Oncogene (2011) 30, 410–422 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Mitogenic Sonic hedgehog signaling drives E2F1-dependent lipogenesis in progenitor cells and medulloblastoma

B Bhatia1, M Hsieh2, AM Kenney1 and Z Nahle´2

1Department of Cardiothoracic Surgery, Weill Cornell Medical College, New York, NY, USA and 2Department of Cancer Biology and Genetics, Memorial Sloan–Kettering Cancer Center, New York, NY, USA

Deregulation of the Rb/E2F tumor suppressor complex patterning and expansion of progenitor cell populations and aberrantion of Sonic hedgehog (Shh) signaling are (Wechsler-Reya and Scott, 2001). Members of the Hh documented across the spectrum of human malignancies. family in mammals include Indian, Desert and Sonic Exaggerated de novo lipid synthesis is also found in hedgehog (Shh). In development, loss of Hh signaling certain highly proliferative, aggressive tumors. Here, we results in severe abnormalities in mice and humans show that in Shh-driven medulloblastomas, Rb is inacti- (Chiang et al., 1996). Conversely, unrestrained Hh vated and E2F1 is upregulated, promoting lipogenesis. pathway activation is implicated in a variety of tumors, Extensive lipid accumulation and elevated levels of the many of which can be traced to Hh-dependent lipogenic enzyme fatty acid synthase (FASN) mark those progenitors (Beachy et al., 2004; Eberhart, 2008; tumors. In primary cerebellar granule neuron precursors Teglund and Toftgard, 2010). Tumors associated with (CGNPs), proposed Shh-associated medulloblastoma cells- Hh aberrations comprise cancers of the skin, pancreas, of-origin, Shh signaling triggers E2F1 and FASN expres- lung and the brain cancer medulloblastoma (Wetmore, sion, whereas suppressing fatty acid oxidation (FAO), in a 2003; Teglund and Toftgard, 2010), the most common smoothened-dependent manner. In the developing cerebellum, solid malignant pediatric tumor, and a leading cause of E2F1 and FASN co-localize in proliferating CGNPs. in vivo cancer deaths in children. Medulloblastoma arises in the and in vitro, E2F1 is required for FASN expression and cerebellum as a result of aberrant activity of devel- CGNP proliferation, and E2F1 knockdown impairs Shh- opmentally critical signaling pathways, including those mediated FAO inhibition. Pharmacological blockade of Rb activated by wnt, Notch and Shh. Of note, Shh inactivation and/or lipogenesis inhibits CGNP proliferation, mitogenic signaling is required for normal cerebellum drives medulloblastoma cell death and extends survival of development (Dahmane and Ruiz-i-Altaba, 1999; medulloblastoma-bearing animals In vivo. These findings Wechsler-Reya and Scott, 1999), which is marked by a identify a novel mechanism through which Shh signaling brief period of rapid proliferation of cerebellar granule links cell cycle progression to lipid synthesis, through neuron precursor (CGNP) cells, proposed to be cells of E2F1-dependent regulation of lipogenic enzymes. These origin for certain genetically defined classes of medullo- findings pertinent to the etiology of tumor metabolism also blastoma (Eberhart, 2008). underscore the key role of the Shh-E2F1-FASN axis Secreted Hh proteins trigger downstream signaling in regulating de novo lipid synthesis in cancers, and as through interactions with the tumor suppressor patched such its value as a global therapeutic target in hedgehog- (Ptc), a trans-membrane protein localized to lipid rafts dependent and/or Rb-inactivated tumors. (Karpen et al., 2001) whose loss or inactivation is Oncogene (2011) 30, 410–422; doi:10.1038/onc.2010.454; documented in many Hh-dependent human tumors. published online 4 October 2010 Indeed, loss of Ptc was first identified in Gorlin’s syndrome that predisposes patients to basal cell Keywords: sonic hedgehog; medulloblastoma; E2F1; carcinoma and medulloblastoma (Hahn et al., 1996; Rb; lipogenesis; metabolism Johnson et al., 1996). In the absence of Shh binding, Ptc inhibits the evolutionarily conserved pathway activated by another transmembrane protein, smoothened (Smo; Ho and Scott, 2002). Shh interaction with Ptc leads to Introduction dis-inhibition of Smo signaling and consequent activa- tion of Smo target genes following translocation from The Hedgehog (Hh) family of mitogens/morphogens has the cytoplasm to the nucleus of Gli transcription factors. a pleiotropic role in cell fate determination, organ Mammals possess three Gli family members, which have activator and repressor functions and are orthologous to Correspondence: Dr Z Nahle´, Department of Cardiothoracic Surgery, Drosophila cubitus interruptus (Hepker et al., 1997). Weill Cornell Medical College, 1300 York Avenue, New York, NY Gli1 and Gli2 primarily activate transcription in 10021, USA. response to activated Smo signaling; in contrast, Gli3 E-mail: [email protected] Received 26 May 2010; revised and accepted 29 August 2010; published serves to antagonize Shh signaling. In addition to Gli1 online 4 October 2010 and Gli2, other targets of Shh mitogenic signaling Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 411 include the oncogenic transcriptional regulators N-myc Results and YAP1, the microRNA miR17/92 and members of the insulin signaling pathway such as IRS1, as well as Exaggerated lipogenesis in Shh-induced medulloblastomas Ptc itself, presumably as a negative feedback mechanism coincides with Rb inactivation and E2F1 induction counteracting excessive Shh signaling (Platt et al., 1997; NeuroD2-SmoA1 mice are transgenic for an activated Kenney et al., 2003; Oliver et al., 2003; Parathath et al., mutant Smo allele driven by the NeuroD2 promoter 2008; Fernandez et al., 2009; Northcott et al., 2009; (Hatton et al., 2008). Approximately 60% of these mice Uziel et al., 2009). develop medulloblastomas by 6 months of age. Ex- Notably, many diseases of cholesterol processing aggerated lipogenesis, indicative of active de novo lipid involve proteins that resemble Shh pathway components synthesis is detected in these tumors. Indeed, the (that is, Neiman–Pick syndrome (Incardona and Eaton, tumors—unlike adjacent non-tumor cerebellar tissue— 2000)) or cause phenotypes reminiscent of Hh pathway can be identified by a striking accumulation of large malfunction (that is, Smith–Lemli–Opitz syndrome lipid droplets (as indicated by Oil Red O stain used to (Kolf-Clauw et al., 1996)). Shh protein itself is modified mark neutral lipids, Figure 1a). Of note, recent studies by cholesterol and palmitoylation, and it has been from our lab have linked the expression of E2F1 or the reported that cholesterol has downstream roles in Shh inactivation of its negative regulator Rb to inhibition of signaling (Corcoran and Scott, 2006). Nonetheless, glucose oxidation, a process typically associated with negative effects of Shh on lipid biosynthesis have also increased lipogenesis especially in pathological contexts been reported, underscoring the complexity of Shh such as obesity and diabetes (Hsieh et al., 2008). Indeed, signaling in the lipid regulatory process (Suh et al., NeuroD2-SmoA1 medulloblastomas have high levels of 2006). In vivo, an understanding of mechanisms through phosphorylated (inactivated) Rb, accompanied by in- which Shh controls the metabolic machinery remains creased levels of E2F1 (Figures 1b and c). E2F1 elusive and whether mitogenic Shh signaling alters expression also correlates with levels of the lipogenic lipogenic metabolic patterns to create a permissive enzyme FASN (Figures 1b and c), as well as the environment for proliferation and tumor development proliferation marker phosphorylated histone H3. These is unknown. Nevertheless, many tumors exhibit exag- results suggest that under Shh pathway deregulation, gerated lipogenesis, a process that could fuel tumor aberrant E2F1 expression can be associated with growth through providing anabolic growth conditions priming the lipogenic machinery, potentially favoring a and critical biomass accumulation, such as cell mem- lipogenic phenotype in this Shh-driven cancer. brane components and other associated lipid molecules, including lipid rafts which themselves are essential for Shh signal transduction. (Karpen et al., 2001; Menen- Shh increases levels of E2F1 and FAS in CGNPs dez, 2009). Above, we showed an association between Shh signaling Here, we demonstrate that Shh signaling is directly and the Rb/E2F1-FASN axis in spontaneous medullo- coupled to lipogenesis in Shh-dependent medulloblasto- blastomas, proposed to arise from CGNPs (Eberhart, mas and in proliferating CGNPs. We show that Shh 2008), whose proliferation during perinatal development promotes de novo fatty acid synthesis (FAS) concomi- requires mitogenic signaling by Shh. To a large extent, tant with suppressing fatty acid oxidation (FAO) or ‘fat primary cultures of CGNPs recapitulate the mitogenic burning’. Shh-regulated lipogenesis requires inactivation signaling seen in the NeuroD2-SmoA1 medulloblasto- of the Rb/E2F1 tumor suppressor complex leading to mas. As shown in Figure 2a, pRb is phosphorylated in the upregulation of fatty acid synthase (FASN), a key Shh-treated wild-type CGNPs (Figure 2a; Kenney and lipogenic enzyme overexpressed in many aggressive Rowitch, 2000). This phosphorylation was blocked by tumors (Menendez, 2009). Down-modulating E2F1 acute treatment with forskolin, which activates protein activity and/or E2F1-regulated lipid synthesis has kinase A, a Shh pathway antagonist (Hammerschmidt profound negative effects on Shh-associated prolifera- et al., 1996), indicating that Rb phosphorylation occurs tion and medulloblastoma cell survival. These results as a result of Shh pathway activation in proliferation- provide insights into the etiology of cancer-associated competent CGNPs. metabolic patterns, demonstrate the functional link Consistent with cdk-mediated Rb inactivation lying coupling the Rb-E2F1 tumor suppressor complex to downstream of Shh signaling, increased levels of cdk2, the lipogenic/lipolytic balance, and underscore the role cdk4 and cdk6 protein were detected in Shh-treated of Shh signaling in regulating the E2F1-FASN axis CGNPs, and correlated with increased activation of in vivo. Findings of this study could have broad cdk2 as determined by in vitro immunoprecipitation implications in cancer, given that unrestrained Hh kinase assays (Figure 2b and c). These results predict signaling and Rb pathway deregulation are widespread that E2F proteins are activated in Shh-treated prolifer- across the spectrum of human malignancies. These ating CGNPs, and indeed, increased levels of E2F1 (and results will influence current treatment paradigms E2F2, data not shown) protein and mRNA are seen in through exploring viable metabolic-based therapeutic the presence of Shh (Figures 2d and e). Of note, levels of modalities targeting E2F-regulated lipogenesis alone or E2F1 mRNA increased over time, suggesting that in combination with modulators of E2F1 activity, in vitro, Shh strongly promotes E2F protein activity, particularly in contexts where Rb is inactivated or in as E2F1 positively regulates its own expression (Johnson Hh-associated tumors. et al., 1994). E2F1 induction also corresponds to Shh

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Figure 1 Shh-induced mouse medulloblastomas have exaggerated lipogenesis, increased levels of FASN and deregulated E2F1. (a) Triglyceride accumulation was analyzed in a NeuroD2-SmoA1 medulloblastoma using Oil Red O staining; lipids appear as red droplets. Upper row, leftmost panel shows hematoxylin and eosin staining (H&E) staining of the tumor and neighboring cortex and cerebellar (Cb) white matter. (b) Protein lysates were prepared from NeuroD2-SmoA1 normal cerebellum adjacent to the tumor, and tumor then analyzed by western blotting for lipogenic markers (FASN) and proliferation markers (Rb phosphorylation, E2F1). 30 mg protein/lane was loaded. (c) NeuroD2-SmoA1 medulloblastoma and adjacent non-tumor cerebellum were subjected to immunofluorescence analyses for E2F1, FASN and the proliferation marker phospho-histone H3. IGL, inner granular layer; MB, medulloblastoma; MOL, molecular cell layer; PC, Purkinje cells.

signaling pathway activation as determined by Gli1 associated with induction of E2F1 and the ACC-FAS expression in CGNPs treated with exogenous Shh lipogenic axis, an effect that can be reversed by (Figure 2f). Importantly, Shh-treatment of CGNPs down-modulating Shh signaling. causes induction of the lipogenic enzyme acetyl CoA carboxylase (ACC) as well as FASN, consistent with the observations in NeuroD2-SmoA1 medulloblastomas. Shh suppresses mitochondrial b-oxidation in CGNPs Co-treatment with the Smo inhibitor cyclopamine (Ber- FAO is intimately coupled to FAS and the lipogenic man et al., 2002) blocked Shh-mediated induction of process. To that extent, FAO and FAS are inversely FASN, ACC and E2F1. We next examined the levels of linked: activation of one typically adversely influences the sterol regulatory element binding protein 1 the other (Dowell et al., 2005). A critical regulatory (SREBP1), a master regulator of FASN transcription component of this balance lies in the activity of the and whose activity and expression could be regulated in aforementioned key enzyme ACC that modifies acetyl- response to mitogens and nutrient sensors (Menendez CoA into malonyl-CoA, which is a potent inhibitor of and Lupu, 2007). As shown in Figure 2h, Shh stimulates mitochondrial b-oxidation and is also a substrate for levels of SREBP1 protein expression, both in the FASN (Figure 3a). We asked whether Shh-triggered precursor and cleaved (active) form as well as its mRNA FASN expression corresponds to reduce FAO capaci- expression (data not shown). Together, our findings ties. First, we examined the expression of key FAO thus far indicate that Shh signaling in proliferating regulators, including ACC, as well as ACOX1 and primary neural precursors and medulloblastoma cells is MCAD, two enzymes whose expression reflects

Oncogene Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 413 CGNPs

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Cyclo -+-+- + Figure 2 Shh signaling inactivates Rb and induces lipogenesis markers in primary CGNP cultures. (a) CGNP cultures were prepared from PN 4/5 mice and incubated with vehicle (Veh), Shh (3 mg/ml) and/or forskolin (10 mM) for up to 48 h. Protein lysates were prepared and assayed for Rb phosphorylation as determined by upward mobility shift of the total protein. 50 mg protein/lane were loaded. (b) CGNP cultures were treated with vehicle or Shh (3 mg/ml) for 24 h, then analyzed for levels of cdk 2, cdk 4 and cdk 6. (c) CGNP cultures prepared and treated as described above were analyzed for cdk2 kinase activity using immune complex kinase assays. In brief, 200 mg protein lysate were incubated with protein A sepharose beads bound with antibodies against cdk2 or p48 MAPK, whose levels of activity are known not to be altered by Shh signaling. Beads were collected by centrifugation, washed and incubated with substrate (cdk 2, histone H1; p48 MAPK, myelin basic protein) in the presence of gP32-ATP. Reactions are separated by SDS–polyacrylamide gelelectrophoresis and visualized by exposure to X-ray film. Each radioactive (that is, phosphorylated) substrate appears as a single band on the gel. (d) Western blot analysis of E2F1 levels in CGNPs treated with vehicle or Shh. (e) E2F1 mRNA expression after 24 or 48 h in CGNPs treated with vehicle or Shh, measured by quantitative reverse transcription–PCR. (f) Shh pathway activation as determined by quantitative reverse transcription–PCR for Gli1 mRNA expression in the same experiment shown in (e). (g) CGNP cultures were treated with vehicle, Shh, or Shh and the smoothened inhibitor cyclopamine (10 mg/ml). mRNA was prepared and analyzed by quantitative reverse transcription–PCR for expression of ACC, FASN and E2F1.(h) Western blot analysis of SREBP1 precursor (pr) and cleaved (cl; active) form in CGNPs treated with vehicle or Shh. enhanced FAO. Indeed, the Shh-induced FASN and functionally inhibits the rates of mitochondrial FAO in ACC expression is accompanied by a reduction in CGNPs. Palmitate oxidation was measured radiome- ACOX1 and MCAD levels, indicating a reduced overall trically to determine FAO rates as described (Djouadi FAO capacity in the presence of Shh (Figure 3b). et al., 2003). Indeed, Shh has a dramatic effect, Notably, treatment of CGNPs with C75, a FASN significantly reducing FAO in primary CGNPs, com- inhibitor (Thupari et al., 2002), resulted in powerful pared with vehicle-treated CGNPs. Importantly, inhi- suppression of FASN expression and corresponding biting Shh signaling with cyclopamine was sufficient to induction of ACOX1 and MCAD (Figure 3b). This is counteract the effect of Shh, restoring FAO rates almost consistent with reported effects of C75 on promoting to basal vehicle-control treated levels (Figure 3c); FAO in cultured cortical neurons (Landree et al., 2004) cyclopamine alone had no effect on expression of FAS and other cells (Loftus et al., 2000; Kim et al., 2004; and FAO enzymes (Figure 2, Supplementary Figure 1). Thupari et al., 2004). Next, we determined whether Shh Taken together, these findings indicate that Shh impedes

Oncogene Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 414 FA Synthesis FA Oxidation and Synthesis markers FAoxidation FAS ACC ACOX-1 MCAD FASN C75 P=0.0365 P=0.01 2 * 2 * 2.5 2.5 1 1.5 Malonyl-CoA 1 0 0.5 0.0 0 Fold Fold Fold -1 Fold -0.5 -2.5 ACC MCD -2 -1 -1.5 -3 -2 -2.5 P<0.0001 * P<0.0001 * * -5.0 AcetylAcetyl CoA P=0.01

CGNPs Fatty Acid Oxidation 6 0.3 Vehicle p=0.0076 Shh 0.2 Shh + Cyclopamine

cell * 0.1

nmole LCFA/hr/10 0.0 Figure 3 Pharmacological inhibition of FASN in Shh-treated CGNPs induces FAO-promoting enzymes, whereas Shh itself reduces FAO rates. (a) Simple schematic demonstrating enzymes and substrates regulating the balance between FAS and FAO. The compound C75 inhibits the function of FASN. (b) CGNP cultures were treated with vehicle or Shh in the presence or absence of the FASN inhibitor C75 (10 mg/ml) for 48 h. mRNA was prepared and analyzed by quantitative reverse transcription–PCR for markers of FAS (ACC, FASN) or FAO (ACOX1, MCAD). (c) Quantification of radiometric analysis of FAO in CGNPs treated with vehicle of Shh in the presence or absence of cyclopamine. Briefly, after 48 h in culture the CGNPs are exposed to H3-palmitate for 2 h. After incubation, 3 the level of H2O, the byproduct of FAO, is measured and normalized to cell number/protein content, to generate a measurement of rate.

the mitochondrial b-oxidation machinery, in a manner shown by hematoxylin and eosin staining (Figure 4b), reversible by an established inhibitor of Shh-mediated the PN7 E2F1À/À EGL was reduced in thickness proliferation. Evidently, Shh signaling modulates the compared with that of age-matched wild-type controls. lipolytic/lipogenic balance in CGNPs, a process that is Reduced EGL thickness and diminished CGNP pro- seemingly coupled to its mitogenic effects, likely through liferation in the absence of E2F1 could underlie the the activation of E2F1. observation that the E2F1-null adult cerebellum is hypocellular (Cooper-Kuhn et al., 2002). To confirm that reduced proliferation and diminished E2F1 is necessary for Shh-mediated proliferation and FASN levels in neonatal E2F1À/À CGNPs was due to FAO suppression in CGNPs loss of E2F1, specifically in these cells and not a result of Next, we took advantage of E2F1-deficient mice to an earlier developmental defect, Shh-treated wild-type address the requirement and/or contribution of E2F1 to CGNPs infected with lentiviruses carrying short hairpin Shh-regulated induction of FASN and the control of RNAs (shRNAs) targeting mouse E2F1 or a scrambled FAO in proliferating CGNPs. We analyzed protein shRNA were evaluated. As shown in Figure 4d, lysates and histological sections from neonatal wild-type uninfected or scrambled shRNA-infected Shh-treated and age-matched E2F1À/À mouse cerebella to character- CGNPs have elevated E2F1, cleaved SREBP1 and ize the relationship between E2F1 and FAS. A striking FASN, in conjunction with increased proliferation as reduction of FASN protein, as well as the proliferation indicated by Rb phosphorylation and cyclin D2 eleva- marker cyclin D2 is found in E2F1-deficient cerebella tion. Importantly, Shh-treated CGNPs also showed (Figures 4a and b), compared with wild-type counter- marked reduction in the levels of MCAD, consistent parts. We also observed a reduction in SREBP1 levels in with Shh ability to suppress FAO and downregulate E2F1-deficient cerebella, consistent with the expression MCAD mRNA expression as shown earlier (Figures 3b, data in CGNPs. Nonetheless, levels of the cleaved c and 4e). In contrast, E2F1 knockdown reduced levels enzyme were not significantly altered and it remains of E2F1, as expected, as well as reduced levels of unclear at this stage whether a change in overall proliferation markers like phosphorylated Rb and cyclin SREBP1 levels could explain the striking abrogation D2. Importantly, E2F1 knockdown markedly blunts of FASN expression seen with E2F1 loss. Significantly, both SREBP1 and FASN protein levels and notably at post-natal day 7, the peak of CGNP proliferation rescues MCAD expression. Consistently, E2F1 knock- in vivo, E2F1 and FASN localized to the external down also reverses Shh-mediated FAO suppression granule layer (EGL) in wild-type pups, where CGNP (Figure 4e). This is also consistent with the effect seen proliferation takes place (Figure 4b, left and center earlier with inhibition of Shh signaling with cyclopamine panels). In contrast, the PN7 E2F1-null EGL has nearly in CGNPs (Figure 3). Thus, genetic ablation of E2F1 undetectable FASN protein. These cerebella also and in vitro lentivirus-mediated E2F1 knockdown reveal exhibited strikingly reduced proliferation (phospho- a requirement for E2F1 in Shh-mediated CGNP histone H3 staining Figure 4b, center panels, 4C). As proliferation and FASN expression, concomitant with

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Figure 4 E2F1 is required for FASN expression, CGNP proliferation and Shh-mediated FAO suppression. (a) Cerebella were collected from wild-type or E2F1-null PN 5 pups, then analyzed by western blotting for levels of E2F1, lipogenic enzymes SREBP1 precursor (pr) and cleaved (cl; active) form and FASN, and the proliferation marker cyclin D2. (b) PN 7 sagital sections of wild-type (bottom) and E2F1-null (top) cerebella were immunostained for FASN, E2F1 and phospho-histone H3. Rightmost panels show hematoxylin and eosin staining (H&E) staining. (c) Graph shows quantification of phospho-histone H3 staining as a measurement of proliferation. (d) Western blot analysis of E2F1, cleaved SREBP1, FASN, MCAD and proliferation markers cyclin D2 and phospho- Rb in CGNPs treated with vehicle, Shh or Shh in the presence of lentiviruses carrying shRNAs targeting E2F1 or a scrambled control shRNA virus. (e) Rates of FAO in CGNPs treated with vehicle, Shh þ scrambled control shRNA, or Shh þ shE2F1, determined by radiometric analysis. suppression of b-oxidation and key associated markers cdk2 and cdk4 (Figure 5a). This is associated with a like MCAD. cessation of proliferation as determined by quantifica- tion of phospho-histone H3 staining. Next, we used C75, an inhibitor of FASN (Thupari et al., 2002; Inhibition of E2F activity or FASN blocks Shh-associated McCullough et al., 2005), shown to block lung tumor medulloblastoma cell proliferation and viability in vitro growth in mice (Orita et al., 2007, 2008). Treatment of The requirement for Shh-driven E2F1-mediated lipo- these cells with C75, like roscovitine, significantly genesis in CGNP proliferation suggests that targeting reduced proliferation (Figure 5b). Moreover, western E2F1 activity and lipid synthesis pathways could be blot analysis reveals that while cdk inhibition, FASN effective in blocking Shh-induced medulloblastoma inhibition and the combination, all result in reduced growth. Pzp53med cells were used to test the effects of levels of the lipogenic enzyme FASN and proliferation E2F1 and lipogenesis inhibition on tumor cells. These markers, C75 potently induces cell death as suggested by cells were derived from a mouse Ptc þ /À/p53À/À medullo- increased levels of cleaved caspase 3 (Figure 5c). This blastoma and they have been used to model Shh was confirmed by quantification of viability (Figure 5d); mitogenic signaling (Berman et al., 2002; Corcoran C75 synergized with increasing doses of roscovitine in and Scott, 2006). Treatment of Pzp53med cells with the driving Pzp53med cell death. Collectively, these findings cdk inhibitor roscovitine (10 nM) resulted in reduced indicate that, in these Shh-associated medulloblastoma levels of E2F1, FASN, Bmi1 (an E2F1 and Shh target cells where E2F1 is deregulated, FASN activity is (Leung et al., 2004; Nowak et al., 2006)), cyclin D2, required for both proliferation and survival.

Oncogene Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 416 Pzp53 med

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Figure 5 Inhibition of E2F1 and FASN is cytotoxic to medulloblastoma cells in vitro.(a) Immunofluorescence analysis of E2F1, FASN, E2F1/Shh target Bmi1, cyclin D2, cdk 2 and cdk 4 in Pzp53med cells treated with dimethyl sulfoxide (DMSO; control) or the cdk inhibitor roscovitine (10 nM) for 18 h. (b) Proliferation quantification in the presence of vehicle, cdk inhibitor roscovitine (10 nM) and FASN inhibitor C75 (10 mg/ml) determined by counting phospho-histone H3-positive cells. (*P-value ¼ 0.0162; **P-value ¼ 0.0188, t-test analysis). (c) Western blot analysis of proteins regulating lipogenesis (FASN), proliferation (E2F1, cdk2, cyclin D2) and apoptosis (cleaved caspase-3) in Pzp53med cells treated with roscovitine (10 nM), C75 (10 mg/ml), or a combination of both drugs. (d) Effects of increasing doses of the FASN inhibitor C75 (0, 3, 10, 30 mg/ml) in the presence of roscovitine, 0 or 10 nM,on Pzp53med cell viability as determined by cell Titer-Glo assay, a bioluminescent analysis which reports ATP concentration.

Cdk inhibition or counteracting lipogenesis reduces tumor pathophysiology in vivo and affect survival of medullo- growth and improves survival of Shh-driven blastoma-bearing mice. First, we examined the effect of medulloblastoma-bearing mice in vivo a short-term treatment regimen with the FASN inhibitor The sensitivity of Pzp53med cells to cdk inhibition C75 (intraperitoneal, 30 mg/kg) on lipid accumulation. suggests a therapeutic potential for cdk inhibition in Medulloblastomas from drug-treated animals have no Shh-associated medulloblastomas in vivo. We therefore detectable neutral lipid staining, in contrast to the carried out a 2-week treatment regimen of the cdk striking accumulation of lipid droplets in tumor-bearing inhibitor olomoucine (intraperitoneal, 3 mg/kg daily) or vehicle-treated mice as determined by Oil Red O vehicle control on NeuroD2-SmoA1-transgenic mice staining (Figure 7a). Western blot analysis of cerebella bearing medulloblastomas as determined by sympto- and medulloblastomas from vehicle or C75-treated mice mology (slight head tilt, minimal seizure), and magnetic also reveals reduced levels of FASN, consistent with resonance imaging. A total of 10 mice were used for abrogated de novo lipid synthesis as well as down- each treatment. Olomoucine was used instead of modulation of proliferation markers like cyclin D2, roscovitine as dose escalation and toxicity studies (data N-myc and cdk2 (Figure 7b). Consistently, a marked not shown) indicated better brain penetration by this reduction in mitosis as reflected by phospho-histone H3 drug. Olomoucine treatment markedly extended survi- quantification was observed in the tumors of C75- val of mice with medulloblastomas (Figure 6a). Im- treated animals (Figure 7c). Importantly, treatment of munocytochemistry and western blotting revealed that NeuroD2-SmoA1-tumor bearing mice with C75 pro- olomoucine effectively reduced E2F1 levels, down- motes a remarkable increase in survival of these animals modulated cdk pathway activity, and impaired prolif- as compared with vehicle-treated tumor-bearing coun- eration as indicated by reduced levels of PCNA, N-myc, terparts (Figure 7d). Evidently, counteracting lipogen- and cyclin D2 (Figures 6b and c). An increase in cleaved esis in Shh-driven medulloblastomas, like inhibition of caspase 3 in tumors of treated mice reflects an increase cdks, reduces tumor growth and improves survival in the apoptotic index. A marked reduction in FASN in vivo. Together, our findings establish a connection, levels with olomoucine is also detected consistent with in vivo and ex vivo, between Shh mitogenic signaling and reduced levels of E2F1 in vivo, and with results shown in the E2F1-FAS axis. This link underscores the coupling Figures 4 and 5. between Hh-dependent proliferation and the regulation We asked whether counteracting FASN-mediated of lipogenic machinery, deregulation of which as shown lipogenesis would be sufficient to influence tumor here is a prominent feature in medulloblastoma.

Oncogene Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 417

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β-tubulin - PCNA

Figure 6 Inhibition of E2F1 is toxic to Shh-mediated medulloblastomas in vivo and reduces levels of FASN. (a) Kaplan–Meyer survival curve of NeuroD2-SmoA1 medulloblastoma-bearing mice in days commencing with initiation of treatment with dimethyl sulfoxide (DMSO) or the cdk inhibitor olomoucine (6 mg/kg, intraperitoneal injection daily; *P-value o0.0001, Mantel–Cox test analysis). (b) Immunofluorescence staining for markers of proliferation and survival in medulloblastomas collected from NeuroD2- SmoA1 mice treated with DMSO (left column) or olomoucine (right column). (c) Western blot analysis of proteins regulating proliferation and lipogenesis in adjacent non-tumor cerebellar material or medulloblastomas from NeuroD2-SmoA1 mice treated with DMSO (À) or olomoucine ( þ ).

Discussion lipolytic balance. These findings provide new mechani- stic insight into how the Rb/E2F1 complex contributes Shh mitogenic signaling is critical in normal develop- to Shh-dependent proliferation and medulloblastoma ment and its aberrant activity is implicated in a wide (Marino et al., 2000; Olson et al., 2007), in that E2F1 range of human cancers like medulloblastoma, basal cell has a pivotal role integrating the cell cycle regulatory carcinoma, lung and pancreatic cancers to name a few. machinery with metabolic pathways essential for cell A better understanding of the mechanisms through growth, downstream of Shh. These novel connections which excessive Shh signaling engages the cell cycle between Shh signaling, E2F1 activation and mitochon- machinery and recruits metabolic pathways to promote drial reprogramming of cellular bioenergetics favoring uncontrolled proliferation would characterize the onco- lipogenesis have broad implications for the treatment of genic networks involved in the biology of cancer and as Shh-associated cancers: they present lipogenesis inhibi- such inform therapeutic modalities. This will also tors as potentially effective pharmacological tools to improve our comprehension of Shh function in normal counteract hyperproliferative signals in tumors, down- development and cell growth. Here, we show—using stream of Shh signaling. in vivo and ex vivo analyses—that Shh mitogenic/ In proliferating CGNPs, Shh pathway activation oncogenic signaling is tightly coupled to the reprogram- stimulates expression of FASN and ACC in an E2F1- ming of mitochondrial bioenergetics: Shh inhibits the dependent manner, and suppresses FAO, concomitant process of FAO while driving increased FAS, an early with a marked reduction of FAO markers like MCAD critical step of lipogenesis to favor tumor growth. and ACOX1. Similarly, high levels of E2F1 are The effects of Shh on regulating this fundamental expressed in conjunction with FASN in CGNPs aspect of lipid metabolism are observed in CGNPs occupying the EGL of the developing cerebellum during in vivo during development, as well as in primary the peak of Shh-mediated CGNP proliferation. More- cultures in vitro. Exaggerated de novo lipid synthesis and over, loss of E2F1 blunts Shh-mediated proliferation intracellular lipid accumulation with striking pathologi- and downregulates expression of lipogenic markers, cal features similar to those observed in the diabetic including FASN. These findings contrast with a report heart or with hepatic steatosis also mark Shh-dependent that Shh inhibits adipogenesis in Drosophila fat bodies (NeuroD2-SmoA1) medulloblastomas. Importantly, and 3T3-L1 cells (Suh et al., 2006). In those studies, Shh signaling engages the Rb/E2F1 tumor suppressor nonproliferating cells in which Shh signaling was complex, inactivating Rb and inducing E2F1, a process activated showed reduced levels of lipid accumulation we found essential for the effect of Shh on the lipogenic/ and reduced expression of adipogenic transcription

Oncogene Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 418 Oil Red O

Adjacent CB SmoA1 MB

20X 20X 40X Cb white matter

-+- +

Vehicle C75: IGL FASN -

Cdk2 - 20X 20X 40X Cyclin D2 - Cb white matter N-myc - C75 β-tubulin - IGL

white matter

IGL Adjacent CB Mol

Vehicle C75 SmoA1 MB 40X 40X Figure 7 Treatment of medulloblastoma-bearing mice with FASN inhibitor C75 prolongs survival, reduces tumoral lipid accumulation and impairs tumor cell proliferation in vivo.(a) Oil Red O staining to mark neutral lipid accumulation in adjacent cerebellum and medulloblastomas of vehicle (dimethyl sulfoxide, DMSO)-treated or C75-treated mice (30 mg/kg, intraperitoneal injection three times a week). (b) Western blot analysis for markers of lipid synthesis (FASN) and proliferation (cyclin D2, N-myc, cdk2) in samples of adjacent cerebella and medulloblastomas from DMSO-treated (À) or C75-treated ( þ ) mice. (c) Immunostaining for phospho-histone H3 in adjacent cerebellum and medulloblastomas in vehicle- or C75-treated mice. Quantification of phospho- histone H3-positive (mitotic) cells is shown in the accompanying graph. (d) Kaplan–Meyer survival curve of NeuroD2-SmoA1 medulloblastoma-bearing mice in days commencing with initiation of treatment with DMSO or the FASN inhibitor C75 (*P-value o0.0001, Mantel–Cox test analysis).

factors. To the contrary, we find that FASN is highly and Thompson, 2009). Recently, Akt-dependent expressed in tumors with activated Shh signaling, SREBP-mediated FASN regulation has also been linked consistent with the highly lipogenic phenotype apparent to survival of glioma cells with activating EGF receptor in the tumors as shown in Figures 1 and 7. The effects of mutations (Guo et al., 2009a, b). We detected increased Shh signaling on lipogenesis could be highly nuanced levels of SREPB1 precursor and cleaved protein in Shh- and may depend on the proliferation competency of the treated CGNPs, and loss of E2F1 in vivo and in vitro led Shh-responsive cells. Nonetheless, active lipogenesis to a reduction in SREPB1 levels. Whether SREPB1 is marks Shh-driven medulloblastomas in vivo, a process responsible for FASN induction in CGNPs remains to that can also be reversed using lipogenisis inhibitors. be seen; we note, however, that changes in Akt activity Altered metabolic patterns are a hallmark of cancer cells. are not observed in Shh-stimulated CGNPs (Parathath Many tumors have elevated glucose uptake and hexokinase et al., 2008), and indeed, EGF receptor levels are activity, a feature associated with poor prognosis and inversely correlated with Gli levels in human medullo- exploited in diagnostic imaging techniques like fluoro- blastomas (Ferretti et al., 2006), indicating that FASN deoxyglucose-positron emission tomography (Jones and expression may be induced in CGNPs and Shh- Thompson, 2009). Conversely, some positive regulators associated medulloblastomas through a novel, E2F1- of proliferation are associated with reduced hexokinase regulated process. activities and reduced glycolysis. For example, antisense- E2F1 directly regulates genes involved in cell cycle mediated cyclin D1 down-modulation has been shown to regulation, nucleotide biosynthesis, DNA replication, as upregulate glycolysis in vivo, by releasing hexokinase II well as apoptotic targets including caspases and Apaf-1 from inhibition (Sakamaki et al., 2006). This reflects the (Helin et al., 1993; Lees et al., 1993; Zhu et al., 1995; complexity of the network underlying cancer-associated Dynlacht et al., 1997; Abrahamowicz et al., 2001; Nahle metabolic patterns and underscores its heterogeneity. It et al., 2002; Ren et al., 2002). E2F1 can also promote is also established that many cancer cells rely heavily on genomic instability and contributes to cancer progres- glutaminolysis (DeBerardinis et al., 2007; Tong et al., sion (Hernando et al., 2004), through induction of the 2009), a process linked to the activity of the myc mitotic regulator Mad2. Previously, we identified oncogene (Wise et al., 2008; Gao et al., 2009). Tumor pyruvate dehydrogenase kinase 4, a key nutrient sensor suppressors such as p53, PTEN and LKB1 also and an inhibitor of pyruvate dehydrogenase, as a direct influence key regulators of cellular metabolism (Jones E2F1 target, establishing a crucial role for E2F1 in

Oncogene Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 419 metabolic regulation of glucose oxidation proper (Hsieh Cell culture et al., 2008). HIF1a was also linked to the induction of CGNP cultures were generated as previously described (Kenney another pyruvate dehydrogenase kinase family member et al., 2003). Where indicated Shh (R&D Systems, Minneapolis, (pyruvate dehydrogenase kinase 1) and the inhibition of MN, USA) was used at a concentration of 3 mg/ml, cyclopamine pyruvate dehydrogenase activity (Kim et al., 2006). (R&D Systems) was used at 10 mg/ml and C75 (Sigma, St Louis, MO, USA) was used at 10 mg/ml. Pzp53med cells, generously Herein, we demonstrate that the Rb/E2F1 axis acts provided by Matt Scott (Stanford University, Stanford, CA, USA) downstream of Shh to control the expression of were grown in Dulbecco0smodiedEagle0s medium/1% fetal calf lipogenic enzymes, promote active lipogenesis, and fuel serum, supplemented with antibiotics. As indicated, these cells were tumor growth in medulloblastoma. This further under- treated with roscovitine (Sigma; 10 nM for 18 h) or C75 compound. scores the intimate link between excessive E2F1 activity and the priming of hyperproliferative metabolic pat- Lentivirus production and infection. E2F1 shRNA and scrambled terns, during both normal development and in tumors. control lentivirus constructs were obtained from The RNAi Shh-stimulated CGNPs and Shh-induced medullo- Consortium library (Sigma). Each construct was transfected into blastomas exhibit high levels of FASN, a key lipogenic Pzp53med cells, and western blotting was used to determine which enzyme. This is functionally linked to high levels of shRNAs effectively and specifically targeted E2F1. These constructs neutral lipid accumulation, as evidenced by striking Oil were used to prepare lentiviruses. 293T cells, grown in 10% FBS 0 0 Red O staining. Using both genetic and pharmacologi- Dulbecco s modied Eagle s medium medium, were transfected cal approaches, we demonstrate a requirement for E2F1 with E2F1 or scrambled control shRNA lentiviral vectors and MISSION lentiviral packaging mix (Sigma) using Fugene 6 in in expression of FASN and for sustained suppression of serum-free OPTI-MEM medium (Invitrogen, Carlsbad, CA, USA). FAO in both normally proliferating CGNPs and tumor Lentiviral supernatants were collected 48 h post-transfection and cells. This connection between E2F1 and the lipogenic/ filtered through a 0.45 mm filter, then pooled. For infection, lipolytoic balance highlights a novel aspect of E2F1 Shh-treated CGNPs were exposed to the lentiviral supernatent for function in cell cycle regulation and tumor cell growth, 4 h. Control scrambled shRNA lentivirus constructs were used to in addition to its well-established roles in regulating cell determine specificity for E2F1 and to rule out off-target effects or cycle progression and apoptosis genes, and highlights non-specific results because of the process of virus infection. The that E2F1-regulated metabolic pathways have impor- lentiviruses were aspirated and replaced with serum-free CGNP tant role in its oncogenic function. Further, direct medium (above) containing Shh. The cells were lysed or fixed 48 h inhibition of FASN by C75 resulted in medulloblastoma post-infection. cell death in vitro and increased survival of medullo- blastoma-bearing mice, associated with reduced lipid Reverse transcription–PCR synthesis and decreased proliferation in the tumors RNA was isolated using the TRIZOL reagent (Invitrogen) in vivo. These data suggest a selective advantage for according to the manufacturer’s protocol. RNA samples were resuspended in 35 ml diethylpyrocarbonate-treated water. Shh-associated tumor cells with active lipogenesis, to Complementary DNA was generated with SuperScript First- which they become ‘addicted’. The dependence of Shh- Strand Synthesis System for reverse transcription–PCR (Invitrogen) associated medulloblastomas on FAS may be exploited as per manufacturer’s instructions. TaqMan Gene Expression in therapeutic approaches aiming at counteracting Arrays (Applied Biosystems, Carlsbad, CA, USA) using TaqMan de novo lipid synthesis downstream of Smo, and may custom designed MGB probes for N-myc and cyclin D2 were reduce the side effects or surmount acquired resistance performed in triplicate according to manufacturer’s protocol on associated with targeting Smo itself (Kimura et al., 2008; an ABI 7000 Sequence Detection System (Applied Biosystems). Yauch et al., 2009). Moreover, given that Hh pathway Data were analyzed with ABI GeneAmp SDS software (Applied activation and Rb inactivation mark the majority of Biosystems). The average threshold cycle (CT) was determined human malignancies; our work may have broad implica- to quantify transcript levels, normalized against b-actin and the results reported as fold changes. Cyber-green reverse tions for therapies targeting the inextricable coupling transcription–PCR primers were used to detect E2F1 (50-CCA between cell cycle control and lipogenesis, especially in AATTCCCAATTCTGGTG-30,50-CAGCGAGGTACTGAT cancers with Rb and/or Shh pathway defects. Finally, GGTCA-30), Gli1 (50-TGGACAACTGCAGGTAAAACC-30, findings of this study are equally relevant to the patho- 50-AATCCGGTGGAGTCAGACC -30), ACC (50-CTAAACCA physiology of chronic diseases like obesity and diabetes, GCACTCCCGATT-30,50-ACTAGGTGCAAGCCAGACAT-30), where lipid signaling and FAO are also dysregulated. FAS (50-GTCTGGAAAGCTGAAGGATCTC-30,50-TGCCT CTGAACCACTCACAC-30), MCAD (50-GAAGGTTGAAC TCGCTAGGC-30,50-GCTAGCTGATTGGCAATGTC-30)and 0 0 0 Materials and methods ACOX (5 -GTATAAACTCTTCCCGCTCCTG-3 ,5-CCAG GTAGTAAAAGCCTTCAGC-30), using a Biorad iCycler and Animal studies companion software for analysis (Bio Rad, Hercules, CA, USA). All mouse work was carried out in compliance with the Weill Cornell Medical College and Memorial Sloan–Kettering Insti- Western blotting tutional animal care and use committee guidelines. Wild-type Protein extracts were prepared as previously described (Kenney and NeuroD2-SmoA1 mice bearing tumors (kindly provided by and Rowitch, 2000). A total of 40 mg murine cerebella and Jim Olson of Fred Hutchinson Cancer Research Center, Seattle, medulloblastoma protein, or 30 mg CGNP protein were run on WA, USA) were administered with 10% dimethyl sulfoxide 8–12% SDS–polyacrylamide gels and transferred to a PVDF (control), olomoucine at 3 mg/kg daily or C75 at 30 mg/kg membrane (Millipore, Billerica, MA, USA). The blots were through intraperitoneal injection. E2F1-null mice were obtained incubated with primary antibodies in 3% bovine serum albumin from the Jackson Laboratory (Bar Harbor, ME, USA). (in phosphate-buffered saline (PBS)-Tween) or 5% milk overnight

Oncogene Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 420 in 4 1C. Blots were washed three times and incubated with three times with separate litters to confirm reproducibility and secondary antibodies in 5% milk in Tris-buffered saline-Tween consistency. for 2 h in room temperature. After washing, the signals were developed using the enhanced chemiluminescence method Oil Red O staining (GE Healthcare, Piscataway, NJ, USA) and the membranes Oil Red O staining of NeuroD2-SmoA1 medulloblastoma were exposed to Kodak Biomax film. Primary antibodies were: frozen sections was performed by the histology core in the E2F1 (Cell Signaling, Danvers, MA, USA), FASN (Cell Signaling), Department of Pathology and Laboratory Medicine in the MCAD (Abcam, Cambridge, MA, USA), Phospho-Rb S608 and Hospital for Special Surgery and by the microcytometry core S720 (Cell Signaling), total Rb (Cell Signaling), SREBP1 (Cell facility at Memorial Sloan–Kettering Cancer Center according signaling), cdk2 (M-2; Santa Cruz, Santa Cruz, CA, USA), cdk4 to established protocols. (C-22; Santa Cruz), cdk6 (C-21; Santa Cruz), N-myc (C19, Santa Cruz), b-tubulin (Sigma) and cyclin D2 (M-20; Santa Cruz). Horseradish peroxidase-conjugated secondary antibodies FAO assay were: goat anti-rabbit IgG (H þ L; Thermo Scientific, Rockford, Radiometric palmitate oxidation assays were carried out IL, USA) and donkey anti-mouse IgG (H þ L; Jackson Immuno- according to established methods (Djouadi et al., 2003). Research, West Grove, PA, USA). A reaction mixture was prepared by adding 60 mCi of [9,10(n)-3H]-palmitic acid to 2 ml of 10 mM unlabeled palmitic Immunoprecipitation kinase assay acid in absolute ethanol. After complete evaporation of the 0 Protein lysates were prepared as described above. Kinase assays solvent, the fatty acid was resuspended in 12 ml Dulbecco s 0 were carried out precisely as described (Sjostrom et al., 2005), modied Eagle s medium containing 3% fatty acid-free bovine using 200 mg of lysate. Antibodies used were: anti-Erk (9102, serum albumin and incubated at 4 1C overnight. The reaction 0 0 Cell Signaling Technology) and anti-Cdk2 (Santa Cruz, sc-163). mixture was further diluted with Dulbecco s modied Eagle s Kinase substrates were: 1 mg histone H1 (Roche Applied Science, medium to a final concentration of 100 mmol/l palmitic acid. Indianapolis, IN, USA), 5 mg MBP (Upstate Biotechnology, Cells were plated in 12-well plates. After overnight incubation, Billerica, MA, USA). Results shown are representative of experi- the monolayer cultures, at 90–100% confluency, were washed ments carried out in triplicate. with PBS before addition of 300 ml of the reaction mixture. The cultures were then incubated at 37 1C for 2 h. At the end of the incubation, the reaction mixture was removed and added to a Immunostaining solution of 10% trichloroacetic acid followed by centrifugation CGNPs and Pzp53med cells were fixed in 4% para-formaldehyde at 4000 g at 4 1C for 10 min. The supernatant is then for 10 min. Cells were then washed with 1 Â PBS and perme- neutralized with 6 M NaOH and transferred to ion exchange abilized in 1% TritonX-100 for 5 min. Cells were blocked in 3 resin columns (DOWEX1 Â 2-400, Sigma) for separating H2O 5% goat serum in PBS-Tween (1 Â PBS and 0.1% TritonX-100) 3 from the unreacted substrate. The eluates, containing H2O, for 1 h in room temperature, washed once with 1 Â PBS, and were collected into scintillation counting vials. After the then incubated with primary antibody in 2.5% goat serum columns were rinsed twice with 1.5 ml deionized water, 5 ml 1 (in PBS-Tween) overnight at 4 C. They were washed three times scintillant was added to each vial, and the samples were with 1 Â PBS and incubated with secondary antibody for 2 h in 0 counted with a Beckman LS6500 scintillation counter room temperature, then washed and mounted in 4 -6-diamidino- (Beckman Instruments, Fullerton, CA, USA). The reaction 2-phenylindole-containing mounting medium (Vector Labs, rate was expressed as (nmol [3H]FA/hr/cell number). Burlingame, CA, USA). Paraffin-embedded tissue slides were processed as previously described before incubation with primary antibodies (Bhatia et al., Viability assay 2009). Primary antibodies used were: E2F1 (H-137; Santa Cruz), Cells were plated in 96-well plates at 90% confluence. Cell ACC (Cell Signaling), FAS (Cell Signaling), P-Histone H3 (Cell viability was assayed after 24 h of treatment with C75 and Signaling), cleaved caspase 3 (Cell Signaling), PCNA (Calbiochem, roscovitine at the indicated concentrations using the CellTiter- Gibbstown, NJ, USA). Secondary fluorescent-tagged antibodies Glo Luminescent Cell Viability Assay (Promega, Madison, were: Alexa Fluor goat anti-rabbit 488/594 (Invitrogen) and Alexa WI, USA) as per manufacturer’s instructions. Fluor goat anti-mouse 488/594 (Invitrogen).

Image capturing Conflict of interest Immunostaining performed on cultured cells or tissue sections was visualized using a Leica DM5000B microscope and images The authors declare no conflict of interest. were captured with Leica FW400 software (Leica Microsystems, Bannockburn, IL, USA). For quantification of phospho-histone H3 immunostaining, TIFF images of four random fields were taken for each experimental group using the  10 objective. The Acknowledgements percentage of phospho-histone-H3-positive cells over the total 0 number of cells, as determined by 4 -6-diamidino-2-phenylindole We thank Dr Michael J Klein and the Pathology and staining, was calculated using Image Pro Plus software (Media Laboratory Medicine group at the Hospital for Special Cybernetics, Bethesda, MD, USA). Surgery and MSKCC microcyteometry facility. We also thank Lori A Mainwaring, Cedric Dray, Cemille Guldal, Elisa Statistics DeStanchina and Fajun Yang for helpful discussions. These Statistical analysis of quantitative immunostaining was per- studies were funded by the Lung Cancer Center and the formed using one-way ANOVA followed by a two-tailed t-test division of thoracic surgery at Weill Cornell Medical College for comparisons between groups. All results are given as (ZN), the NIH (NINDS R01NS061070) (AMK) and the mean±s.e.m. All in vitro experiments were performed at least Memorial Sloan–Kettering Brain Tumor Center (BB).

Oncogene Hedgehog signals via E2F1 to promote lipogenesis B Bhatia et al 421 References

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