Sox10 Has a Broad Expression Pattern in Gliomas and Enhances Platelet-Derived Growth Factor-B–Induced Gliomagenesis

Maria Ferletta, Lene Uhrbom, Tommie Olofsson, Fredrik Ponte´n, and Bengt Westermark

Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala, Sweden

Abstract without any known intermediate stages of the tumor, whereas In a previously published insertional mutagenesis screen secondary glioblastoma arises by progression from a lower for candidate brain tumor genes in the mouse using a grade to a higher tumor grade. Common genetic alterations in Moloney mouse leukemia virus encodingplatelet-derived primary glioblastoma are amplification of EGFR and MDM2 growth factor (PDGF)-B, the Sox10 gene was tagged in and deletions of the INK4A gene (1, 2). During the progression five independent tumors. The proviral integrations of secondary glioblastoma, mutations accumulate over time suggest an enhancer effect on Sox10. All Moloney murine and more than 65% of the tumors have mutations in TRP53. leukemia virus/PDGFB tumors had a high protein Overexpression of the platelet-derived growth factor receptor-a expression of Sox10 independently of malignant grade (PDGFRA) and PDGFA are characteristic for secondary glio- or tumor type. To investigate the role of Sox10 in blastoma (3). Constitutive expression of growth factors and gliomagenesis, we used the RCAS/tv-a mouse model in their receptors has been shown to be necessary for the develop- which the expression of retroviral-encoded genes can be ment of brain tumors resulting in autocrine stimulation and directed to glial progenitor cells (Ntv-a mice). Both Ntv-a increased activity of downstream pathways (4). Inactivation transgenic mice, wild-type, and Ntv-a p19Arf null mice of the tumor suppressor gene phosphatase and tensin homo- were injected with RCAS-SOX10 alone or in combination logue (PTEN; refs. 5, 6) is another common trait of glio- with RCAS-PDGFB. Infection with RCAS-SOX10 alone blastoma. PTEN signals through Akt and many glioblastomas did not induce any gliomas. Combined infection of have an increased activity of Akt (7). RCAS-SOX10 and RCAS-PDGFB in wild-type Ntv-a mice To better understand the role of PDGF in gliomagenesis, we yielded a tumor frequency of 12%, and in Ntv-a ArfÀ/À have generated a mouse glioma model in which a Moloney mice the tumor frequency was 30%. This indicates that murine leukemia virus (MMLV)/PDGFB–containing retrovirus Sox10 alone is not sufficient to induce gliomagenesis but (MMLV/PDGFB) together with a replication-competent helper acts synergistically with PDGFB in glioma development. virus was injected into newborn mouse brains, which resulted All induced tumors displayed characteristics of in malignant brain tumors (8). All tumors grew invasively and PNET-like structures and oligodendroglioma. The tumors diffusely, and most of them had areas of necrosis and had a strongand widely distributed expression of angiogenesis. The tumors stained positively for nestin, Sox10 and PDGFR-A. We investigated the expression suggesting that they had evolved from an immature neuroglial of Sox10 in other human tumors and in a number of progenitor cell (8). We have postulated that the tumors gliomas. The Sox10 expression was restricted to gliomas developed through an autocrine PDGF receptor activation in and melanomas. All glioma types expressed Sox10, combination with insertional mutagenesis through proviral and tumors of low-grade glioma had a much broader integrations. A number of common proviral integration sites distribution of Sox10 compared with high-grade gliomas. were identified, targeting candidate tumor-causing genes (9). (Mol Cancer Res 2007;5(9):891–7) One of the tagged genes was Sox10. Sox10 is a transcription factor and belongs to the Sox superfamily, which all contain a Introduction DNA binding motif known as the high-mobility group domain. Glioblastoma is the most common and malignant primary During development, Sox10 first appears in the developing brain tumor in the adult, with a mean survival time after neural crest and is expressed during the formation of the diagnosis of 1 year. Primary glioblastoma develops de novo peripheral nervous system (10). In central nervous system, Sox10 was first found on glial progenitor cells but later also detected in oligodendrocytes in the adult brain (11). Sox10 Received 3/6/07; revised 5/7/07; accepted 5/31/07. precedes the expression of PDGFR-a in oligodendrocyte Grant support: The Swedish Cancer Society and the Swedish Children’s Cancer precursors, but once the PDGFR-a is expressed, they are Foundation. Sox10 null The costs of publication of this article were defrayed in part by the payment of found in the same cells (12). Homozygous mice die page charges. This article must therefore be hereby marked advertisement in before or at birth. The entire peripheral nervous system is accordance with 18 U.S.C. Section 1734 solely to indicate this fact. defective and motor neurons are absent (13). The levels of Requests for reprints: Maria Ferletta, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory C11, Dag Hammarskjoldsv 20, S-751 PDGFR-a are reduced, suggesting that Sox10 influences the 85 Uppsala, Sweden. Phone: 46-18-611-1174; Fax: 46-18-55-89-31. E-mail: expression of PDGFR-a (12). In human, mutations in the [email protected] SOX10 Copyright D 2007 American Association for Cancer Research. gene have been linked to Waardenburg-Shah syndrome doi:10.1158/1541-7786.MCR-07-0113 type IV, which is characterized by depigmentation of hair and

FIGURE 1. Schematic illustration of proviral integrations upstream of Sox10. Five proviral integrations were tagged upstream of the transcriptional start site of Sox10 in five different tumors. Arrows illustrate the position and the transcriptional orientation of the proviral genome. Data were adapted from the Ensembl Mouse Genome Browser and exact positions of the integrations can be found in the Retrovirus Tagged Cancer Gene Database (http:// RTCGD.ncifcrf.gov/).

skin, and Hirschsprung’s disease (megacolon; ref. 14). PDGFB–induced tumors (9). The proviral integration sites Mutations in SOX10 in combination with Waardenburg-Shah were at both transcriptional orientations and located upstream syndrome type IV have also been associated with severe of Sox10 within an area of 60 kb (Fig. 1). The upstream dysmyelination syndromes (PCWH; ref. 14). Further, Sox10 is localization of the proviral insertions suggests an enhancer expressed in, and during the development of, melanocytes, effect on Sox10. which also are derived from the neural crest. During The brain tumors induced by the MMLV/PDGFB viruses melanocyte specification, Sox10 is responsible for activating were of different malignancy grades and could be divided the melanocyte transcription regulator Mitf, which controls into glioblastoma-like, oligodendroglioma-like, and PNET-like melanocyte survival and differentiation (15, 16). (primitive neuroectodermal tumor) tumors. When analyzing the In the present investigation, we have studied the role of MMLV/PDGFB tumors for Sox10 expression, we found that all Sox10 in human and mouse gliomas. For studies in the mouse, analyzed tumors had a high protein expression of Sox10, we used the RCAS/tv-a model (replication-competent avian independent of the tumor type. The protein was expressed also leukemia virus splice acceptor/avian leukemia virus receptor) in in tumors without integrations in Sox10 (Fig. 2). In addition, which the expression of retrovirus-encoded genes can be real-time PCR analysis has previously shown that Sox10 is directed either to glial progenitor cells (Ntv-a mice; mice strongly up-regulated in both early and late MMLV/PDGFB– expressing the tv-a receptor behind the nestin promoter) or induced tumors (17). astrocytes (Gtv-a mice; mice expressing tv-a by the glial fibrillary acidic protein promoter). We have found that Sox10 Sox10 Enhances PDGF-Induced Gliomagenesis has a broad distribution in different types of gliomas in both To investigate the role of Sox10 in gliomagenesis, we used human and mouse tumors. Sox10 was not able to initiate the RCAS/tv-a mouse model. Cells infected with RCAS virus tumorigenesis by itself, but in combination with an RCAS virus containing SOX10 (RCAS-SOX10) were injected intracerebral- expressing PDGFB, the tumor incidence was increased. ly in newborn mice. Both Ntv-a transgenic mice, wild-type (wt), and Ntv-a p19Arf null mice were injected with RCAS- Results SOX10 alone or in combination with RCAS-PDGFB (Table 1). Sox10 Is Highly Expressed in PDGFB-Induced Mouse Infection with RCAS-SOX10 alone did not induce any gliomas, Gliomas neither in Ntv-a wt nor in Ntv-a ArfÀ/À mice. Injections with In our previous screen for candidate glioma genes in the RCAS-PDGFB together with an empty RCAS vector (RCAS-X) mouse, Sox10 wastaggedinfiveindependentMMLV/ gave rise to 3 tumors from 37 injected mice in the Ntv-a ArfÀ/À

FIGURE 2. Sox10 expression in PDGFB-induced tumors. Immunohistochemical staining of Sox10 in mouse gliomas induced with MMLV/PDGFB. A. A representative PNET-like tumor. B. A glioblastoma-like tumor. C. A PNET-like tumor in which one of the Sox10 integrations was identified.

Table 1. Tumor Incidence in Mice Injected with Different All the tumors grew invasively and had vessel formation. The RCAS Virus in Different Combinations tumor cells were relatively small and displayed perineuronal satellitosis (Fig. 3A). All tumors showed a strong and widely Genotype RCAS No. No. Incidence P a Mice Tumors (%) distributed expression of Sox10 and PDGFR- (Fig. 3B and C). Only scattered cells in the tumors were positive for the Ntv-a wt SOX10 28 0 0 astrocytic marker GFAP (Fig. 3D), suggesting that the main SOX10 + PDGFB 25 3 12 0.0428 tumor cell population is not of astrocytic origin. The tumors RCAS-X + PDGFB 33 0 0 Ntv-a ArfÀ/À SOX10 29 0 0 were NG2 positive, indicating a more immature cell of origin SOX10 + PDGFB 33 10 30 0.0143 (Fig. 3E); NG2 expression in central nervous system is PDGFB RCAS-X + 37 3 8 normally found on a class of glia cells with properties of NOTE: P values are given for pairwise comparison with mice of the same oligodendrocyte type II astrocyte precursor cells (18). Figure 3F genotype injected with RCAS-X + PDGFB. shows that the staining for nestin was more or less restricted to the vessels. The tumors also stained positive for one of the earliest expressed transcription factors, Sox2. Sox2 has been background, whereas no tumors were detected in Ntv-a wt suggested to maintain neural cells in an immature and mice (Table 1). These injections were used as controls for the proliferative stage (neural stem cell) both during embryogenesis combined injections of RCAS-SOX10 plus RCAS-PDGFB. and in the adult brain (19, 20). The expression of Sox2 and The injection of RCAS-SOX10/RCAS-PDGFB in wt Ntv-a Sox10 were colocalized in most tumor cells but there were also mice yielded a tumor frequency of 12%, and in Ntv-a ArfÀ/À cells positive only for Sox2 or Sox10 (Fig. 3G and H). mice the tumor frequency was even higher, 30% (Table 1). When we compared the results of injection of RCAS-SOX10/ The Expression ofSox10 in Mouse Gliomas Is High Even RCAS-PDGFB with that of RCAS-X/RCAS-PDGFB,we in Akt/Kras–Induced Tumors found a significantly higher tumor frequency in both Ntv-a wt The high expression of the Sox10 protein both in the and ArfÀ/À mice (Ntv-a wt: P = 0.0428, RCAS-SOX10/ MMLV/PDGFB and in the RCAS-SOX10/RCAS-PDGFB – RCAS-PDGFB versus RCAS-X/RCAS-PDGFB;Ntv-a induced tumors prompted us to investigate the expression of ArfÀ/À:P= 0.0143, RCAS-SOX10/RCAS-PDGFB versus Sox10 in other types of experimental mouse glioma. We RCAS-X/RCAS-PDGFB). These results indicated that the therefore studied the expression of Sox10 in tumors induced Sox10 expression enhanced PDGFB-induced glioma develop- with other oncogenes such as RCAS-Akt and RCAS-Kras, both ment and that the glioma incidence increased even more when in combination or alone, and in tumors induced with RCAS- injecting RCAS-SOX10 and RCAS-PDGFB in the ArfÀ/À PDGFB using Ntv-a– and Gtv-a–expressing Arf null or background. InkÀ/À mice (Fig. 4 and data not shown). Figure 4A and B The histology of the tumors induced by RCAS-SOX10/ shows an Akt/Kras–induced brain tumor in a Gtv-a ArfÀ/À RCAS-PDGFB or RCAS-X/RCAS-PDGFB was examined. All mouse with a mixed histology containing areas that are the tumors, independent of the presence or absence of p19Arf, glioblastoma- or gliosarcoma-like. In this tumor Sox10 is displayed morphologic characteristics of PNET-like tumors. expressed in the glioblastoma areas but not in the sarcoma

FIGURE 3. Immunohistochemical staining of a RCAS-SOX10 – and RCAS-PDGFB – induced PNET-like tumor. Immunohistochemical staining of a representative tumor induced by injection of a combination of RCAS-SOX10 and RCAS-PDGFB in Ntv-a transgenic Arf null mice. A. H&E staining. Arrows, typical perineuronal satellitosis. B. Strong positive staining for Sox10. C. Expression of PDGFR-a. D. Scattered expression of GFAP (i.e., presence of astrocytes in the tumor). E. NG2 expression, indicating that the tumor is of immature nature. F. Nestin, expressed mainly in the vessels. G. Presence of the stem cell marker Sox2. H. Sox10 and Sox2 (yellow) are mainly coexpressed in the tumor cells, but there are cells that only express Sox10 (green) or Sox2 (red).

All tested mouse gliomas thus expressed Sox10 independent of genetic background or oncogenes used for induction. When evaluating the presence of PDGFRa, we found a regional hetero- geneity in the Akt/Ras-induced gliomas. In the Sox10/PDGFB– or PDGFB-induced tumors, the expression of PDGFRa was broader and coincided with a higher expression of Sox10.

Sox10 Expression Is High in Human Low-Grade Gliomas Because Sox10 is known to be expressed normally in oligodendrocyte precursor cells and in oligodendrocytes (11, 21), we wanted to examine if it is possible to distinguish human oligodendroglioma from astrocytoma based on Sox10 expression. We investigated the expression of Sox10 on a number of human gliomas (Table 2). Surprisingly, most tumors expressed Sox10 independent of tumor type. Tumors with a low cell density, such as grade 2 astrocytomas and grade 2 oligodendrogliomas, had a broad distribution of Sox10, and grade 3 and grade 4 tumors such as grade 4 glioblastoma, grade 3 oligodendroglioma, and grade 3 ependymoma contained a lower number of Sox10-positive cells (Fig. 5). The expression of Sox10 in pediatric gliomas showed the same pattern with higher expression in pilocytic astrocytomas (grade 1) compared with glioblastoma (Table 3). Among the typical child brain tumors, medulloblastoma and PNET, we found detectable staining for Sox10 only in one PNET (no. 4), which was diagnosed as a PNET with astrocytic and neural differentiation. In the remaining PNET tumors, there was no detectable staining and all the medulloblastomas were negative. Both astrocytomas and oligodendrogliomas express Sox10, consistent with the findings in the Akt/Kras mouse tumors where astrocytic areas were also positive for Sox10. Sox10 had a high expression in low-grade gliomas independently of tumor origin both in FIGURE 4. Sox10 is expressed in mouse glioma induced by Akt and pediatric and adult gliomas. There are reports (22, 23) Kras in the RCAS/Tv-a mouse model. A, C, E, and G. H&E staining suggesting a different cell of origin for medulloblastomas and of the tumor. B, D, F, and H. Corresponding tumor stained for Sox10. The injections are done in Arf null mice. A and B. A tumor induced by the RCAS viruses expressing the oncogenes Akt and Kras. Typical areas of glioblastoma and sarcoma are present. C and D. A fibrillar astrocytoma Table 2. Expression of the Sox10 Protein in Human with sarcoma-like areas induced by injections of RCAS-Akt and RCAS- Gliomas of Different Grade Kras. E and F. A tumor containing typical areas of oligodendroglioma, PNET, and sarcoma induced by the Akt and Kras viruses. G and H. Typical PNET tumor with perineuronal satellitosis induced by the RCAS- Case No. Glioma Type Grade Cell Density Sox10 Expression PDGFB virus. The tumors in (E)to(H) contain small immature cells, neuroepithelial-like cells, with blast-like nuclei that are typical for PNET 1 Astrocytoma 2 ++ +++ and oligodendroglioma. The tumors in (A), (C), and (G) are induced in 2 Astrocytoma 2 + +++ G-tva transgenic mice and the tumor in (E) is induced in an Ntv-a 3 Astrocytoma 2 + +++ transgenic mouse. All tumors are positive for Sox10 but to different extent. 4 Astrocytoma 2 + +++ It seems like it is the glioma-like parts that are positive for Sox10. S, 5 Glioblastoma 4 ++ ++ sarcoma-like areas; GB, glioblastoma-like areas; F, fibrillar areas; vp, 6 Glioblastoma 4 +++ + vessel proliferation; arrows, perineuronal satellitosis. 7 Glioblastoma 4 +++ + 8 Glioblastoma 4 +++ À 9 Glioblastoma 4 +++ + 10 Glioblastoma 4 +++ + areas. Figure 4C and Dshows another Akt/Kras–induced 11 Mixed oligoastrocytoma 3 +++ + 12 Oligodendroglioma 2 ++ ++ tumor with a histology of mixed fibrillar astrocytoma and 13 Oligodendroglioma 2 ++ +++ gliosarcoma. This tumor contains Sox10-positive cells but only 14 Oligodendroglioma 3 ++ + 15 Oligodendroglioma 3 +++ + in the astrocytoma areas. The tumor depicted in Fig. 4E and F 16 Oligodendroglioma 3 +++ À was induced by Akt/Kras in an Ntv-a ArfÀ/À mouse and has 17 Ependymoma 3 +++ + the histologic features of a mixed oligodendroglioma, PNET 18 Ependymoma 3 +++ ++ 19 Ependymoma 3 +++ À and gliosarcoma. This tumor was negative for Sox10 20 Normal brain + ++ expression in the sarcoma areas. Figure 4G and H shows a 21 Normal brain + ++ tumor induced with RCAS-PDGFB in a Gtv-a ArfÀ/À mouse NOTE: Tumor cell density: +, not cell dense; ++, cell dense; +++, very cell dense. with a PNET-like histology, which was strongly positive for Sox10 expression in the tumor: +, occasionally positive cells; ++, tumor is Sox10. positive to a larger extent; +++, the whole tumor is positive for Sox10.

and expressed mainly in brain/gliomas and in skin/melanoma and basal cell carcinoma.

Discussion In the previously published series of MMLV/PDGFB– induced brain tumors, five integrations were located upstream of the Sox10, indicating that Sox10 could be a potential oncogene (9). In the present study, we have investigated if Sox10 is able to initiate tumorigenesis on its own or in cooperation with PDGF. We used the RCAS/tv-a system where gene(s) of interest can be expressed in specific cell types in the brain. We found that Sox10 alone is not able to induce gliomagenesis, neither in Ntv-a wt nor in ArfÀ/À transgenic mice. Although Arf is one of the major suppressor genes in gliomagenesis (25), overexpression of Sox10 in Arf null mice did not initiate gliomagenesis within 12 weeks. However, the RCAS-SOX10 virus significantly enhanced the gliomagenic activity of RCAS- PDGFB in Arf null mice; RCAS-SOX10 caused an almost 4-fold increase in tumor incidence, as compared with RCAS- PDGFB alone. Our data therefore indicate that Sox10 is not an oncogene by itself but may enhance the gliomagenic activity of PDGF in experimental mouse glioma. Sox10 was uniformly expressed in all mouse gliomas induced by PDGFB vectors, without co-injection with RCAS- FIGURE 5. Human brain and gliomas express the Sox10 protein. A. SOX10. This finding is in line with the notion that the PDGFB- Scattered expression of Sox10 in oligodendrocytes and a few unstained induced tumors originate from transformed, immature cells of neurons in human hippocampus. B. Sox10 expression in human cerebellum. The different layers of cerebellum are indicated: molecular the oligodendrocyte lineage (i.e., oligodendrocyte precursors). layer (ML), Purkinje cell layer (PCL), and granular layer (GL). All three This view is further supported by the finding that the tumors layers contain occasional Sox10-positive oligodendrocytes. C. Glioblas- induced by PDGFB, plus or minus Sox10, were NG2, toma grade 4 (case no. 10). D. Astrocytoma grade 2 (case no. 3). E. Oligodendroglioma grade 2 (case no. 13). F. Ependymoma grade 3 (case PDGFRa, and Sox2 positive; both NG2 and of PDGFRa are no. 17). All tumors are positive for Sox10 to varying extent. markers of oligodendrocyte precursors (18) and Sox2 is a marker of stem cells. Further, the tumors were, to some extent, nestin positive, nestin being another marker for immature cells PNETs compared with gliomas, which could explain the lack of of the central nervous system (26). One mechanism by which Sox10 expression in these tumors. RCAS-SOX10 enhances PDGFB-induced gliomagenesis could therefore be to increase the Sox10-expressing, PDGFB- Sox10 Expression in Human Tumor Tissue Is Restricted responsive immature target cell population. to Glioma and Melanoma Sox10 expression was higher and more widely distributed The expression of Sox10 in human tissues and tumors was in PNET- and oligodendroglioma-like tumors, induced by further analyzed using human tissue microarrays. As expected, Sox10 was expressed in oligodendrocytes in the cerebellum, Table 3. Sox10 Expression in Human Pediatric Tumors of cortex, lateral ventricles, and hippocampus of the brain (Fig. 5). Different Grade In the basal portion of epidermis, scattered cells, most likely melanocytes, were positive as well as cells connected to the hair Case no. Tumor Type Grade Cell Density Sox10 follicles. In salivary gland, the acinic cells most probably were positive. In a few tissues, epithelial cells stained positive; in Expression 1 Medulloblastoma 4 ++ À breast, a subset of the ducts were positive as well as certain 2 Medulloblastoma 4 +++ À ductal glands in respiratory mucosa and in head and neck areas. 3 Medulloblastoma 4 +++ À In pancreas and prostate, a few smooth muscle cells stained 4 PNET 4 +++ ++ 5 PNET 4 +++ À positive for Sox10. All other examined tissues were negative or 6 PNET 4 +++ À contained only occasionally positive cells. 7 Pilocytic astrocytoma 1 ++ ++ 8 Pilocytic astrocytoma 1 ++ ++ Analysis of the tumor tissue microarray showed that the 9 Pilocytic astrocytoma 1 ++ ++ malignant gliomas were positive as expected. Sox10 was also 10 Glioblastoma 4 +++ À very strongly expressed in malignant melanomas as reported 11 Glioblastoma 4 +++ À 12 Glioblastoma 4 +++ ++ (24). Essentially all tumor cells in the 11 tested malignant 13 Ependymoma 3 +++ ++ melanomas were Sox10 positive. Six of 12 basal and squamous 14 Ependymoma 3 +++ À skin carcinomas had scattered positive cells, most likely NOTE: Tumor cell density: +, not cell dense; ++, cell dense; +++, very cell dense. melanocytes. Taking these data together, Sox10 seems to have Sox10 expression in the tumor: +, occasionally positive cells; ++, tumor is a restricted distribution pattern both in normal and tumor tissues positive to a larger extent; +++, the whole tumor is positive for Sox10.

PDGFB alone or in combination with Sox10, as compared with Nu¨rnberg University; kindly provided by Dr. Olle Ka¨mpe, the tumors induced by Akt and Kras. The latter tumors resembled Uppsala University, Uppsala, Sweden). The 1,591-bp fragment, human gliosarcoma as they were biphasic tumors composed of which includes the entire coding sequence of SOX10 plus the two distinct histologic parts, one glioblastoma-like and one V5 tag, was subcloned into the RCAS-X vector by digestions sarcoma-like (27). Interestingly, Sox10 was virtually absent with the restriction enzymes ClaI and NotI to construct from the sarcoma-like elements but heavily expressed in the the RCAS-SOX10 virus vector. The PDGFB-containing vector glioblastoma-like regions. The origin of the sarcoma-like is RCAS-PDGFB-IRES-EGFP (ref. 33; here referred to as elements in gliosarcoma has not been determined, although it RCAS-PDGFB). RCAS-X is empty vector. is generally believed that the cells, despite their sarcoma-like morphology, have a common origin with the glioblastoma- Transfection of DF-1 Cells like cells. In addition, the genetic aberrations in glioblastoma Chicken DF-1 fibroblasts were grown in DMEM comple- and gliosarcoma indicate that the genomic changes are similar mented with 12% FCS. The RCAS-SOX10 and RCAS-X (28). Further studies are required to resolve if loss of Sox10 vectors were transfected into DF-1 cells with the FuGENE 6 expression is one of the events that result in a sarcomatous transfection reagent (Roche). The vectors did replicate within phenotype. the producer cell populations. The expression pattern of Sox10 in human was restricted both in normal and tumor tissues. The normal tissues that Mice stained convincingly positive were brain, epidermal melano- The generation of the Ntv-a wt mouse line expressing the tv- cytes, and salivary gland. On the tumor tissue microarray, malignant gliomas and all malignant melanomas and scattered a receptor behind the nestin promoter has been described earlier (34, 35) as well as the Ntv-a ArfÀ/À mouse line (35, 36). The cells in basal cell carcinomas expressed Sox10. Sox10 is known brain tumor sections induced by RCAS-Kras and RCAS-Akt to be expressed in melanocytes, and inactivation of one Sox10 allele leads to melanocyte defects, often presented as the have been described earlier (36). Waardenburg syndrome (depigmentation of skin, hair, and iris; refs. 14, 29). We confirmed the findings of Bannykh et al. (30) Tumor Surveillance that Sox10 is expressed in all human glioma subtypes, even in Neonatal mice were injected in the right cerebral hemis- low-grade astrocytomas, compared with the mouse tumors. phere with 2 AL of DF-1 cells producing the appropriate Despite the fact that the expression of Sox10 in the human adult RCAS viruses (35). The mice were sacrificed when ill or at brain is restricted to oligodendrocytes, Sox10 cannot be used as 12 weeks of age. Statistical analysis was done with the a marker for oligodendroglioma. The finding of Sox10 software GraphPad Prism 4 using the Kaplan-Meier survival expression in a wide range of human gliomas suggests that analysis. oligodendroglioma and astrocytoma may have a common precursor. In the adult brain, type B cells, putative neural stem Brain Sectioning and Immunochemical and Histochem- cells that express both Sox2 and GFAP, have been identified ical Staining in the subventricular zone of the lateral ventricles and in the Brain sectioning and immunochemical and histochemical subgranular layer of hippocampus (19, 31). These type B cells staining were done as described (33). The following antibodies could be the cell of origin for human gliomas and give rise to an were used: polyclonal anti-Sox10 (Chemicon; Santa Cruz immature/progenitor tumor cell population expressing Sox10. Biotechnology), polyclonal anti-PDGFRa (Santa Cruz Bio- Alternatively, human gliomas are derived from bipotential technology), monoclonal anti-GFAP (Chemicon), polyclonal oligodendrocyte precursors, as discussed above with regard anti-NG2 (Chemicon), monoclonal anti-nestin (BDBioscien- to the experimental mouse gliomas. In childhood medulloblas- ces), and polyclonal anti-Sox2 (Chemicon). toma and PNET, there was no or little expression of Sox10 compared with the gliomas. The PNET cell of origin is sug- Human Glioma Tissue Collection gested to be an undifferentiated or less differentiated neuro- Human glioma tissue sections were obtained from patients epithelial cell present in any part of the brain (23, 32), and, for diagnosed at the Department of Genetics and Pathology, at least a subset of medulloblastomas, the cell of origin has been Uppsala University Hospital, Uppsala, Sweden. The classifica- suggested to be granule neuron precursor cells originated from tion of the glioma type and grade were reevaluated by a the external granular layer of the cerebellum (22, 32). The dif- neuropathologist (T.O.). ferences in tumor cell of origin could be a reason why these tumor types show different Sox10 staining patterns. Further studies will address this important issue. Normal and Tumor Human Tissue Microarray The normal and tumor tissue microarray staining was done in collaboration with the Swedish Human Proteome Resource Materials and Methods Center (37).1 The normal tissue microarray contained 47 tissues DNA Constructs ofRCAS Vectors and the tumor tissue microarray contained 21 different tumor A V5 tag was added to the 3¶ end of the human SOX10 types (38).1 cDNA clone (originally from Dr. Michael Wegner, Erlangen-

Acknowledgments 1 http://www.proteinatlas.org We thank Marianne Kastemar for skillful technical assistance.

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Mol Cancer Res 2007;5(9). September 2007 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Sox10 Has a Broad Expression Pattern in Gliomas and Enhances Platelet-Derived Growth Factor-B−Induced Gliomagenesis

Maria Ferletta, Lene Uhrbom, Tommie Olofsson, et al.

Mol Cancer Res 2007;5:891-897.

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