Review Boards. Snap-frozen tissue specimens from surgically resected meningiomas were histologically reviewed for speci- Introduction men adequacy (at least 80% tumor) and subsequent 50 Am serial sections from each banked frozen specimen were cut, placed immediately into Meningiomas account for approximately one-fourth of all Trizol reagent (Invitrogen, Carlsbad, CA), and homogenized for RNA primary central nervous system neoplasms. Although most are preparation. Total RNA was isolated from Trizol homogenates using the benign, as many as 20% display clinically aggressive features, manufacturer’s protocol. RNA was then further purified using RNeasy spin leading to increased patient morbidity or mortality (1, 2). The columns (Qiagen, Valencia, CA). For RNA isolation from paraffin- 2000 WHO grading scheme has improved the ability to predict embedded tissues, Ambion’s (Austin, TX) formalin-fixed paraffin-embedded clinical behavior (3), though significant variability remains, RNA isolation kit was used according to the manufacturer’s protocol. All particularly within the subset of atypical meningiomas (WHO RNAs were quantified by UV absorbance at 260 and 280 nm and qualitatively assessed using an RNA Nano Assay and Bioanalyzer 2100 grade II). The most clearly established early genetic alteration in (Agilent, Palo Alto, CA). sporadic meningioma is biallelic inactivation of the neurofibro- Microarray analysis. Five micrograms of tumor RNA was converted matosis 2 (NF2) gene located on chromosome 22q, with to double-stranded cDNA using a dT-T7 promoter primer. Purified, associated loss of merlin (schwannomin) expression. Recently, double-stranded cDNA was then used as a template to create we have shown that loss of expression of other protein 4.1 family biotinylated aRNA. The labeled aRNA target was quantified, fragmented, members and interacting proteins, such as 4.1B (DAL-1), 4.1R, and 15 Ag was used for microarray hybridization. The complete target preparation protocol was done according to the manufacturer’s recommendations (Affymetrix, Santa Clara, CA) and as previously published (9). Labeled targets were hybridized to Affymetrix U133A Requests for reprints: Arie Perry, Division of Neuropathology, Washington and U133B GeneChip microarrays for 16 hours and washed following University School of Medicine, Campus Box 8118, 660 South Euclid Avenue, St. Louis, standard protocols. Microarray images were processed using Affymetrix MO 63110. Phone: 314-362-9130; Fax: 314-362-4096; E-mail: [email protected]. I2005 American Association for Cancer Research. Microarray Analysis Suite version 5.0. Each array was scaled so that the doi:10.1158/0008-5472.CAN-05-0043 average probe set hybridization signal intensity value (target intensity) www.aacrjournals.org 7121 Cancer Res 2005; 65: (16). August 15, 2005

Figure 1. Histogram demonstrating the location of 83 genes whose expression was significantly lower (P < 0.01) in anaplastic (WHO grade III) meningiomas relative to benign (WHO grade I) meningiomas. x-axis, the cytogenetic location; y-axis, percentage of differentially expressed probe sets located on each chromosome arm, relative to the total number of probe sets on that arm that are represented on the GeneChip U133A and U133B microarrays. Overrepresentation of probe sets at chromosome 1p and 14q (black column) was statistically significant (1p, Z score = 1.7288, P = 0.0419; 14q, Z score = 5.4000, P < 0.0001). NA, probe sets for which chromosomal localizations are not known.

was 1,500. Scaled data for each array was exported to the Siteman Immunohistochemistry. Immunohistochemistry using NDRG-specific Cancer Center Bioinformatics Server,6 merged with updated gene affinity-purified rabbit polyclonal antisera (1:10,000 dilution) was done on annotation data for each probe set on the array, and downloaded for formalin-fixed paraffin-embedded sections, using a recently developed further data visualization and analysis. The completely annotated, antibody (11). Paraffin blocks from surgically resected meningiomas were MIAME-compliant data set can be found at the above noted URL. Basic retrieved from the surgical pathology files at Washington University microarray data visualization, data filtering, and hierarchical clustering were School of Medicine in St. Louis, MO. Positive controls included normal done using DecisionSite for Functional Genomics (Spotfire, Somerville, MA). human and rodent brain, as well as human leptomeningeal rolls prepared Average fold differences in gene expression between benign and anaplastic from autopsy specimens as described previously (4). Cases with strong meningiomas were calculated and Student’s t test (uncorrected for multiple diffuse staining were scored as +, those with either patchy immunoreac- comparisons) was used to generate a list of differentially expressed genes tivity or decreased staining intensity compared with adjacent vessels were between the two tumor groups. scored as F (i.e., partial loss), and those with lack of tumoral staining in Quantitative reverse transcription-PCR analysis. Oligonucleotide >90% of the specimen were scored as À (i.e., complete loss). Regions lacking sequences corresponding to the NDRG2 gene transcript were designed vascular staining were not scored due to the possibility of technical failure. using Primer Express software (Applied Biosystems, Foster City, CA) and NDRG2 promoter methylation analysis. The NDRG2 promoter was are available on request from the authors. Five hundred nanograms of analyzed for CpG site methylation using sequencing of sodium bisulfite- total cellular RNA from tissue specimens was subjected to reverse modified DNA. Sodium bisulfite treatment of genomic DNA was carried out transcription using Omniscript reverse transcriptase (Qiagen) and oligo- as described by Herman et al. (12). Two overlapping fragments from the dT, following the manufacturer’s protocol. After first strand synthesis, an NDRG2 promoter region (covering 32 CpG sites between nucleotides equivalent of 50 ng of starting total cellular RNA (1/10 of the cDNA 20,564,110 and 20,562,576, Genbank accession no. NC_000014) were reaction) was added to two duplicate PCR reactions containing 1Â PCR-amplified from sodium bisulfite–modified DNA using the following SybrGreen master mix (Applied Biosystems), 100 nmol/L forward primer, primers: P1-sense, 5V-TTTTCGAGGGGTATAAGGAGAGTTTATTTT-3V and and 100 nmol/L reverse primer in a final volume of 20 AL. Each sample P1-antisense, 5V-CCAAAAACTCTAACTCCTAAATAAACA-3V(320 bp product); was used in a single reaction that cycled at 95jC for 10 minutes (to P2-sense, 5V-TTTAGGATATTGCGTTTTTTTTAAGTTTTTATTTT-3Vand P2-anti- activate enzyme), followed by 40 cycles of 95jC for 30 seconds, and 60jC sense, 5V-AAAATTCCGACTCCCTCGTACCCAAAA-3V (335 bp product). PCR for 1 minute on an ABI SDS 7000 sequence detection system (Applied amplification was carried out for 43 cycles and the PCR products were then Biosystems). Fluorescent data were converted into cycle threshold (CT) purified using the High Pure PCR Product Purification Kit (Roche, Penzberg, measurements using the SDS system software and exported to Microsoft Germany). Sequencing was done with the BigDye Cycle Sequencing Kit and an Excel. Thermal dissociation plots were examined for biphasic melting ABI PRISM 377 semiautomated DNA sequencer (Applied Biosystems). curves, indicative of whether primer-dimer formation or other nonspecific DNA extracted from four different normal leptomeningeal tissue speci- product could be contributing to amplification signal. The DDCT method mens obtained at autopsy served as nonneoplastic references. As a positive (10) was used to calculate fold expression, relative to the lowest tumor control, we methylated reference DNA in vitro using SssI (CpG) methylase grade in the specific analysis and using glyceraldehyde-3-phosphate (New England Biolabs, Beverly, MA). To calculate the degree of CpG site dehydrogenase and h-glucuronidase as reference transcripts. The Mann- methylation in the NDRG2 promoter region, the methylation status at each Whitney rank sum and Kruskall-Wallis ANOVA by ranks were used to of the 32 CpG sites analyzed was semiquantitatively rated using the compare relative expression levels between the different groups and following scale: 0, completely unmethylated; 1, a weak methylated signal results <0.05 were considered significant. detectable in the sequence; 2, methylated signal approximately equal to unmethylated signal; 3, methylated signal markedly stronger than unmethylated signal. For each tumor, a methylation score was then calculated by 6 http://bioinformatics.wustl.edu. adding the figures determined at the individual CpG sites. Based on this

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. NDRG2 in Aggressive Meningiomas score, the tumors were then subdivided into three groups: low/absent representation of probes along the genome, the percentage of promoter methylation (methylation score <20), moderate promoter down-regulated transcripts localized to chromosomes 1p and 14q methylation (methylation score 21-30), and strong methylation (methylation was statistically more frequent than that at any other cytogenetic score >30). location. Thirty-four of the 83 genes (41%) with attenuated expression in anaplastic meningiomas mapped to the 1p (P = Results and Discussion 0.0419) or 14q (P < 0.0001) arms, both previously shown to be To date, only a limited number of studies have examined the frequently lost during meningioma progression. transcriptome of meningiomas (13, 14). Therefore, we compared This finding led us to further define the exact cytogenetic gene expression in a set of 10 anaplastic and benign meningiomas location of these transcripts, hypothesizing that a cluster of down- using high-density oligonucleotide microarrays. Initially, we regulated transcripts might represent a common region of selected transcripts whose average expression between the two chromosomal loss. The 21 transcripts on 14q and the 13 tumor groups varied by at least 2-fold and by a standard t test, transcripts on 1p were mapped according to their absolute showed a statistical difference in expression at the level of P < nucleotide location, along with all other transcripts represented 0.01 (uncorrected for multiple comparisons). Using these criteria, on the microarray. Figure 2 illustrates the cytogenetic locations of we identified 153 transcripts and subsequently focused on a these 34 genes. Down-regulated transcripts were distributed subset of 83 transcripts with reduced expression in anaplastic randomly along the 1p and 14q chromosomal arms, and were meningiomas. not clustered at any single cytogenetic locus, arguing against a Transcripts were next mapped to their corresponding chromo- single region of chromosome loss that would account for all of the somal locations, and the number of differentially expressed coordinate decreases in gene expression. We further reasoned that transcripts localized to a specific chromosome arm was normalized potential tumor suppressor genes critical to progression would to the total number of probe sets on the array representing that exhibit biallelic inactivation, with a consequent complete loss of chromosomal arm (Fig. 1). After controlling for the unequal gene expression. Accordingly, we identified one gene, NDRG2,

Figure 2. Cytogenetic locations of 34 genes with a statistically significant decrease in expression in anaplastic versus benign meningiomas. Each column represents one tumor sample (five benign versus five anaplastic meningiomas), whereas each row represents a unique probe set on the microarray. Filled boxes indicate probe sets scored detected (‘‘P’’) in the particular tumor by the Affymetrix Microarray Analysis Suite 5.0 software, whereas open boxes indicate probe sets whose signal was below the threshold of detection. A, cytogenetic location of 21 genes on chromosomal arm 14q that are differently expressed. Arrow, loci where these transcripts are found. B, cytogenetic location of 13 genes on chromosomal arm 1p that are differently expressed. Arrow, loci where these transcripts are found. MI, benign meningioma (WHO grade I); MIII, anaplastic meningioma (WHO grade III). www.aacrjournals.org 7123 Cancer Res 2005; 65: (16). August 15, 2005

Figure 3. Differential NDRG2 expression in human meningiomas as measured by real-time reverse transcription-PCR. A, relative gene expression among 15 benign (WHO grade I), 11 atypical (WHO grade II), and 8 anaplastic meningiomas (WHO grade III) shows significantly reduced NDRG2 mRNA expression in the anaplastic meningiomas (Kruskall-Wallis ANOVA by ranks, P = 0.0015). B, relative NDRG2 mRNA expression in 16 atypical meningiomas (WHO grade II), stratified by clinical behavior. Levels of NDRG2 expression were normalized to the average expression level in the lowest grade of tumor. NDRG2 expression in the ‘‘aggressive’’ meningiomas was significantly lower as compared with the ‘‘indolent’’ tumors (Mann-Whitney U test, P = 0.0379). C, relative NDRG2 mRNA expression in 45 meningiomas stratified by NDRG2 promoter methylation. NDRG2 mRNA levels and promoter methylation were determined by real-time reverse transcription-PCR and sequencing of sodium bisulfite–treated DNA, respectively. Depending on each tumor’s promoter methylation score (see Materials and Methods), the cases were subdivided into three groups, i.e., low/absent promoter methylation (n = 14 tumors), moderate promoter methylation (n = 17 tumors), and strong promoter methylation (n = 14 tumors). Note significantly lower mean NDRG2 mRNA expression in the tumors with strong promoter methylation (P < 0.01, two-sided t test). which completely lacked detectable mRNA expression in all five Next, we examined corresponding NDRG2 protein expression by anaplastic meningiomas examined by gene expression microarray immunohistochemistry in a cohort of 49 tumors selected based on (Fig. 2). Other genes flanking NDRG2 on 14q11.2 (e.g., FLJ20859 well-characterized biological behavior. This third independent and ZNF219) retained expression regardless of tumor grade, tumor set included 17 clinically stratified atypical meningiomas, suggesting that NDRG2 represents a specific target of transcrip- 10 benign meningiomas with no recurrence after at least 10 years of tional inactivation in anaplastic meningiomas. follow-up, 10 benign meningiomas with recurrence despite gross To confirm differential expression of NDRG2, quantitative total resection, and 12 anaplastic meningiomas. Representative reverse transcription-PCR analysis was done on an independent examples of protein expression are shown in Fig. 4. NDRG2 protein set of 34 meningiomas, which included 15 benign, 11 atypical, and expression was retained in all but one benign tumor, whereas 8 anaplastic tumors. Although benign meningiomas showed complete or partial loss of expression was seen in all 12 anaplastic considerable variability in NDRG2 expression, anaplastic meningi- tumors. In intermediate grade tumors, loss of protein expression omas consistently showed attenuated expression (P = 0.0015) with was seen in only three of nine (33%) clinically indolent cases, as a mean of 4.2-fold lower expression levels (Fig. 3A). It was not compared with six of eight (75%) aggressive tumors. In total, 4 of 19 possible to determine whether the ‘‘benign’’ tumors with attenu- (21%) biologically benign meningiomas versus 23 of 30 (77%) ated NDRG2 expression were more aggressive, as the majority of biologically aggressive meningiomas showed partial or complete these tumors were only recently resected and there was insufficient loss of protein expression (P < 0.001; Fisher’s exact test). clinical follow-up. Therefore, to determine whether NDRG2 Finally, to investigate the mechanistic basis of NDRG2 gene expression correlates with clinical behavior independent of tumor inactivation, we determined the methylation status of the NDRG2 grade, we examined an additional set of atypical (WHO grade II) promoter in 45 meningiomas (24 benign, 11 atypical, and 10 meningiomas. Total RNA was isolated from 16 atypical meningi- anaplastic meningiomas) using sequencing of sodium bisulfite– omas, previously stratified by clinical behavior. Eight patients with modified DNA. These analyses revealed strong methylation, clinically ‘‘indolent’’ tumors were defined by a neurosurgeon (M.R. preferentially involving CpG sites 1 to 12 and 16 (located between Chicoine) as patients with no recurrence or death (median follow nucleotides 20,564,169 and 20,564,309, Genbank accession no. up 9.9 years), whereas eight patients with ‘‘aggressive’’ disease had a NC_000014) in 7 of 10 (70%) anaplastic meningiomas examined median time to tumor recurrence of 2.2 years and a median (Fig. 5). In normal leptomeningeal tissue, these CpG sites were only survival of 8.2 years. Expression of NDRG2 was consistently partially methylated or completely unmethylated. Among the attenuated in the clinically aggressive subset and differed nonanaplastic meningiomas, strong NDRG2 promoter methylation significantly from the indolent subset (P = 0.0379), with an average was detected in only 7 of 35 cases (20%; P < 0.01, m2 test). The of 5.8-fold decreased expression (Fig. 3B). remaining tumors showed either low (14 tumors) or moderate

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Figure 4. Immunohistochemical analysis of NDRG2 protein expression. A, strong, diffuse staining in both normal cortex and overlying leptomeninges. B, benign meningioma with strong and diffuse immunoreactivity, consistent with retained expression. C, anaplastic meningioma scored as partial loss of expression based on decreased staining intensity compared with intratumoral blood vessels. D, anaplastic meningioma with complete loss of NDRG2 expression, but retained immunoreactivity in adjacent blood vessel.

(17 tumors) levels of NDRG2 promoter methylation. Correlation of mechanism of transcriptional down-regulation of NDRG2 in the promoter methylation data with NDRG2 mRNA expression aggressive meningiomas is NDRG2 promoter hypermethylation. revealed a significantly lower mean expression level in the 14 Nevertheless, the presence of low expression levels in some tumors with strong promoter methylation as compared with the meningiomas lacking promoter hypermethylation suggests that tumors with low/absent or moderate promoter methylation other mechanisms of inactivation may also be taking place in a (Fig. 3C). In total, these data suggest that the most frequent subset of cases.

Figure 5. Demonstration of NDRG2 promoter methylation by sequencing of sodium bisulfite–modified DNA from one anaplastic meningioma (MN212; A), normal leptomeningeal tissue (B), and in vitro methylated (SssI methylase-treated) normal leptomeningeal tissue (C). Note methylation of the six depicted CpG sites (CpG sites 3-8) in the anaplastic meningioma (A) and the positive control (C). In contrast, normal leptomeningeal tissue (B) either shows partial or no methylation at the depicted CpG sites. Arrowheads, CpG sites in the depicted sequences (noncoding strand sequences).

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N-myc downstream regulated gene 2 (NDRG2) is normally tumor types. Although our study clearly shows that NDRG2 expressed in brain, heart, and muscle and is one of four members of inactivation is common in clinically aggressive meningioma, further the NDRG family (15). Although the NDRG1 gene is regulated by studies will be necessary to validate this biomarker using larger N-myc, other members of this gene family are not similarly cohorts of patients with more extensive follow-up data. In addition, regulated, with each member demonstrating distinct patterns of future genomic and functional studies will be required to define the expression during development (15, 16). The precise biological precise mechanism of NDRG2-associated growth regulation in function of this protein family is unknown, but an a/h hydrolase fold meningioma, which could potentially elucidate additional targets for domain of 220 amino acids suggests possible enzymatic function biologically based meningioma therapy. (15). NDRG2 has been implicated in cell growth (17), differentiation (18), apoptosis (19), and is rapidly responsive to mineralocorticoid Acknowledgments stimuli in the kidney (20). Most recently, NDRG2 has been found to be up-regulated in the brains of Alzheimer’s patients (11), suggesting Received 1/6/2005; revised 3/14/2005; accepted 5/4/2005. Grant support: Doris Duke Charitable Foundation (E.A. Lusis), the James S. roles in both cell growth and neurodegeneration. McDonnell Foundation (D.H. Gutmann) and the Deutsche Krebshilfe (G. Reifenberger). The analytic approach described here, combining a priori The costs of publication of this article were defrayed in part by the payment of page knowledge of tumor-associated cytogenetic alterations with tran- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. scriptome profiling, represents a potentially unique and powerful We thank Dr. Niels A. Jensen from The Panum Institute 6.5, University of method to identify clinically relevant molecular biomarkers in other Copenhagen N, Denmark for providing the NDRG2 antibody used in this study.

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Eriks A. Lusis, Mark A. Watson, Michael R. Chicoine, et al.

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