Functional Evaluation of p53 and PTEN Gene Mutations in Gliomas 1 Hideaki Kato, Shunsuke Kato, Toshihiro Kumabe, Yukihiko Sonoda, Takashi Yoshimoto, Satoshi Kato, Shuang-Yin Han, Takao Suzuki, Hiroyuki Shibata, Ryunosuke Kanamaru, and Chikashi Ishioka 2 Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575 [H. K., Sh. K., Sa. K., S-Y. H., T. S., H. S., R. K., C. I.], and Department of Neurosurgery, Tohoku University School of Medicine, Sendai 980-8574 [H. K., T. K., Y. S., T. Y.], Japan ABSTRACT We screened mutations of two major tumor suppressor genes, p53 and PTEN, in 66 human brain tumors using a yeast-based functional assay and cDNA-based direct se- quencing, respectively. The frequency of p53 mutations was 28.8% (19 of 66) and was higher in anaplastic astrocytoma (9 of 14, 64.3%,) than in glioblastoma multiforme (GBM; 7 of 27, 25.9%,), supporting previous speculation that there are at least two genetic pathways leading to GBM, a de novo pathway without p53 mutation and a “progressive” pathway with p53 mutation. PTEN mutation was observed in 8 of 64 tumors (12.5%), mainly GBMs (7 of 26, 26.9%), both with and without p53 mutation. These results suggest that muta- tion of the PTEN gene is a later event than that of the p53 gene in glioma progression and is associated with both the genetic pathways. All of the detected PTEN missense muta- tions and an in-frame small deletion inactivated PTEN phos- phoinositide phosphatase activity in vitro. Because the tu- mors containing PTEN mutations also showed loss of heterozygosity in the chromosome 10q23 region flanking the PTEN gene, our data clearly indicate that inactivation of both PTEN alleles occurs in a subset of high-grade gliomas, therefore confirming the previous idea that PTEN acts as a tumor suppressor gene. INTRODUCTION GBM 3 is the most common as well as the most aggressive primary brain tumor and is clinically separable into two sub- types. One type is primary or de novo GBM, which is charac- terized by later onset, rapid tumor growth and a short clinical course. The other type is secondary or “progressive” GBM, which arises from a less malignant precursor lesion, including astrocytoma or AA, and is characterized by earlier onset, slow tumor growth, and less aggressive clinical features (for reviews, see Refs. 1–3). Although the two types are generally indistin- guishable histologically, recent molecular genetic analyses have provided evidence to support at least two distinct pathways contributing to the tumorigenesis of GBM. Primary GBM is closely associated with the absence of p53 mutation and the presence of gene amplification such as that of EGFR, whereas secondary GBM is associated with the presence of p53 mutation and the absence of gene amplification (2– 4). As well as these alterations, loss of chromosome 10q and/or 10p occurs in the majority of GBMs and AAs and is associated with both de novo and progressive GBMs (5– 8). This suggests that there may be unknown tumor suppressor gene(s) on chromosome 10 that may be involved in the tumorigenesis of either of the two GBM subtypes. At chromosome 10q23, the PTEN gene (also called MMAC1 and TEP1) was recently identified as a putative tumor suppressor gene, and mutations of this gene have been reported in human glioma and other tumors (9 –18). In addition, germ- line PTEN mutations have been found in the dominant cancer susceptibility syndromes Cowden disease and Bannayan- Zonana syndrome (19, 20). Furthermore, enforced expression of PTEN cDNA suppresses tumor cell growth both in vitro and in vivo (21–23). These results strongly suggest that PTEN acts as a tumor suppressor gene in GBM and other tumors, although the functional significance of the detected mutations has not been tested. Recent studies have shown that PTEN protein acts as a phosphoinositol phosphatase and negatively controls the phos- phatidylinositol 3'-kinase/Akt pathway by dephosphorylating phosphoinositides at the 3 position (23–27). This biochemical action may contribute to the regulation of cell growth and survival (23, 26, 28). To investigate how PTEN mutations are involved in the tumorigenesis of glioma, we examined glioma samples for both p53 and PTEN mutations and evaluated the functional significance of PTEN mutations by in vitro phosphoi- nositide phosphatase assay and examination of LOH at chromo- some 10q23. Received 5/30/00; revised 7/26/00; accepted 7/26/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture and the Ministry of Health and Welfare, Japan. 2 To whom requests for reprints should be addressed, at Department of Clinical Oncology, Institute of Development, Aging and Cancer, To- hoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8547; Fax: 81-22-717-8548; E-mail: chikashi@ idac.tohoku.ac.jp. 3 The abbreviations used are: GBM, glioblastoma multiforme; AA, anaplastic astrocytoma; LOH, loss of heterozygosity; RT-PCR, reverse transcription-PCR; Ins(1,3,4,5)P 4 , inositol 1,3,4,5-tetrakisphosphate; nt, nucleotide. 3937 Vol. 6, 3937–3943, October 2000 Clinical Cancer Research Research. on December 6, 2021. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from