Somatic mutations of WNT/wingless signaling pathway components in primitive neuroectodermal tumors
Article first published online: 10 MAY 2001
Copyright © 2001 Wiley-Liss, Inc.
International Journal of Cancer
Volume 93, Issue 3, pages 445–449, 1 August 2001
How to Cite
Koch, A., Waha, A., Tonn, J. C., Sörensen, N., Berthold, F., Wolter, M., Reifenberger, J., Hartmann, W., Friedl, W., Reifenberger, G., Wiestler, O. D. and Pietsch, T. (2001), Somatic mutations of WNT/wingless signaling pathway components in primitive neuroectodermal tumors. Int. J. Cancer, 93: 445–449. doi: 10.1002/ijc.1342
- Issue published online: 14 JUN 2001
- Article first published online: 10 MAY 2001
- Manuscript Accepted: 2 MAR 2001
- Manuscript Revised: 28 FEB 2001
- Manuscript Received: 17 OCT 2000
- German Research Council. Grant Number: SFB400
- Turcot's syndrome
Primitive neuroectodermal tumors (PNETs) represent the most frequent malignant brain tumors in childhood. The majority of these neoplasms occur in the cerebellum and are classified as medulloblastomas (MB). Most PNETs develop sporadically; however, their incidence is highly elevated in patients carrying germline APC gene mutations. The APC gene encodes a central component of the WNT/wingless developmental signaling pathway. It regulates the levels of cytoplasmic β-catenin protein that plays a central role in neural development and cell proliferation. We analyzed 87 sporadic PNETs and 10 PNET cell lines for mutations of the APC gene and β-catenin (CTNNB1) gene using single strand conformational polymorphism (SSCP) and sequencing analysis. We examined the mutation cluster region of APC (codons 1255–1641) for germline variants and somatic mutations. The medulloblastoma cell line MHH-MED-2 carried a Glu1317Gln missense germline variant and a sporadic MB sample showed a somatic Pro1319Leu substitution. Mutational analysis of exon 3 of CTNNB1 uncovered 4 PNETs (4.8%) with somatic missense mutations. These mutations caused amino acid substitutions in 3 of 80 medulloblastomas (Ser33Phe, Ser33Cys and Ser37Cys) and 1 of 4 supratentorial PNETs (Gly34Val). All mutations affected GSK-3β phosphorylation sites of the degradation targeting box of β-catenin and resulted in nuclear β-catenin protein accumulation. Deletions of CTNNB1 were not detected by PCR amplification with primers spanning exons 1–5. Our data indicate that inappropriate activation of the WNT/wingless signaling pathway by mutations of its components may contribute to the pathogenesis of a subset of PNETs. © 2001 Wiley-Liss, Inc.
Primitive neuroectodermal tumors (PNETs) most frequently develop in the cerebellum where they are classified as medulloblastomas (MBs). In children, MBs are the most common tumors of the central nervous system with an incidence of 5 cases/1 million children.1 Most MBs develop as sporadic tumors but their incidence is markedly elevated in Turcots syndrome, a familial cancer syndrome. This syndrome is characterized by colorectal polyposis and central nervous system tumors, in particular glioblastomas and MBs.2
The APC gene product is involved in the WNT/wingless signal transduction pathway.3, 4 This pathway is important in the embryonal development of the central nervous system. The presence of a WNT/wingless signal in normal embryonic cells stabilizes β-catenin that then enters the cell nucleus and forms a complex with members of the Tcf-lymphoid enhancer factor family of transcription factors.5, 6 This complex regulates transcription and expression of target genes, including the c-myc- and cyclin D1 (CCND1) gene.7, 8
One of the most important functions of the APC protein is the regulation of the cytoplasmic β-catenin levels by direct binding to β-catenin and targeting it for degradation.9 Binding of β-catenin by APC results in NH2-terminal phosphorylation of β-catenin by the GSK-3β kinase at serine and threonine residues encoded in exon 3 of CTNNB1.10
Oncogenic activation of the β-catenin protein by amino acid substitutions and interstitial deletions has been demonstrated in various tumors such as melanomas and colorectal cancers.5, 11, 12, 14, 15 More than 80% of primary colorectal tumors carry APC mutations.16
To define the role of the WNT/wingless signaling pathway in the pathogenesis of sporadic PNETs/MBs we performed a systematic mutational analysis of the APC- and β-catenin (CTNNB1) genes in a large panel of these neoplasms.
MATERIAL AND METHODS
Patients, tumors and cell lines
A total of 87 PNETs from 84 patients, including 80 patients with MB and 4 with supratentorial PNETs were analyzed. In addition, 10 MB cell lines were studied, including 5 that have been reported previously.17 All tumors were classified according to the revised WHO classification of brain tumors.18 The MBs contained 64 classic MBs, 18 desmoplastic MBs and 2 medullomyoblastomas. The age of patients at diagnosis ranged from 1 month to 59 years.
DNA and cDNA preparation
DNA was extracted from tumor samples, cell lines and peripheral blood leukocytes by standard proteinase K digestion and phenol/chloroform extraction.19 From 50 cases, total cellular RNA was prepared, either by lysis in guanidinium isocyanate and ultracentrifugation20 or by Trizol reagent according to the protocol of the supplier (Life Technologies, Bethesda, MD). Contaminating residual genomic DNA was removed by digestion with RNase-free DNase (Boehringer-Mannheim, Indianapolis, IN) before reverse transcription. The RNAs were reverse transcribed using the Super-Script Preamplification System (Life Technologies) with random hexamers as primers. Individual tissue samples for DNA and RNA extraction were preexamined by frozen section histology to document the histopathological appearance of the specimen and exclude contamination by normal central nervous tissue.
Amplification of APC gene segments
Six overlapping primer pairs were used to amplify exon 15 (codon 1255 to codon 1641) of the APC gene that includes the mutation cluster region (MCR). PCR was carried out in a final volume of 10 μl with 10–50 ng of genomic DNA in a buffer containing 1.0–1.5 mM MgCl2 (Life Technologies), 200 mM of each deoxynucleoside triphosphate, 5 pmol of each forward and reverse primer and 0.25 U Taq polymerase (Life Technologies). Thirty-five cycles of PCR were performed for all primers with a denaturation step of 94°C for 35 sec, an annealing step of 55°C for 40 sec and extension step of 72°C for 40 sec. The primer sequences may be provided by request of the reader.
Amplification of CTNNB1 gene segments
The PCR-conditions and primer sequences for SSCP analysis of exon 3 of the β-catenin gene and the conditions of the detection of deletions are described previously.21
Single strand conformation polymorphism (SSCP) and sequencing analysis of the APC and CTNNB1 genes
PCR products were loaded on 10% and 14% polyacrylamide gels with different acrylamide:bisacrylamide ratios with and without 5% glycerol. The single and double strands of the PCR products were visualized by silver staining, as described previously.22, 23 PCR products that showed a gel mobility shift were excised from the wet gel, eluted, and reamplified. The resulting PCR products were purified (QIAquick PCR purification kit; Qiagen, Chatsworth, CA) and sequenced in both directions.
The cycle sequencing reaction was performed in a final volume of 20 μl using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) with 20 ng of PCR product as template. The sequencing products were analyzed on an ABI 373A sequencer (Applied Biosystems, Foster City, CA).
Western blot analysis
Seventeen MB samples were analyzed for β-catenin protein expression by Western blotting using antibodies that detects wild-type and mutant β-catenin protein (clone 14, IgG1, 0.5 μg/ml in PBS, 0.1% BSA, Transduction; and clone 7D11, IgG2a, 1 μg/ml in PBS, 0.1% BSA, Alexis). The protein-separation by electrophoresis on SDS-polyacrylamide gels, blotting onto nitrocellulose and the immunohistochemical staining by alkaline phosphatase anti-alkaline phosphatase (APAAP)24 are described elsewhere.21
Sections from formalin-fixed, paraffin-embedded tumor samples were cut at 4 μm, mounted on positively charged slides (Superfrost + Menzel), air-dried in an incubator at 42°C overnight, and deparaffinized in xylene. After rehydration in a graded alcohol series, the slides were incubated in 1% hydrogen peroxide diluted in methanol for 30 min to block endogenous peroxidase activity, and then rehydrated in distilled water, followed by PBS. After microwave treatment for 30 min in 0.1 M sodium citrate (pH 6.0), the slides were incubated in a blocking solution (PBS with 5% nonfat dry milk and 2% normal rabbit serum) for 30 min at RT. This was followed by a 2 × 15-min incubation with avidin-biotin blocking solutions (avidin-biotin blocking kit; Vector Laboratories, Inc., Burlingame, CA). The solution was removed from the slides and the monoclonal anti-β-catenin antibody 14 (IgG1, 0.25 μg/ml in PBS, 0.1% BSA; Transduction) was added to the samples overnight at 4°C. After removing unbound antibody by several rinses with PBS and PBS containing 0.1% Triton X-100, the bound antibody was detected using the avidin-biotin complex method (DAKO) and visualized by diaminobenzidine tetrahydrochloride. Slides were lightly counterstained with hematoxylin.
APC mutation analysis in PNETs
Eighty-three samples of sporadic MBs and 4 supratentorial PNETs were analyzed for mutations in the MCR encoded by exon 15. SSCP analysis and sequencing uncovered a point mutation in codon 1317 in the MB cell line MHH-MED-2 that has been derived from a classic MB. The mutation resulted in an amino acid substitution from glutamic acid to glutamine (E1317Q). Among the primary tumors analyzed, 1 classic MB (D289) demonstrated an aberrant SSCP pattern. This case carried a somatic mutation in codon 1319 (CCTCTT) resulting in an amino acid substitution from proline to leucine (P1319L). This mutation was absent in the patient's constitutional DNA (Table I, Fig. 1c). Thirty-eight patients were informative for a known polymorphism at codon 1453, but none of the corresponding tumors showed loss of heterozygosity.
|Tumor sample||Gender/age||Specimen||Histology||Gene||Nucleotide change||Effect of mutation||Mutations in patched|
|D230II||F/13||RT||Classic||CTNNB1||TCT TGT||Ser 33 Cys||No|
|D366||M/10||PT||Classic||CTNNB1||TCT TTT||Ser 33 Phe||No|
|D322||M/51||PT||Desmoplastic||CTNNB1||TCT TGT||Ser 37 Cys||Yes|
|stP4||F/2||PT||Suprat. PNET||CTNNB1||GGA GTA||Gly 34 Val||No|
|D289||M/5||PT||Classic||APC||CCT CTT||Pro 1319 Leu||No|
|D83II||F/6||CL MHH-MED-2||Classic||APC||GAA CAA||Glu 1317 Gln||No|
CTNNB1 mutation analysis
Eighty-seven PNET biopsies and 10 MB cell lines were examined for mutations in exon 3 by SSCP analysis. In 3 MBs and 1 supratentorial PNET aberrant bands were detected that were absent in the patient's constitutional DNA. Sequence analysis confirmed the presence of somatic point-mutations.
In MB D322, a desmoplastic primary tumor of a 51-year-old male patient, the mutation in codon 37 (TCTTGT) resulted in a SerCys substitution (S37C). Two classic MBs, D230II a recurrent and D366 a primary tumor, had point mutations in codon 33. In D230II, a TCT to TGT transversion occurred resulting in a SerCys substitution (S33C). Tumor sample D366 displayed a TCTTTT transition leading to a SerPhe substitution (S33F) (Table I, Fig. 1a). All 3 mutations resulted in the loss of serine residues as targets for phosphorylation by GSK-3β in the NH2-terminal regulatory domain of the CTNNB1 gene in codons 29–45. Of the 4 supratentorial PNETs in our analysis, 1 (stP4) carried a somatic mutation in codon 34 (GT transversion; G34V).
For the detection of larger deletions we analyzed by PCR/RT-PCR a region spanning exon 1 to exon 5 of the CTNNB1 gene. This analysis revealed no deletions in any of the PNETs or cell lines.
β-Catenin protein analysis
Additional Western blot analysis of 17 MBs showed no additional shorter bands representing deleted β-catenin proteins. Immunohistochemical staining of those PNETs carrying somatic mutations of the CTNNB1 gene showed cytoplasmic and nuclear accumulation of β-catenin protein in the tumor cells (Fig. 2). The tumors with APC missense mutation/variant and tumors without any mutations displayed cytoplasmatic staining but no nuclear accumulation (not shown).
Although PNETs represent the most frequent malignant brain tumors in childhood, their molecular pathogenesis is only partially understood. The most frequent molecular alteration is allelic loss of the short arm of chromosome 17 indicating the presence of one or more yet unidentified tumor suppressor gene(s) at this site.25, 26 A subset of medulloblastomas, preferentially of the desmoplastic subtype shows allelic loss on chromosome arm 9q and inactivating mutations of the PTCH gene at 9q22.23, 27, 28 Although most MBs occur sporadically, patients carrying germline mutations in either PTCH (Nevoid basal cell carcinoma syndrome) or APC (Turcots syndrome) have a strikingly increased risk for the development of MBs. The finding of loss of the second APC allele in a medulloblastoma from a Turcots patient with APC germline mutation is in line with the 2-hit hypothesis of tumor suppressor inactivation in hereditary tumor syndromes and strongly argues for a role of APC inactivation in the genesis of MBs.29 Two earlier studies addressed the question whether APC may be altered also in sporadic PNETs, but neither point mutations nor allelic loss were detected by ribonuclease protection assay and microsatellite analysis, respectively.30, 31 This would argue against a major role of APC in the pathogenesis of sporadic PNETs. A similar observation was made in hepatoblastoma, a childhood liver tumor with high incidence in patients with APC germline mutations, but infrequent APC mutations in sporadic cases.21, 32
In the present study we were able to show that a small subset of PNETs carry mutations of either the APC or the CTNNB1 gene. Only 2 APC sequence changes were found in exon 15 including the mutation cluster region (MCR). At present, we cannot exclude the possibility of individual mutations in other domains of the gene.
Tumor D289 was a medulloblastoma of classic histology and carried a somatic missense mutation at codon 1319. The second allele appeared to be intact. In colorectal cancer, most APC mutations are truncating leading that cause a loss of function.33 The biological consequences of missense mutations are not established in detail. Missense mutations, however, have been described in other tumors including gastric carcinomas.34 Codon 1319 is located close to but is not directly involved in β-catenin binding domains defined by distinct 15 amino acids and 20 amino acids repeats.13 Missense APC mutations have also recently described in PNETs by Huang et al.35 Further functional analysis will be necessary to elucidate the significance of such mutations. Immunohistochemistry of Case D289, however, failed to demonstrate a nuclear accumulation of β-catenin protein suggesting that this mutation does not lead to an inappropriate activation of WNT/wingless signaling.
The second alteration identified in DNA D83II from the MB cell line MHH-MED-2 was a missense mutation of codon 1317 (E1317Q) located only 2 codons apart. This sequence variant was also present in the constitutive DNA of the patient who had a classic type medulloblastoma. We were not able to find this variant in more than 100 control alleles. This subpolymorphic variant has been described to be associated with a predisposition to colorectal tumors in patients without the florid phenotype of classical FAP.36 The E1317Q variant was detected in 4 of 164 patients with multiple colorectal tumors but not in 160 control alleles. In our medulloblastoma patient, however, no family history for brain tumors or colorectal tumors was apparent. Furthermore, the wild-type allele was present in the tumor, and nuclear accumulation of β-catenin protein was absent.
Allelic loss of APC was absent in 38 cases informative for the APC polymorphism at codon 1453, consistent with the finding of Yong et al.31 Taken together, our data indicate that sporadic PNETs show neither allelic loss involving the APC locus nor truncating APC mutations. A small subset of tumors, however, carry missense mutations the functional significance of which remains to be elucidated.
As a second component of the WNT/wingless pathway, we analyzed the CTNNB1 gene encoding exon 3. Four PNETs with missense point mutations of codons 33, 34 and 37 of the protein degradation targeting box of β-catenin were identified. In all cases, these mutations were somatic and resulted in a strong nuclear accumulation of β-catenin protein as shown by immunohistochemistry (Fig. 2). Such a nuclear accumulation was not present in tumors without CTNNB1 mutations (data not shown). Two classic medulloblastomas had S33C and S33F mutations, a desmoplastic MB showed a S37C mutation and a supratentorial PNET a G34V missense mutation. Zurawel et al.37 recently described 2 MBs with a S33C and 1 with a S37C mutation. Therefore, the serine residues at positions 33 and 37 may constitute specific hot spots in PNETs. This was also illustrated by a recent study of Huang et al.35 The residues T41 and S45 frequently affected in many other cancer entities seem not to be a major target in PNETs (Fig. 3 and references therein38). We searched for deletions of exon 3 encoding this crucial degradation targeting box and larger deletions by PCR of genomic DNA and cDNA that was available in 50 cases. Such deletions are frequent in several tumor types including melanoma cell lines and hepatoblastomas.15, 21 We did not detect a single case with a deletion suggesting that PNETs do not carry this type of mutation. The reason for the described hot-spots for the point mutations and the absence of deleting mutations in PNETs remains unclear. These findings may hint to tissue specific mutagenetic events.
In contrast to PTCH mutations that were only identified in infratentorial PNETs (medulloblastomas), we were able to show the occurrence of CTNNB1 mutations in both, supratentorial and infratentorial PNETs. They were also present in desmoplastic as well as classic variant that appear to originate from different precursor cells.39 In addition, alterations of the APC or CTNNB1 genes do not bear any apparent relationship to mutations of components of the Sonic hedgehog/Patched signaling pathway. All tumors investigated here, have also been previously screened for PTCH and SMOH mutations, and a single case (D322) carried both a PTCH and a CTNNB1 mutation theoretically leading to an overactivation of both the patched and WNT/wingless signaling pathways in the same tumor. The other cases with mutant APC or CTNNB1 had no PTCH or SMOH mutations.
In summary, we show that a small subset of sporadic cerebellar and cerebral PNETs carries mutations of the CTNNB1 gene leading to a nuclear accumulation of the β-catenin protein. This alteration may lead to an increased transcription of growth-related target genes and enhanced proliferation of neural progenitors, finally resulting in tumor development. The presence of CTNNB1 mutations in PNETs suggests an involvement of the WNT/wingless signaling pathway in the molecular pathogenesis of these cases. Further studies will address the question if other components of this pathway may also show genetic abnormalities in PNETs.
We would like to thank the patients and their parents for their contribution to this study.
- 1Posterior fossa brain tumors in children. In: YoumansJR. Neurological surgery. Philadelphia: WB Saunders, 1982:2733–52..
- 18WHO classification of tumors: pathology and genetics of tumors of the nervous system. Lyon: IARC Press, 2000., .
- 20Molecular cloning: a laboratory manual. New York: Cold Spring Harbor, 1989., , .