Oncogene (2007) 26, 6289–6296 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Loss of Arf causes tumor progression of PDGFB-induced oligodendroglioma E Tchougounova1, M Kastemar1, D Bra˚ sa¨ ter1,3, EC Holland2, B Westermark1 and LUhrbom 1 1Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden and 2Departments of Surgery (Neurosurgery), Neurology and Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA In a subset of gliomas, the platelet-derived growth factor The tumors are believed to originate from a glial (PDGF) signaling pathway is perturbed. This is usually an progenitor cell or an astrocyte based on the expression early event occurring in low-grade tumors. In high-grade of various glial-specific mRNAs and proteins in the gliomas, the subsequent loss of the INK4a-ARF locus tumors. Gliomas are diagnosed based on malignancy is one of the most common mutations. Here, we dissected into four grades (I–IV). Owing to infiltrative growth and the separate roles of Ink4a and Arf in PDGFB-induced a strong tendency of malignant progression of tumor oligodendroglioma development in mice. We found that cells, the prognosis is poor even when diagnosed with a there were differential functions of the two tumor low-grade tumor. The oligodendrogliomas are divided suppressor genes. In tumors induced from astrocytes, both into grade II oligodendroglioma and grade III anaplas- Ink4a-loss and Arf-loss caused a significantly increased tic oligodendroglioma. They commonly display over- incidence compared to wild-type mice. In tumors induced expression of the epidermal growth factor receptor from glial progenitor cells there was a slight increase in (EGFR) (Reifenberger et al., 1996) and of ligands and tumor incidence in Ink4aÀ/À mice and Ink4a-ArfÀ/À mice receptors of the platelet-derived growth factor (PDGF) compared to wild-type mice. In both progenitor cells and family (Di Rocco et al., 1998). In high-grade oligoden- astrocytes, Arf-loss caused a pronounced increase in tumor drogliomas, the malignant progression is often asso- malignancy compared to Ink4a-loss. Hence, Ink4a-loss ciated with homozygous deletion of the INK4a-ARF contributed to tumor initiation from astrocytes and Arf-loss locus (Cairncross et al., 1998; Bigner et al., 1999), which caused tumor progression from both glial progenitor cells is one of the most frequent mutations found in human and astrocytes. Results from in vitro studies on primary glioma (Jen et al., 1994; Schmidt et al., 1994). INK4a- brain cell cultures suggested that the PDGFB-induced ARF encodes the tumor suppressor proteins p16INK4A activation of the mitogen-activated protein kinase pathway and p14ARF (p19Arf in mouse), which regulates the RB via extracellular signal-regulated kinase was involved in the and P53 pathways, respectively. Their separate roles in initiation of low-grade oligodendrogliomas and that the suppression of spontaneous tumor development in vivo additional loss of Arf may contribute to tumor progression have been studied in mice with targeted deletions of the through increased levels of cyclin D1 and a phosphoinositide corresponding genes (Serrano et al., 1996; Kamijo et al., 3-kinase-dependent activation of p70 ribosomal S6 kinase 1997a; Sharpless et al., 2001, 2004). Arf-null mice causing a strong proliferative response of tumor cells. showed a high frequency of spontaneous tumors in Oncogene (2007) 26, 6289–6296; doi:10.1038/sj.onc.1210455; diverse tissue types, whereas Ink4a-null mice demon- published online 16 April 2007 strated a low frequency of spontaneous tumors with a more restricted tissue distribution. The combined loss Keywords: oligodendroglioma; PDGF; mouse model; of Ink4a-Arf displayed a phenotype similar to that of Ink4a; Arf; p70S6K Arf-null mice. The somatic cell gene transfer model replication- competent avian leukemia virus splice acceptor/avian Introduction leukemia virus receptor (RCAS/tv-a) has been used to study oncogenic mechanisms of tumor genes during Gliomas, comprising astrocytomas and oligodendro- gliomagenesis. Infection with RCAS retroviruses carry- gliomas, are the most common primary tumors of the ing specific genetic mutations can be directed to specific central nervous system (Kleihues and Cavenee, 1997). brain cell populations using transgenic mice expressing tv-a from cell type-specific promoters. In Ntv-a mice, the Correspondence: Dr LUhrbom, Rudbeck Laboratory,Department of nestin promoter that supports transfer to neural/glial Genetics and Pathology, Uppsala University, SE-751 85, Uppsala, progenitor cells controls expression of tv-a.InGtv-a, Sweden. mice tv-a is expressed by the glial fibrillary acidic protein E-mail: [email protected] (GFAP) promoter that is active primarily in astrocytes 3Current address: Ludwig Institute for Cancer Research, Karolinska Institutet, Box 240, SE-171 77 Stockholm, Sweden. in the newborn mouse brain. In the Ntv-a mice, we have Received 29 June 2006; revised 6 February 2007; accepted 2 March 2007; previously shown that various types of gliomas (astro- published online 16 April 2007 cytomas and oligodendrogliomas) can be induced in Role of Ink4a and Arf in PDGF-induced gliomagenesis E Tchougounova et al 6290 wild-type mice by infecting with KRas þ Akt or PDGFB more of the following features had to be present: high retrovirus (Dai et al., 2001; Uhrbom et al., 2002). When tumor cell density with mitotic figures (Figure 1c), these oncogenic stimuli were combined with the loss of cellular and nuclear pleomorphism, microvascular pro- Ink4a-Arf tumor incidence, latency and malignancy liferation and pseudopalisading necrosis (Figure 1d). were increased (Dai et al., 2001; Uhrbom et al., 2002). Grade III tumors normally displayed grade II tumor Furthermore, for KRas-induced astrocytomas, we have histology in some parts of the tumor. Grade II tumors shown that there were functionally independent roles of were mostly found in asymptomous 12-week-old ani- loss of either Ink4a or Arf (Uhrbom et al., 2005). mals whereas grade III tumors usually presented earlier In the present study, we have investigated the than 12 weeks. Some tumors in Gtv-a mice had the individual contributions of loss of p16Ink4a and p19Arf histopathology of mixed oligo-astrocytomas. All tumors in PDGFB-induced oligodendroglioma development. were positive for the oligodendroglial cell marker NG2 We found that the main consequence of Ink4a-loss (Figure 1e) and the neural stem cell marker Sox2 was to render astrocytes susceptible to PDGFB-induced (Figure 1f). In addition, most tumors showed areas oncogenesis and that the primary result of Arf-loss was positive for Gfap (Figure 1g) and nestin (Figure 1h). increased tumor malignancy. Results from in vitro In Ntv-a mice, significant increase in tumor incidence studies on primary brain cell cultures supported these was found upon loss of either Ink4a (Fischer’s exact findings and suggested a mechanism by which test, P ¼ 0.0496 (*)) or Ink4a-Arf (Fischer’s exact test, tumor initiation was caused by activation of the P ¼ 0.0159 (*)) compared to wild-type mice (Table 1). In mitogen-activated protein kinase (MAPK) pathway via Gtv-a mice, the separate loss of Ink4a or Arf had a more extracellular signal-regulated kinase (Erk), and tumor striking effect (Table 1). There was a significant and progression occurred upon loss of p19Arf resulting in similar increase in tumor incidence in Ink4aÀ/À and increased levels of cyclin D1 and activation of the ArfÀ/À mice compared to wild-type mice (Fischer’s phosphoinositide 3-kinase (PI3K) pathway via p70 exact test, Po0.0001 (***) for both comparisons), and ribosomal S6 kinase (p70S6K). the combined loss of Ink4a-Arf did not further increase the tumor incidence. In line with what has previously been shown for Kras-induced gliomagenesis (Holland Results et al., 2000; Uhrbom et al., 2002), the incidence of PDGFB-induced transformation was significantly high- To dissect the effect of loss of each of the Ink4a-Arf gene er in Ntv-a wild-type mice than in Gtv-a wild-type mice products in vivo, Ntv-a and Gtv-a wild-type mice and (Fischer’s exact test, P ¼ 0.0159 (*)). However, upon mice lacking p16Ink4a, p19Arf or both were injected with loss of either Ink4a or Arf, or combined loss of both RCAS–PDGFB–EGFP (Table 1). As previously des- Ink4a-Arf the difference in tumor incidence between cribed (Dai et al., 2001), the PDGFB-induced tumors Ntv-a and Gtv-a mice disappeared. Thus, the major displayed a histopathology similar to human oligoden- effect of Ink4a-loss in PDGFB-induced oligodendro- droglioma (grade II) (Figure 1a and b) or anaplastic glioma formation was to increase the susceptibility of oligodendroglioma (grade III) (Figure 1c and d). The the target cells in Gtv-a mice to PDGFB-induced tumors consisted of small tumor cells with regular, transformation. round nuclei and some tumors displayed the ‘peri- The main consequence of Arf-loss was increased nuclear halo’ feature where the cytoplasm was cleared malignancy and shorter survival of tumor-bearing mice. from the tumor cell nuclei (Figure 1a and c). They grew This was most obvious in Gtv-a mice but the trend could diffusely into the normal brain parenchyma and also be seen in Ntv-a mice. In Gtv-a mice, there was a displayed occurrence of the ‘secondary structures of significant difference in survival between Ink4aÀ/À mice Scherer’ including tumor cells lining up in the white and ArfÀ/À mice, which