Publication of the International Union Against Cancer
Int. J. Cancer: 101, 198 –201 (2002) © 2002 Wiley-Liss, Inc.
SHORT REPORT ROLE OF WNT PATHWAY IN MEDULLOBLASTOMA ONCOGENESIS Naoki YOKOTA1, Shigeru NISHIZAWA1*, Seiji OHTA1, Hiroaki DATE3, Haruhiko SUGIMURA4, Hiroki NAMBA1 and Masato MAEKAWA2 1 Department of Neurosurgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan 2 Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan 3 Department of Neurosurgery, Chiba Children’s Hospital, Midoriku, Chiba, Chiba, Japan 4 1st Division of Pathology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan To clarify the roles of Wnt pathway in medulloblastoma oncogenesis, immunohistochemical staining of -catenin and Wnt-1 and genomic analyses of CTNNB1 (-catenin) and AXIN1 (axin 1) were examined in 23 sporadic cases. Accumulation of -catenin in tumor cells was immunohistochemically proven in 5 cases; 2 cases showed positive immunoreactivity for Wnt-1 and another 2 showed mutation of either CTNNB1 or AXIN1. AXIN1 mutation was in exon 3, corresponding to GSK-3 binding site and CTNNB1 mutation was in exon 3, corresponding to its phosphorylation site. Disruption of these proteins could result in upregulation of the Wnt signaling and accumulation of -catenin, followed by cell proliferation and medulloblastoma oncogenesis. © 2002 Wiley-Liss, Inc. Key words: medulloblastoma; -catenin; axin; Wnt; oncogenesis
Medulloblastoma occurs most frequently in the cerebellum of children.1–5 Despite its poor prognosis, the mechanism of oncogenesis in medulloblastoma remains still unclear. Turcot syndrome and Gorlin syndrome are both inherited syndromes that associate with occurrence of medulloblastoma.1 Genetical analysis for oncogenesis in these syndromes has been undergoing clarification.1 In Turcot syndrome, medulloblastoma is combined with colonic cancer. Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is present in Turcot syndrome.1,2 Mutation of APC has not been detected in sporadic medulloblastoma and ⬇5% of sporadic medulloblastoma have been reported to contain mutations in the Wnt pathway member, -catenin.3 The Wnt pathway plays important roles in development, cellular proliferation and differentiation in various species.6,7 The Wnt pathway is also considered to be involved in carcinogenesis of medulloblastoma, as well as in other tumors such as uterus, hepatic and colon carcinomas.1–5,8 –11 The components of the Wnt pathway, including disheveled, glycogen synthase kinase-3 (GSK3), -catenin and APC interact with each other and form a complex with Axin.12 Axin plays a role in regulating phosphorylation of -catenin. Failure of such phosphorylation leads to stability of -catenin, resulting in accumulation of -catenin.13,14 Based on this evidence, the etiology of sporadic medulloblastoma has been reported recently.3–5 To further confirm the mechanism of oncogenesis in medulloblastoma, we examined 23 sporadic cases of medulloblastoma. The purpose of our study was to investigate the roles of Wnt pathway members in oncogenesis of sporadic medulloblastomas by examining immunohistochemical expression of Wnt-1 and -catenin and genetic alterations in CTNNB1 and AXIN1 and to detect the alterations of these genes. MATERIAL AND METHODS
Surgical specimens were obtained from the patients of sporadic medulloblastoma in our University Hospital. Clinical information of these 23 cases is shown in Table I. No cases of Turcot or Gorlin syndrome were included. Immunohistochemistry Immunohistochemical staining was carried out as described previously.15 Formaldehyde-fixed paraffin-embedded sections (6
TABLE I – CLINICAL PROFILE OF THE CASES Case
Age
Gender
Location
Histological finding
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
13 2 1 1 5 13 1 2 18 4 15 1 2 6 2 1 7 5 9 35 19 21 3
F M F M M F M F M M M F F F M M M F F F F F M
Vermis Vermis Vermis Vermis Vermis Vermis Vermis Vermis Vermis Vermis Hemisphere Vermis Hemisphere Vermis Vermis Vermis Vermis Hemisphere Vermis Vermis Hemisphere Hemisphere Vermis
Classical Desmoplastic Classical Desmoplastic Classical Desmoplastic Desmoplastic Classical Classical Classical Classical Desmoplastic Classical Classical Classical Classical Classical Classical Classical Classical Classical Classical Desmoplastic
m) were prepared using standard procedure. The sections were dewaxed in xylene and hydrated in a series of ethanol. Because tissue antigens were decreased markedly in the tumor specimens, they were autoclaved in 0.01% citrate buffer (pH 6.5) at 110°C for 2 ⫻15 min as a means of antigen retrieval. In the sections used for Wnt-1 immunostaining this step was omitted. The sections were incubated with hydroperoxide and blocking solution (DAKO Japan, Kyoto, Japan) at room temperature, followed by incubation with anti--catenin MAb (Transduction, 1,000-fold) or anti-Wnt-1 Ab (Santa Cruz, 100-fold) at room temperature for 1 hr. Immunoreactivity was detected by an immunoperoxidase reaction using Emvision kit (DAKO Japan, Kyoto, Japan) for -catenin and those in anti-goat immunostaining kit (Santa Cruz) for Wnt-1. 3-3⬘Diaminobenzidine tetrahydrochloride was used as the substrate of the peroxidase reaction. Immunoreactivity of each sample was Grant sponsor: Hamamatsu University School of Medicine. *Correspondence to: Shigeru Nishizawa, Department of Neurosurgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192 Japan. Fax: ⫹81-53-435-2282. E-mail:
[email protected] Received 14 December 2001; Revised 18 February 2002, 23 May 2002; Accepted 24 May 2002 DOI 10.1002/ijc.10559 Published online 26 July 2002 in Wiley InterScience (www.interscience. wiley.com).
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TABLE II – IMMUNOHISTOCHEMICAL AND GENETICAL FINDINGS OF THE CASES Case
-catenin
Wnt-1
Gene alteration
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫹
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ CTNNB1 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ AXIN1 ⫺ ⫺ ⫺
1 ⫺, no immunoreactivity; ⫹, occasional and faint immunoreactivity; ⫹⫹, focal immunoreactivity; ⫹⫹⫹, diffuse immunoreactivity.
FIGURE 2 – CTNNB1 mutation in Case 9. -Catenin phosphorylation sites by GSK3 are shown in bold. Single point mutation from cytosine to guanine was detected in this case.
expressed as follows: ⫺, no immunoreactivity; ⫹, occasional and faint immunoreactivity; ⫹⫹, focal immunoreactivity; ⫹⫹⫹, diffuse immunoreactivity. Mutation analysis of the -catenin and AXIN1 genes We screened the coding of sequence of the -catenin and AXIN1 genes. The primers used to amplify exon 3 of -catenin from genomic tumor DNA were (5⬘–3⬘): forward, ATTTGATG-
FIGURE 1 – Imuunohistochemical findings of Cases 12 (upper) and 9 (lower). In Case 12, immunohistochemical study showed diffuse immunoreactivity both in -catenin and Wnt-1. Moreover, -catenin was positive both in the nucleus and cytosol. In Case 9, immunohistochemical study showed diffuse immunoreactivity only in -catenin, but not in wnt-1. Left: -catenin. Right: Wnt-1. Magnification: ⫻400 (upper); ⫻100 (lower).
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FIGURE 3 – AXIN1 mutation in Case 20. In the exon 3 of AXIN1, corresponding to GSK-3 binding site, the last nucleic acid “Cytosine” was deleted. In this case, additional single point mutation from guanine to cytosine was also detected.
GAGTTGGACA-TGGC; and reverse, CCAGCTACTTGTTCTTGAGTG-AAGG. For the AXIN1 gene, we used 23 sets of primers designed from cosmids with GenBank accession numbers Z99754, Z81450, Z69667 and Z982721.16 Gene alterations of AXIN1 and CTNNB1 were examined. DNAs from formaldehyde-fixed paraffin embedded medulloblastoma tumor sections and those from peripheral blood mononuclear cells were extracted. Loss of heterozygosity (LOH) analyses of AXIN1 were carried out with a fluorescence DNA sequencer using microsatellite markers, D16S521.16 –18 Sequence analyses of CTNNB1 and AXIN1 were carried out using direct PCR sequencing. The following amplification was used: 35 cycles of denaturation at 95°C for 30 sec; 55°C for 30 sec; and extension at 72°C for 2.5 min. After PCR, PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. Single bands were cut out and DNA was purified by spin column (Super 01, Takara, Japan) and DNA sequencing was carried out by the dideoxy chain terminal method using a Dye Terminator Cycle Sequencing FS Ready Reaction Kit and PRISM 310 Genetic Analyzer (PE Biosystems, Branchburg, NJ). Sequence analyses were carried out using DNASIS program (Hitachi, Ibaraki, Japan).
RESULTS
Immunohistochemical and genetical findings of the present cases were summarized in Table II. Immunohistochemical findings of -catenin and Wnt-1 showing diffuse immunoreactivity (Fig. 1, upper) and that of only -catenin without Wnt-1 (Fig. 1, lower) are shown. Samples of 5 cases among 23 cases (Cases 9, 12, 13, 20, 21) showed diffuse immunoreactivity of -catenin, a down stream effecter of Wnt signaling. Furthermore, -catenin accumulated both in the cytosol and nucleus of the tumor cells (Fig. 1, upper). In 2 of the 5 cases (Cases 12, 13) with positive staining for -catenin, Wnt-1 signal was also overexpressed as shown in Figure 1. In Case 9, mutation analysis showed the single point mutation of CTNNB1 in exon 3 (codon 33), which corresponded to a -catenin phosphorylation site by GSK-3 (Fig. 2). In another case (Case 20), AXIN1 mutation was found in exon 3, which corresponded to GSK-3 binding site (Fig. 3). Neither Wnt-1 overexpression nor gene alteration of CTNNB1 or AXIN1 was found in 18 cases with no or weak expression of -catenin.
DISCUSSION
Axin mutation in tumor oncogenesis was first detected in hepatocellular carcinoma,16 however, the role of such mutation has not been precisely clarified yet in oncogenesis of medulloblastoma. In 2 inherited syndromes with occurrence of medulloblastoma (Turcot syndrome and Gorlin syndrome), genetical analysis has been reported.1,2 Based on such evidence, the mechanism of oncogenesis in sporadic medulloblastoma has been reported very recently.1,3,4,5 To further confirm such mechanism, we analyzed 23 sporadic cases of medulloblastoma using immunohistochemical and genetical analysis. -Catenin accumulation was detected in 5 (22%) of 23 cases with sporadic medulloblastoma. In Cases 12 and 13, immunohistochemical study showed abnormalities of both Wnt pathway and its downstream effecter, -catenin. It is suggested that increased Wnt signaling might promote cell cycle progression through Wnt signaling effectors, such as TCF or LEF-1.19,20 Disruption of genes such as axin and -catenin was found in Cases 9 and 20. In these 2 cases, sequence analysis showed single nucleic acid deletion at the end of exon 3. This part of the protein is functionally indispensable for interaction with other proteins in complex formation.14,16 Disruption of such part of the genes could result in failure of complex formation to regulate -catenin phosphorylation, thereby stabilization of -catenin leading to promote cell proliferation.12 In Case 21 with -catenin accumulation, abnormality in Wnt pathway was not found. Novel Axin family protein, Axin2, or Axin-associated protein were isolated recently.21,22 Disruption of these Axin-like or Axin-associated proteins might have resulted in activation of signal in this case. We conclude that activation of Wnt pathway by disruption of axin, -catenin as well as APC, which interact with GSK-3, results in upregulation of Wnt pathway and accumulation of -catenin. As a result, cell proliferation and medulloblastoma oncogenesis occur in certain parts of sporadic medulloblastomas. ACKNOWLEDGEMENTS
We express sincere appreciation to Professor Y. Koide, Department of Microbiology and Immunology, Hamamatsu University School of Medicine and Dr. M. Uchijima and other members of the Koide’s Laboratory for helpful advise, technical assistance and valuable discussions and Dr. M. Taylor, Toronto University, for critical reading of this manuscript.
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