SHORT REPORT Alterations in the rap1 signaling pathway are common in human gliomas David H Gutmann 1 , Susan Saporito-Irwin 1 , Jerey E DeClue 2 , Ralf Wienecke 2 and Abhijit Guha 3 1 Department of Neurology, Washington University School of Medicine, Box 8111, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA; 2 Laboratory of Cellular Oncology, Building 36, Room 1D-32, National Cancer Institute, Bethesda, Maryland 20892, USA; 3 Lunenfeld Research Institute, Mount Sinai Hospital, Division of Neurosurgery, The Toronto Hospital, University of Toronto, 2-415 McLaughlin; 399 Bathurst, Toronto, Ontario M5T 2S8, Canada Several inherited predisposition to cancer syndromes are associated with the development of nervous system tumors. Tuberous sclerosis complex (TSC) is an autosomal dominant disorder in which aected indivi- duals are at risk for developing astrocytomas. One of the genes responsible for this disorder is TSC2, located on chromosome 16p, and encoding a 180 kDa protein (tuberin) that functions in part as a negative regulator of rap1. Previous studies from our laboratory demon- strated that 30% of sporadic astrocytomas have reduced or absent tuberin expression. In addition to loss of tuberin in sporadic astrocytomas, aberrant rap1 mediated signaling may also result from overexpression of rap1. In this study, we test the hypothesis that alterations in the rap1 signaling pathway are frequently observed in certain subsets of gliomas compared to other tumors of the nervous system. Analysis of sporadic astrocytomas and ependymomas demonstrated either increased rap1 or reduced/absent tuberin protein expression in 50 ± 60% of dierent cohorts of these gliomas, compared to 30 ± 33% of sporadic schwannomas and meningiomas and none of eight oligodendrocyte tumors. These results suggest that alterations in the rap1 signaling pathway are important in the development of certain sporadic human gliomas. Keywords: astrocytomas; tumor suppressor gene; tuberous sclerosis complex 2; tuberin Studies on the molecular genetic events associated with the development of human malignant astrocytic tumors (the most common subtype of glioma and primary human brain cancer) have demonstrated alterations in the p21-ras signaling pathway. Ampli®cation of the epidermal growth factor receptor (EGF-R) has been detected in a high proportion of the most malignant astrocytoma or glioblastoma multiforme (GBM; Olson et al., 1995), contributing to increased activation of p21-ras. Previously, we demonstrated elevated levels of p21-ras activity in astrocytoma cell lines as well as fresh GBM surgical specimens (Gutmann et al., 1996; Guha et al., manuscript submitted). Inhibition of p21- ras activity using either a dominant inhibitory p21-ras mutant or farsenyltransferase inhibitors resulted in reduced astrocytoma cell proliferation in vitro and tumor growth in vivo. In addition to the p21-ras signaling pathway, activation of other small GTPase proteins may also result in increased cell proliferation in astrocytomas. To this end, the TSC2 gene product, tuberin, functions as a negative regulator of rap1. Loss of tuberin expression has recently been demonstrated by our laboratory in one third of sporadic gliomas, suggesting that alterations in the rap1 signaling pathway may be associated with the development of human astrocyto- mas (Wienecke et al., in press). In this study, we test the hypothesis that alterations in the rap1 signaling pathway, either due to rap1 overexpression or reduced levels of the rap1 negative regulator, tuberin, are frequently associated with gliomas compared to other tumors of the nervous system. Tuberin has been demonstrated to suppress cell growth and this activity resides in the C-terminus of tuberin where the domain that functions as a GTPase activating protein (GAP) for rap1 is located (Jin et al., 1996). Biochemical studies demonstrated that this GAP activity is speci®c for rap1 and not other related small GTPase proteins, including rap2, H-ras and rho (Wienecke et al., 1995). Loss of tuberin expression would therefore be predicted to lead to elevated rap1 activity in these tumors. At present, there is no biochemical method available to determine rap1 activity in cells to directly test this hypothesis. In addition to loss of tuberin expression, another mechanism for elevated rap1 signaling might result from increased rap1 expression. To this end, amplifica- tion of a segment of chromosome 12q containing the rap1B gene has been detected in many dierent tumor types, including astrocytomas (Cheng et al., 1995; He et al., 1994, 1995; Khatib et al., 1993; Reifenberger et al., 1996). In these tumors, there is ampli®cation of the 12q chromosomal region variably containing the CDK4/SAS, MDM2, GLI, AM2R, GADD153, IFNG and RAP1B genes. To determine whether alterations in the rap1 signaling pathway are observed in human astrocyto- mas, Western blot analysis was performed on twenty adult astrocytomas (Figure 1a). Tumor samples were obtained from patients undergoing surgery in the Division of Neurosurgery at the University of Toronto or Washington University School of Medicine. All tumors were obtained in accordance with the respective institutions' Institutional Review Board Human Studies Protocols and classi®ed according to the World Health Organization (WHO) classi®cation of tumors of the central nervous system. For control purposes, normal cortex tissue was obtained from patients who underwent cerebral resection because of contusion. Mouse neocortical astrocyte cultures were generated as previously described (Hewett et al., 1995). The tumor samples were coded in order to conduct the experiments in a blind fashion. Brie¯y, specimens were minced and Correspondence: DH Gutmann Received 14 March 1997; revised 28 May 1997; accepted 28 May 1997 Oncogene (1997) 15, 1611 ± 1616  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00