Oncogene (2005) 24, 4026–4036 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice Hiromichi Kimura1, Daniel Stephen2, Alexandra Joyner2 and Tom Curran*,1 1Department of Developmental Neurobiology, St Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA; 2Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA Germline mutations in the human homolog of the pathway signaling genes, PTCH1 (10%), smoothened, patched1 (PTCH1) are associated with basal cell nevus SMOH (6%)and suppressor-of-fused, SUFU (9%)have carcinoma syndrome (BCNS or Gorlin syndrome), which been reported to occur in sporadic medulloblastoma is characterized by developmental anomalies, radiation (Raffel et al., 1997; Vorechovsky et al., 1997; Wolter hypersensitivity and a predisposition to medulloblastomas et al., 1997; Xie et al., 1997; Reifenberger et al., 1998; and skin tumors. Patched1 (Ptc1) functions as a receptor Dong et al., 2000; Zurawel et al., 2000a; Taylor et al., for Sonic hedgehog (Shh) in a wide range of biological 2002). Mice heterozygous for Ptc1 display several processes. Binding of Shh to Ptc1 results in activation of features of Gorlin syndrome and they develop both Smoothened (Smo), which in turn stimulates expression of rhabdomyosarcoma and medulloblastoma (Goodrich downstream target genes including Ptc1 and Gli1. Gli1 is et al., 1997). Previously, we reported that Ptc1 þ /À mice a member of a family of DNA-binding zinc-finger on a C57Bl/6 background exhibit a 14% incidence of proteins, including Gli2 and Gli3, that function in medulloblastoma by 10 months of age (Wetmore et al., transcription control. Here, we report that inactivation 2000). All medulloblastomas examined from Ptc1 þ /À of both Gli1 alleles in Ptc1 þ /À mice significantly reduces mice expressed Gli1 and the remaining wild-type Ptc1 spontaneous medulloblastoma formation. Therefore, Gli1 allele (Wetmore et al., 2000; Zurawel et al., 2000b). is not only a marker of pathway activation but also plays a Thus, medulloblastoma formation in Ptc1 þ /À mice functional role in medulloblastoma formation. Interest- occurs as a consequence of haploinsufficiency of Ptc1. ingly, Gli2 levels were elevated in medulloblastoma cells Since Ptc1 functions to repress Smo, these results imply but not in normal granule neuron precursors during that reduced expression of Ptc1 leads to increased cerebellar development in mice lacking Gli1. In cultured pathway signaling and ultimately tumorigenesis. fibroblasts, Gli1 was more potent than Gli2 at inducing Hedgehog (Hh)is a secreted protein that is highly cell transformation. These results demonstrate that Gli1 conserved from flies to mammals (Ingham and McMa- plays a central role in medulloblastoma formation in hon, 2001). The three mammalian Hh family members, Ptc1 þ /À mice and that Gli2 may also contribute to Shh, Indian hedgehog (Ihh)and Desert hedgehog oncogenesis. (Dhh), are involved in cell growth, proliferation and Oncogene (2005) 24, 4026–4036. doi:10.1038/sj.onc.1208567 differentiation (Ingham and McMahon, 2001). Shh- Published online 4 April 2005 deficient mice exhibit embryonic lethality with skeletal abnormalities, defects in neuronal development and Keywords: medulloblastoma; Patched1; Gli1 small body size (Chiang et al., 1996). In the cerebellum, Shh is detectable in Purkinje cells as early as embryonic day (E)17.5 in mice (Lewis et al., 2004). Ptc1-null mice also show lethality at E 9.5 as a consequence of ectopic activation of Smo (Goodrich et al., 1997). Binding of Introduction Shh to Ptc1 results in derepression of Smo and increased downstream signaling, which results in elevated expres- Basal cell nevus syndrome (BCNS or Gorlin syndrome), sion of Gli1 (Ingham and McMahon, 2001). The Gli which is associated with heterozygous germline muta- family of zinc-finger transcription factors is composed of tion of patched1 (PTCH1), is characterized by a large three members, Gli1, Gli2 and Gli3 in mice and humans. body size, skeletal abnormalities, radiation sensitivity, Targeted disruption of the zinc-finger region together soft tissue sarcoma, basal cell carcinoma and medullo- with the adjacent 43 amino acids (Gli1zfd)or disruption blastoma (Gorlin, 1987). Medulloblastoma, the most in the entire Gli1 N-terminus and zinc-finger region by common malignant pediatric brain tumor, is a primitive lacZ insertion (Gli1lZ)in mice shows no obvious neuroectodermal tumor that arises in the developing biological effect (Park et al., 2000; Bai et al., 2002). In cerebellum. Mutations in the sonic hedgehog (SHH) contrast, disruption of Gli2 or Gli3 in mice results in severe skeletal and neural defects and embryonic or *Correspondence: T Curran; E-mail: [email protected] perinatal lethality (Hui and Joyner, 1993; Mo et al., Received 24 November 2004; revised 19 January 2005; accepted 28 1997). Double mutation of Gli1 and Gli2 causes more January 2005; published online 4 April 2005 severe developmental defects than loss of Gli2 alone Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4027 (Park et al., 2000; Bai et al., 2002). These findings Substitution of the transactivation domain in Gli1 suggest that Shh signaling is critical for normal with the VP16 transactivation domain (NF-Gli1VP16) development and that Gli1 and Gli2 have overlapping resulted in higher transcriptional activity than wild- functions. This hypothesis was confirmed directly by type Gli1. Mutants lacking the entire Zn-finger motif substitution of Gli1 into the Gli2 locus (Bai and Joyner, (amino acids 240–394)(Gli1 zfd240–394)did not show any 2001). luciferase activity (NF-Gli1zfd240–394). The expression level Increased activity of the Shh pathway is associated of the Gli protein constructs was confirmed using anti- with oncogenesis in many cell types. For example, FLAG and anti-Gli1 C-terminal antibodies (data not overexpression of either Shh, Gli1, Gli2 or a constitu- shown). tively active mutant of Smo under the control of a keratin promoter in mice causes basal cell carcinoma Gli1 and Gli2, but not Gli3 transform cultured fibroblasts (Oro et al., 1997; Grachtchouk et al., 2000, 2003; Nilsson et al., 2000; Oro and Higgins, 2003). Further- We used a retroviral vector system to examine the more, in primary rat fibroblasts, overexpression of Gli1 transforming activity of Gli family members and Gli1 induces morphological transformation in cooperation mutants in NIH 3T3 cells and 208F rat fibroblasts. The with the oncoprotein E1A (Ruppert et al., 1991), Gli constructs that were active in the Gli-luc reporter suggesting that deregulation of Smo signaling promotes assay, F-Gli1, NF-Gli1 and NF-Gli1VP16 were all tumorigenesis. Recently, it was shown that ectopic capable of inducing expression of the endogenous Ptc1 expression of Shh in granule cells in the developing and Gli1 genes in NIH 3T3 cells (Figure 1B). Gli2 also cerebellum of Gli1lZ mice induced medulloblastoma induced both Gli1 and Ptc1 expression at lower levels formation (Weiner et al., 2002), suggesting that Gli1 is than those induced by Gli1, whereas NF- Gli1zfd240–394 not required for tumorigenesis. However, since Gli and Gli3 did not activate the endogenous targets. These family members exhibit functional redundancy and data confirm the results of the luciferase assay that Gli2 compensation, and mice with targeted deletions in is a weaker transactivator than Gli1 (Figure 1A). We different Gli genes show distinct phenotypes, it is found that Shh induces Gli transcriptional activity in important to investigate the specific role of each family 208F cells as determined by the Gli-luc reporter assay member in a mouse model of spontaneous tumor (data not shown). We next examined the morphological formation. Here we investigate the contribution of transformation potential of Gli family members and Gli1 to oncogenesis in mice heterozygous for Ptc1.We Gli1 mutants in 208F cells (Miao and Curran, 1994) found that medulloblastoma formation in Ptc1 þ /À mice (Figure 1C). Only the constructs capable of causing was dramatically reduced but not completely eliminated transactivation (F-Gli1, NF-Gli1, NF-GliVP16 and F- in the absence of Gli1. Thus, Gli1 is a crucial Gli2)were able to induce morphological transformation contributor to medulloblastoma formation in Ptc1 þ /À (Figure 1C, b, c, f and g). These results imply that mice, but it is likely that Gli2 shares this oncogenic activation of target genes by Gli1 or Gli2 is required for function. morphological transformation. Gli3 did not induce transformation (Figure 1C, h), and F-Gli2 exhibited less transforming activity than F-Gli1 (Figure 1C, g). Furthermore, NIH3T3 cells expressing F-Gli1 or NF- Results Gli1VP16 were able to form colonies in soft agar (Figure Transcription properties of Gli proteins 1D, b and c). Cells expressing F-Gli2 grew in agar, but they formed smaller colonies than those expressing F- As a first step in investigating the oncogenic potential of Gli1 or NF-Gli1VP16 and they arose after a longer Gli proteins, we compared their transcriptional activity period of culture (Figure 1D, d). Cells expressing F-Gli1 using a Gli luciferase (Gli-luc)reporter construct that or F-Gli2 grew more readily in soft agar in the presence provides a convenient and sensitive marker for tran- of Shh than in the absence of Shh, whereas F-Gli3- scription activity (Sasaki et al., 1999). Comparison of expressing cells did not form colonies in soft agar even the transcriptional activities of Gli family members when cultured in the presence of Shh (data not shown). revealed that Gli1 exhibited the strongest transcriptional Taken together, these results confirm that Gli1 is the activity, Gli2 possessed approximately half the activity strongest activator among Gli family members and that of Gli1, whereas Gli3 was not active in this assay. Since Gli1 induces cell transformation through its transcrip- Gli1 is localized in the cytoplasm in 293 T and NIH 3T3 tion activation function.