Cancer Therapy: Preclinical

Statins Synergize with Hedgehog PathwayInhibitors for Treatment of MedulloblastomaRenata E. Gordon1,2, Li Zhang3, Suraj Peri4, Yin-Ming Kuo5, Fang Du1,Brian L. Egleston4, Jessica M. Y. Ng6, Andrew J. Andrews5, Igor Astsaturov2,7,Tom Curran6, and Zeng-Jie Yang1,3

Abstract

Purpose: The role of cholesterol biosynthesis in hedgehogpathway activity and progression of hedgehog pathway medul-loblastoma (Hh-MB) were examined in vivo. Statins, commonlyused cholesterol-lowering agents, were utilized to validate cho-lesterol biosynthesis as a therapeutic target for Hh-MB.

Experimental Design: Bioinformatic analysis was performedto evaluate the association between cholesterol biosynthesis withhedgehog group medulloblastoma in human biospecimens.Alterations in hedgehog signaling were evaluated inmedulloblas-toma cells after inhibition of cholesterol biosynthesis. The pro-gression of endogenous medulloblastoma in mice was examinedafter genetic blockage of cholesterol biosynthesis in tumor cells.Statins alone, or in combination with vismodegib (an FDA-

approved Smoothened antagonist), were utilized to inhibitmedulloblastoma growth in vivo.

Results: Cholesterol biosynthesis was markedly enhanced inHh-MB from both humans and mice. Inhibition of cholesterolbiosynthesis dramatically decreased Hh pathway activity andreducedproliferation ofmedulloblastoma cells. Statins effectivelyinhibited medulloblastoma growth in vivo and functioned syn-ergistically in combination with vismodegib.

Conclusions: Cholesterol biosynthesis is required forSmoothened activity in the hedgehog pathway, and it is indis-pensable for the growth of Hh-MB. Targeting cholesterol bio-synthesis represents a promising strategy for treatment ofHh-MB. Clin Cancer Res; 24(6); 1375–88. �2018 AACR.

IntroductionCholesterol is a key component of cellular membranes and a

precursor for steroid hormones and bile acids. The brain is themost cholesterol-rich organ, containing approximately 20% oftotal body cholesterol. In the brain, cholesterol is primarilygenerated by de novo synthesis, as the blood–brain barrier invertebrates prevents its uptake from the bloodstream (1). Almostall cholesterol in the brain is present in an unesterified, free form.Cholesterol is synthesized through a series of approximately30 enzymatic reactions, starting with the substrate acetyl CoA(Fig. 1A). The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A

reductase (HMGCR) is rate limiting for cholesterol biosynthesis.HMGCR converts HMG-CoA to mevalonate and is the targetof statins (2). NAD(P) steroid dehydrogenase-like (NSDHL), a3b-hydroxysterol dehydrogenase, is involved in the removal oftwo C-4 methyl groups in one of the later steps of cholesterolbiosynthesis. Deficiency in NSDHL dramatically impairs choles-terol biosynthesis in both humans and mice (3). In particular,conditional deletion ofNsdhl gene in cerebellar granule neuronalprecursors inhibits cholesterol synthesis and dramaticallyrepresses proliferation (4). The final step in cholesterol synthesisis catalyzed by the enzyme 24-dehydrocholesterol reductase(DHCR24), which converts desmosterol to cholesterol by satu-rating the C-24,25 double-bond in the side chain. Triparanol, thefirst synthetic cholesterol-lowering drug, works by antagonizingDHCR24 (5).

In addition to its function as a component of cell membranes,cholesterol regulates many signaling pathways, including thehedgehog (Hh) pathway. The Hh pathway plays a critical role inbrain development and function. In the absence of Hh ligand, theantagonizing receptor Patched1 (Ptch1) suppresses the seven-transmembrane protein, Smoothened (Smo), thus precludingdownstream signaling. The interaction between Hh and Ptch1relieves inhibition of Smo, which then triggers a cascade of eventsculminating in activation of the glioma-associated oncogenes 1and 2 (Gli1 andGli2). Gli proteins translocate into cell nuclei andpromote transcription of Hh pathway target genes, includingPtch1 and Gli1 (6, 7). Extensive evidence suggests that someexogenous oxysterols, such as 20 (S)-hydroxycholesterol and25-hydroxycholesterol, generated by cholesterol oxidation, canactivate Smo by binding to a site located in the extracellularcysteine-rich domain (CRD). This indicates that cholesterol deri-vatives are capable of triggering Hh pathway activation in

1Cancer Biology Program, Fox Chase Cancer Center, Temple University HealthSystem, Philadelphia, Pennsylvania. 2Institute of Fundamental Medicine andBiology, Kazan Federal University, Kazan, Russia. 3Jiangsu Key Laboratory ofNeuropsychiatric Diseases and College of Pharmaceutical Sciences, SoochowUniversity, Suzhou, Jiangsu, China. 4Biostatistics and Bioinformatics ResearchFacility, Fox Chase Cancer Center, Temple University Health System, Philadel-phia, Pennsylvania. 5Cancer Epigenetics Program, Fox Chase Cancer Center,Temple University Health System, Philadelphia, Pennsylvania. 6Children'sResearch Institute, Children's Mercy Kansas City, Missouri. 7Molecular Thera-peutics Program, Fox Chase Cancer Center, Temple University Health System,Philadelphia, Pennsylvania.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Cancer Therapy: Preclinical

Corresponding Author: Zeng-Jie Yang, Fox Chase Cancer Center, 333 CottmanAvenue, Philadelphia, PA 19111. Phone: 215-214-1545; Fax: 215-728-2741; E-mail:zengjie.yang@fccc.edu

doi: 10.1158/1078-0432.CCR-17-2923

�2018 American Association for Cancer Research.

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recipient cells (8–11). Two recent studies revealed that cholesterolitself synergizes with Hh ligand to activate Smo through bindingto the CRD (12, 13). The physical interaction between Smo andcholesterolwas further confirmed in studies of the crystal structureof Smo (14). These findings suggest that cholesterol functions as anative ligand for Smo activation and that Hh signaling may beinfluenced by the local availability of cholesterol.

Insufficient Hh signaling can lead to birth defects, whereasexcessive activity of the Hh pathway is associated with the for-mation of humanmalignancies, including medulloblastoma, themost common malignant brain tumor in children. The Hhpathway is activated in approximately 30% of human medullo-blastoma. Although the cellular and molecular basis for medul-loblastoma tumorigenesis has been widely studied, the role ofcholesterol in medulloblastoma tumorigenesis has yet to beelucidated.

Previously, several studies claimed that inhibition of choles-terol synthesis promoted apoptosis in medulloblastoma cells,and this effect was greater during cotreatment with the naturallyoccurring Hh pathway inhibitor cyclopamine (15–18). However,these effects required the use of statins at levels 1,000-fold greaterthan the physiologic concentrations required to block cholesterolbiosynthesis (15–18). In addition, these studies relied on the useof established medulloblastoma cell lines in which the Hh path-way is now known to be downregulated (19). An additionalconfounding problem with these studies is that they utilizedcyclopamine to inhibit the Hh pathway in cultured tumor cells,and it has now been demonstrated that, at the concentrationsused, cyclopamine causes apoptosis by promoting ceramideproduction independently of any effects on the Hh pathway(20). Thus, the question remains open as to whether cholesterolpathway inhibitors, alone or in combination with Hh pathwayinhibitors, have potential as therapeutic agents in the treatment ofHh-medulloblastoma.

Here, we demonstrate that there is enhanced cholesterol bio-synthesis in Hh pathway–associated medulloblastoma (Hh-MB)and that cholesterol is required for proliferation of tumor cells

in vitro as well as tumor growth in vivo. Furthermore, we show thatcholesterol is necessary for maintaining Smo activity in theabsence of Ptch1. Statins, antagonists of cholesterol biosynthesis,exhibited promising therapeutic effects in mouse medulloblas-toma models. Moreover, statins synergized with the clinicallyapproved Smo antagonist, vismodegib, to prevent medulloblas-toma progression in vivo. These findings elucidate an importantrole for endogenous cholesterol in medulloblastoma tumorigen-esis and demonstrate that cholesterol biosynthesis represents anovel therapeutic target for the treatment of medulloblastomaand, potentially, other Hh pathway–associated malignancies.

Materials and MethodsAnimals

All mice were maintained under barrier conditions in theLaboratory Animal Facility at Fox Chase Cancer Center (Phila-delphia, PA), and all experiments were performed in accordancewith procedures approved by the Fox Chase Cancer CenterAnimal Care and Use Committee. Nsdhlfl/fl mice were kindlyprovided by Dr. Gail Herman (The Ohio State University,Columbus, OH). Math1-Cre mice, Math1-CreERT2 mice,GFAP-CreERT2 mice, Ptch1fl/fl mice, Ptch1þ/� mice, R26R-eYFPmice, and SmoM2 (R26SmoM2) mice were purchased from TheJackson Laboratory. CB17/SCID mice were generated by theanimal facility laboratory at Fox Chase Cancer Center.

Tamoxifen (Sigma-Aldrich) was dissolved in corn oil andadministrated by oral gavage at a dosage of 200 mg/kg dailyduring 3 subsequent days. Animals were sacrificed for furtheranalyses after 7 days from initial tamoxifen treatment.

CompoundsThe following compounds were used for our in vitro and in vivo

studies: simvastatin (Cayman Chemicals), atorvastatin (CaymanChemicals), triparanol (Sigma-Aldrich), vismodegib (Chemlea-der Biochemical), and water-soluble cholesterol (cholesterol-methyl-beta-cyclodextrin, Sigma-Aldrich).

Hh conditioned media (Hh-CM) was prepared by transfectionof pcDNA3.1 Shh-N (Addgene) plasmid into 293FT cells. Super-natant collected at 48 hours after transfection was used at 20% fortreating NIH 3T3 cells.

Subcutaneous allograftsCB17/SCID male mice of age 6 to 8 weeks were used for

subcutaneous allograft establishment. Cells isolated fromtumor-bearing mice in a single-cell suspension in PBS at concen-tration 2 � 106 cells were mixed with reduced growth factorMatrigel (Corning) in a 20:80 ratio and injected subcutaneouslyon the flank of CB17/SCIDmice. Four weeks following injection,tumor-bearing mice that satisfied initial tumor volume require-ment (200–400 mm3) were randomized into experimentalgroups. Simvastatin DMSO stock was dissolved in a mixture ofethanol, 0.1 mol/L NaOH, and PBS and administrated in desig-nated dose by intraperitoneal injection. Atorvastatin and vismo-degib DMSO stock was dissolved in 0.5% methylcellulose 0.2%Tween-80 and administrated per os. Mixture of DMSO, ethanol,0.1 mol/L NaOH, and PBS or DMSO and 0.5% methylcellulose0.2%Tween-80wasused as a vehicle substance.Compoundswereadministrated in half of the indicated dose twice a day and tumorsize measurements were taken every 2 days and tumor volumecalculated as described previously (21).

Translational Relevance

Medulloblastoma is the most common malignant braintumor in children. Approximately 30% of human medullo-blastoma arise from aberrant activation of hedgehog (Hh)pathway. Vismodegib and sonidegib are two FDA-approvedHh pathway inhibitors that have demonstrated efficacy inclinical trials of Hh pathway–associated medulloblastoma(Hh-MB). Despite a dramatic response to these inhibitors ininitial clinical trials, drug resistance and developmental toxi-cities arise in humans as predicted bymousemodels. Here, wedemonstrate that cholesterol is required forHhpathway signaltransduction in tumor cells. Statins, inhibitors of cholesterolbiosynthesis, block hedgehog pathway activity in medullo-blastoma and synergize with vismodegib to inhibit tumorgrowth. Given the excellent safety profile, low cost and readyavailability of cholesterol inhibitors, targeting cholesterolbiosynthesis represents a promising strategy for treatment ofHh-MB and potentially other malignancies caused by Hhpathway activation.

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Cell cultureMedulloblastoma cells were suspended in NB-B27 culture

medium (Neurobasal, 1 mmol/L sodium pyruvate, 2 mmol/LL-glutamine, B27 supplement, and 1% penicillin/streptomycin,all from Invitrogen) and plated on poly-D-lysine–coated cover-slips (Millipore). For bromodeoxyuridine (BrdUrd) incorpo-ration assay, cells were pulsedwith 10 mmol/L BrdUrd (Millipore)for 2 hours,fixed in 4%PFA, and incubated in 4.2mmol/LMgCl2/0.15 mol/L NaCl pH 5.0 buffer in the presence of 50 U/mL ofDNase I (Sigma-Aldrich) at 37�C for 30 minutes.

Primary mouse embryonic fibroblasts (MEF) were isolatedfrom E13.5 of Ptch1fl/fl and SmoM2 mouse embryos. DissociatedMEFs were cultured in DMEM supplemented with 15% FBS(SH30071.03, HyClone, General Electric), 1 mmol/L sodium

pyruvate, 2 mmol/L L-glutamine, 1 g/L glucose, penicillin/strep-tomycin 100 U/mL, and 1% nonessential amino acids (all fromLife Technologies, unless noted otherwise).

NIH3T3 andHEK293T cell lines were acquired fromATCC andcultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mmol/L L-glutamine (all from Life Technol-ogies). All of the above cell lines were authenticated and tested formycoplasma by the Cell Culture Facility at Fox Chase CancerCenter. NIH 3T3 cells andMEFs were starved in serum-free mediafor 24 hours before virus infection or Hh treatment. All in vitrostudies were performed in serum-free environment.

For synergistic effects experiments, NIH 3T3 cells were starvedfor 24 hours before treatment with 20%Hh-CM. Hh-treated NIH3T3 cells and freshly isolated Ptch1þ/� medulloblastoma cells

Figure 1.

Cholesterol biosynthesis is enhanced in Hh subtype of medulloblastoma. A, A schematic diagram of cholesterol biosynthesis pathway with genes encodingcorresponding enzymes listed along arrows. Enzymes: HMGCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1; HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase;MVD mevalonate diphosphate decarboxylase; FDPS farnesyl diphosphate synthase; FDFT1 farnesyl-diphosphate farnesyltransferase 1; SQLE squalene epoxidase;LSS lanosterol synthase; NSDHL NAD(P) dependent steroid dehydrogenase-like; DHCR7 7-dehydrocholesterol reductase; DHCR24 24-dehydrocholesterolreductase. Statins inhibit conversion of HMG-CoA to mevalonate through competitive inhibition of HMG-CoA reductase (HMGCR). Triparanol blocks the last step ofcholesterol biosynthetic pathway through inhibition of 24-dehydrocholesterol reductase (DHCR24). B and C, Boxplots showing the enrichment scores fromGSVA. x and y-axes illustrate enrichment statistics across 4 different subtypes of medulloblastoma. Positive and negative scores indicate positive and negativeenrichment, respectively. Pink dots represent the enrichment score for each sample in that subtype. Black bar in the middle of the box indicates median. D,Heatmaps showing expression of representative genes in the Hh and cholesterol synthesis pathways. z-scores calculated for each gene are plotted on a red (higherexpression) and blue (low expression) scale. Top color bar, subtype. E–G, IHC analysis of NSDHL (E), ABCA1 (F), and SREBP2 (G) proteins in mousePtch1þ/� medulloblastoma. Normal adjacent tissue represents a cerebellar lobe with differentiated granule neurons used as a control. Scale bar, 100 mm;inset, 10 mm.

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were then incubated with 0.001 to 1 mmol/L water-soluble cho-lesterol (WSC) for 18 to 20 hours to saturate Smo, and 1 nmol/Lvismodegib for additional 48 hours.

Lentiviral productionLentiviral productionwas performed by utilizingNsdhl shRNAs

and scrambled shRNA (Genocopiea, MSH029806), pLUT Gli1-HA, pMD2.G, and psPAX2 (Addgene) plasmids in accordancewith the standard protocol.

Histology and IHCFor histologic analyses, cerebella were removed frommice and

fixed in 10% neutral-buffered formalin overnight. Samples wereembedded in paraffin; 5 mm sagittal sections were prepared andstained for ABCA1 (Abcam, ab18180, 1:500), NSDHL (Protein-tech, 15111-1-AP, 1:100), and SREBP2 (Abcam, ab30682, 1:100).

Frozen sections were prepared according to standard protocol(22). Briefly, tissue was fixed in 4% PFA overnight, cryoprotectedin 30% sucrose overnight, and frozen in O.C.T. (Thermo FisherScientific). Sagittal sections (12 mm)were preparedwith a cryostat(Leica). Culturedmedulloblastoma cells were fixed in 4% PFA for15 minutes.

Tissue sections or cultured cells were permeabilized, blockedwith 10% normal goat serum in PBS, 0.1% Triton X-100, andincubated overnight in primary antibody mix against Ki67(Abcam, ab15580, 1:1,000), NeuN (Abcam, ab104224, 1:200),cleaved caspase-3 (Cell Signaling Technology, CST9661, 1:500),BrdUrd (Sigma-Aldrich, B8434, 1:500), mCherry (Abcam,ab167453, 1:500), Zic1 [generous gift from Dr. Rosalind Segalat Dana Farber Research Institute (Boston, MA), 1:200], S100b(Sigma-Aldrich, S2532, 1:500), GFP (Life Technologies, A11122,1:500), GFAP (BD, 556330, 1:500). After 2 hours of incubation insecondary antibodies, samples were counterstained with DAPIand mounted with Fluoromount-G. For fillipin staining experi-ments, medulloblastoma sections were incubated with 2 mg/mLfilipin (Sigma-Aldrich) for 2 hours, counterstained with DRAQ5,and mounted with Fluoromount-G.

Western blottingCells and tissues were lysed in RIPA buffer (Thermo Fisher

Scientific) supplemented with protease (Roche) and phosphatase(Thermo) inhibitors cocktail. Total protein lysates were separatedin 8% SDS-PAGE and transferred on PVDF membrane. Mem-branes were probed with antibodies against Gli1 (Cell SignalingTechnology, CST2534, 1:1,000), NSDHL (Proteintech, 15111-1-AP, 1:1,000), Ptch1 (Novus Biologicals, MAB41051, 1:1,000),GAPDH (Sigma-Aldrich, G8795, 1:1,000). Western blot signalswere detected with SuperSignal West Pico Substrate and exposedon films.

qPCRRNA was isolated using TRI reagent (Sigma-Aldrich) in RNase-

free conditions. cDNAwas synthesized using oligo(dT) and Super-script II reverse transcriptase (Invitrogen). Quantitative PCR reac-tions were performed in triplicates using iQ SYBRGreen Supermix(Bio-Rad) and the Bio-Rad iQ5 Multicolor Real-Time PCR Detec-tion System. Primer sequences are available upon request.

Mass spectrometry and fluorometric cholesterol assayCells or tissue samples were washed with ice-cold DPBS,

and lipids were extracted using a Bligh/Dyer procedure, in which

6mL of 1:2 (v/v) chloroform:methanol was added to the sample.2H6-26,26,26,27,27,27-cholesterol (Sigma-Aldrich) was used asthe internal standard spiked in individual samples. The choles-terol-containing chloroform layer was collected and evaporatedunder a gentle, dried nitrogen stream at 35�C. The dried sampleswere redissolved in 200 mL methanol and analyzed by ultraper-formance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). The peaks of samples were processed and ana-lyzed using Xcalibur, version 2.1.

Statistical analysisAll studies were performed at least in three independent repli-

cates with outcomes reflected on photographs, graphs, or filmscans. Material collection time points were designed in accor-dance to previously published literature. During data analysis,no exclusion criteria were applied. Two-sided Student t tests wereperformed to determine the statistical significance of the dif-ference in means between samples in the experiments reported.P < 0.05 was considered statistically significant. Error barsrepresent the SEM. Data handling and statistical processingwas performed using Microsoft Excel.

The raw gene expression data used for analyzing subtype-specific pathway alterations in medulloblastoma were obtainedfrom GEO (GSE21140) and normalized using RMA. Gene setvariation analysis (GSVA; ref. 23) on the RMA normalized expres-sion data was applied to identify pathways that are enriched ina single sample (method arguments: function¼'gsva'; mx.diff¼'FALSE'; verbose¼FALSE). Over 4,900 gene sets compiledfrom all gene sets available in MsigDB, including KEGG andBiocarta pathways, were used for enrichment analysis. To identifypathways that differ significantly among subtypes, Kruskal–Wallistest was used to analyze the enrichment scores resulting fromGSVA.

To compare the progression rates (Figs. 5A and 6A and C), weused generalized linear regression models assuming gamma fam-ily and log links.Weused the following growth curvemodel to testfor synergy:

log E YjV; S;D½ �ð Þ ¼ b0 þ b1V þ b2Sþ b4V � S

þ b3 þ b5V þ b6Sþ b7V � Sð Þ �D;

where Y ¼ tumor volume, D ¼ day, V ¼ vismodegib, S ¼ statin,andE[Y|V,S,D]¼ conditional expectation (i.e., average).b5 andb6test for additive drug growth effects, and the interaction term, b7,tests for synergistic/antagonistic drug growth effects. Synergismwas concluded if the direction of b7 enhanced drug effects andP < 0.05 (24).

ResultsHh-MB exhibit dysregulated cholesterol metabolism

Human medulloblastoma comprises at least four subgroups,including Hh, Wnt, group 3, and group 4, based on distinct geneexpression profiles (25, 26). Hh-MB arises from aberrant activa-tion of theHh pathway in the developing cerebellum. To examinepathway alterations among medulloblastoma subgroups, weanalyzed transcriptomes of 103 primary human medulloblasto-ma by GSVA (23). This analysis uncovered 363 gene sets andpathways (GSP) that exhibit selective enrichment across the foursubtypes. The significantGSPs rankinghigh in the list included theHh and Netrin pathways known to play roles in Hh-MB tumor-igenesis (Supplementary Table S1; Fig. 1B–D; ref. 27), In addition,

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we observed that cholesterol and sterol biosynthesis GSPs wereassociated with Hh-MB but not the other 3 medulloblastomasubgroups. The enrichment scores of both steroid and cholesterolbiosynthesis pathways were higher in Hh-MB than the other 3subtypes (Fig. 1C). Most of the genes in these two pathways areidentical, and their expression levels are remarkably elevated inHh-MB (Fig. 1D). These data suggest an unexpected associationbetween cholesterol biosynthesis and Hh-MB.

Mice heterozygous for Ptch1 (hereafter referred as Ptch1þ/�

mice) are widely used in preclinical studies for Hh-MB as 15%to 20% of mice spontaneously develop medulloblastoma incerebella after 3 to 4 months of age (28), and this is acceleratedto 100% in the absence of p53 (29). Constitutive activation of theHh pathway as a consequence of loss of Ptch1 heterozygosity iscritical for medulloblastoma formation in Ptch1þ/� mice. Toinvestigate the possible association between cholesterol biosyn-thesis and tumorigenesis in Hh-MB, we examined the expressionof proteins involved in cholesterol homeostasis in medulloblas-toma tissue from Ptch1þ/�mice by IHC. Cholesterol homeostasisis tightly regulated by a complex protein network responsible forits import, synthesis, and export (30). As shown in Fig. 1E,expression of NSDHL was significantly upregulated in medullo-blastoma tissue compared with adjacent normal cerebellar tissue,suggesting there is increased cholesterol synthesis in tumor tissue.Transcription of ATP-binding cassette transporter 1 (ABCA1), anestablished regulator of cellular cholesterol homeostasis, is nor-mally driven by increased accumulation of intracellular choles-terol (31) and sterol regulator element-binding protein 2(SREBP2) is well known to upregulate genes involved in choles-terol biosynthesis and uptake (32). The levels of both ABCA1 andSREBP2 were substantially elevated in tumors, compared withadjacent normal cerebellum (Fig. 1F–G). These data imply thatthere is enhanced synthesis of cholesterol in Hh-MB.

Medulloblastoma growth relies on de novo cholesterolbiosynthesis

Having observed upregulation of cholesterol biosynthesisgenes in Hh-MB, we investigated the possible function of cho-lesterol in medulloblastoma tumorigenesis. Medulloblastomacells isolated from Ptch1þ/� mice were infected with a lentiviruscarrying mCherry-tagged Nsdhl shRNAs or scrambled shRNA. At48hours following viral infection,NSDHLprotein expressionwaseffectively repressed byNsdhl shRNAs (Fig. 2A). ExpressionofGli1was significantly reduced in NSDHL-deficient medulloblastomacompared with scrambled shRNA-infected medulloblastomacells (Fig. 2A), indicating that Nsdhl knockdown impaired Hhpathway activity. We next examined proliferation of medullo-blastoma cells at 48 hours following viral infection. As shownin Fig. 2B–D, approximately 15% of scrambled shRNA-infectedmedulloblastoma cells were positive for BrdUrd, compared withonly 4% to 6% BrdUrd-positive cells among NSDHL-deficientmedulloblastoma cells. These data suggest that cholesterol syn-thesis is critical for proliferation and Hh pathway activity inmedulloblastoma cells.

We next examined proliferation of medulloblastoma cellsin vivo after genetic ablation of cholesterol biosynthesis in tumorcells. Hh-MB cells specifically express the helix-loop-helix tran-scription factor Math1 (22). To establish an appropriate assaysystem, Math1-CreERT2 mice, capable of expressing a tamoxifen-inducible Cre recombinase only in Math1-expressing cells, werecrossed with Ptch1þ/� mice and R26R-eYFP mice carrying a loxP-

flanked STOP cassette upstream of the eYFP gene (33). Aftertumors were established, Math1-CreERT2/R26R-eYFP/Ptch1þ/�

mice were treated with tamoxifen by oral gavage. As shownin Fig. 2E, most of the tumor cells positive for Zic1 (34) alsoexpressed YFP, but no astrocytes (S100b positive) were positivefor YFP (Fig. 2F). These data indicate thatMath1-CreERT2mice canbe utilized to target medulloblastoma cells in vivo.

To disrupt cholesterol biosynthesis in medulloblastoma cells,we utilized conditional Nsdhlfl/fl knockout mice, in which aportion of the Nsdhl gene is flanked by loxP sites (4), crossedwith Math1-CreERT2 mice and Ptch1þ/� mice. Math1-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice and Nsdhlfl/fl/Ptch1þ/� mice were orallytreated with tamoxifen after tumors were fully established. At 7days after tamoxifen treatment, medulloblastoma tissues fromMath1-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice and Nsdhlfl/fl/Ptch1þ/� lit-termates were examined for expression levels of NSDHL and Gli1as well as the amount of intracellular cholesterol present in thetumor. As shown in Fig. 2G, after tamoxifen treatment, NSDHLwas depleted in medulloblastoma tissues obtained from Math1-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice. Moreover, Gli1 expression wasdecreased in medulloblastoma tissue from Math1-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice compared with tumor tissue fromNsdhlfl/fl/Ptch1þ/� mice, suggesting that Hh signaling was com-promised in Nsdhl-deficient medulloblastoma. In addition, theamount of cholesterol in tumor tissuewas reduced by 50%� 10%after tamoxifen treatment, as revealed by mass spectrometryanalysis (Fig. 2H), indicating that Nsdhl deletion effectively sup-pressed cholesterol biosynthesis in tumor cells.Wenext examinedthe proliferation and survival of cholesterol-deficient medullo-blastoma cells in vivo. As shown in Fig. 2I–I00, cell proliferationwas reduced by 50% � 5% in Nsdhl-deficient medulloblastomatissue compared control medulloblastoma tissue from Nsdhlfl/fl/Ptch1þ/� mice, as shown by immunostaining for Ki67. Medullo-blastoma cells that remained proliferating were predominatelylocated in regions of tumor tissue in which cholesterol was notcompletely depleted based on filipin staining (SupplementaryFig. S1). It appeared that most of the Nsdhl-deficient tumor cellsdifferentiated based on the expression of neuronal differentiationmarker NeuN (Fig. 2J–J00). Increased apoptosis was also detectedby cleaved caspase-3 (CC3) immunostaining of tumor tissueafter Nsdhl deletion compared with that in control tumor tissue(Fig. 2K–K00). Consistent with our findings in NSDHL-deficientmedulloblastoma tissues, expression of Hh pathway target genes,includingGli1, Myc-N, and Ptch1, was significantly repressed afterNsdhl deletion (Fig. 2L). The above data demonstrate that de novobiosynthesis of cholesterol in medulloblastoma cells is requiredfor proliferation and tumor growth.

Astrocytes expressing glial fibrillary acidic protein (GFAP) wereabundantly present and intermingled with tumor cells in medul-loblastoma tissue (Zic1 positive, Supplementary Fig. S2A). To testwhether cholesterol from astrocytes is required for medulloblas-toma progression, we examined medulloblastoma cell prolifer-ation after disrupting cholesterol synthesis in astrocytes by usingGFAP-CreERT2 mice (35). For this purpose, we generated GFAP-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice, and Nsdhlfl/fl/Ptch1þ/� mice as acontrol. These mice were treated with tamoxifen by oral gavageafter tumorswere established at approximately 30weeks of age. Asshown in Supplementary Fig. S2B–S2D00, no obvious alterationsin the proliferation, differentiation, or apoptosis were foundin medulloblastoma tissue after Nsdhl deletion in astrocytes inGFAP-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice, compared with that in

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Figure 2.

Deficiency ofNSDHL inhibitsPtch1þ/�medulloblastoma cell proliferation.A,Western blot analysis ofNSDHL andGli1 protein expression inPtch1þ/�medulloblastomacells following infection with lentivirus carrying Nsdhl shRNA (#3 and #4) or scrambled shRNA and uninfected control after 48 hours in culture. GAPDHwas used as a loading control. B and C, Immunofluorescence analysis of BrdUrd among Ptch1þ/� medulloblastoma cells infected with scrambled control shRNA(mCherryþ,B) orNsdhl shRNA#4 (mCherryþ,C) after 48 hours in culture.D,Percentage of BrdUrdþ cells in infectedmedulloblastoma cells (mCherryþ) in Fig. 2B andC. E and F, Sagittal sections of medulloblastoma tissue from Math1-CreERT2/R26R-eYFP/Ptch1þ/� mice were immunostained against Zic1 and YFP (E), S100b,and YFP (F).G,Western blot analysis of NSDHL andGli1 protein expression inmedulloblastoma tissue fromMath1-CreERT2/Nsdhlfl/fl/Ptch1þ/� andNsdhlfl/fl/Ptch1þ/�

mice after tamoxifen treatment. GAPDHwas used as a loading control.H,Mass spectrometry analysis of cholesterol content (mmol/L) inmedulloblastoma tissue fromMath1-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice and Nsdhlfl/fl/Ptch1þ/� mice after tamoxifen treatment. I–K00, Immunofluorescence analysis of Ki67 (I–I00), NeuN(J–J00), and CC3 (K–K00) in tumor tissue fromNsdhlfl/fl/Ptch1þ/�mice (I–K),Math1-CreERT2/Nsdhlfl/fl/Ptch1þ/�mice (I0–K0) after tamoxifen treatment, and percentageof Ki67, NeuN, and CC3-positive cells in above sections, respectively (I00–K00). Medulloblastoma tissues were counterstained with DAPI. L, qPCR analysisof relativemRNA expression of Hh pathway genes (Gli1, N-Myc, Ptch1) inmedulloblastoma tissue fromMath1-CreERT2/Nsdhlfl/fl/Ptch1þ/�mice andNsdhlfl/fl/Ptch1þ/�

mice after tamoxifen treatment. Scale bar, 10 mm. Significance asterisk key: P < 0.05 (�), P < 0.01 (��), P < 0.001 (���).

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Nsdhlfl/fl/Ptch1þ/� mice. These data suggest that astrocyte-derivedcholesterol is dispensable for medulloblastoma growth.

Cholesterol is required for Smo activationTo investigate the role of cholesterol in Hh pathway acti-

vation, we treated medulloblastoma cells from Ptch1þ/� micewith simvastatin in short-term cultures. Under these condi-tions, the Hh pathway remains active, whereas it is repressedin long-term cultures (19). At 48 hours after treatment, cellswere harvested to determine the level of Gli1 expression byWestern blot analysis. As shown in Fig. 3A, the expressionlevels of Gli1 declined in medulloblastoma cells as the con-centration of simvastatin was increased. To confirm thatcompromised Hh pathway activity in medulloblastomacells after treatment with simvastatin is a consequence ofreduced cholesterol levels, we added WSC to the tumor cellcultures. As shown in Fig. 3A, the addition of exogenouscholesterol rescued Gli1 expression in simvastatin-treated

medulloblastoma cells. These data suggest that simvastatininhibits Hh pathway activity in medulloblastoma cells bydisrupting cholesterol biosynthesis.

To identifywhich componentof theHhpathway is regulatedbycholesterol, we investigated the effect of cholesterol on Hh ligandsignaling inNIH3T3 cells. As expected,Hh treatment significantlyupregulated Gli1 expression in NIH 3T3 cells. The induction ofGli1 by Hh ligand was blocked by simvastatin or triparanol, andthis was overcome by the addition of exogenous WSC to theculture medium (Fig. 3B). These data confirm that cholesterol isnecessary for transduction of Hh pathway activity from ligand totarget gene expression.

We next generated primary MEFs from Ptch1fl/fl mice carryingexons 1 and 2 of the Ptch1 gene flanked by loxP sites, and fromSmoM2 mice carrying loxP-flanked STOP cassette placedupstream of constitutively active Smo (36). MEFs were theninfected with a lentivirus carrying Cre recombinase to deletePtch1 or activate Smo, or with a control lentivirus carrying an

Figure 3.

Cholesterol is required for Smo activation in Hh signal transduction. A, Western blot analysis of Gli1 protein expression in Ptch1þ/� medulloblastoma (MB) cellsfollowing treatment with simvastatin alone or in combination with WSC for 48 hours. B, Western blot analysis of Gli1 protein expression in NIH 3T3 cellsfollowing treatment with Hh-CM, simvastatin, triparanol, or together withWSC for 48 hours.C andD,Western blot analysis of Gli1 protein expression in Ptch1fl/flMEFs(C) or SmoM2 MEFs (D) infected with a lentivirus carrying Cre recombinase or control empty vector following treatment with simvastatin, triparanol, orWSC for 48hours.E,Western blot analysis of Gli1 andPtch1 proteins inNIH3T3 cells infectedwith a lentivirus carrying aGli1-HAconstruct or an empty vector followingtreatment with simvastatin, triparanol, or WSC for 48 hours. GAPDH was used as a loading control. F, A schematic diagram of Hh signaling pathway. In theabsence of Hh ligand, its receptor Ptch1 tethers Smo, preventing the downstream activation of Hh pathway. After interaction with Hh, Ptch1 releases Smo, causingactivation of Gli1/2 transcription factor and leading to transcription of Hh pathway genes (Gli1, Ptch1, etc.). Cholesterol is required for Smo activation inHh signal transduction.

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empty vector. At 48 hours after infection with a Cre-bearingvirus, Hh pathway activity was elevated in both models, asdetermined by increased levels of Gli1 expression (Fig. 3C andD). Importantly, treatment with simvastatin or triparanoldramatically inhibited Gli1 expression in Ptch1-deficientMEFs, and this inhibition was overcome by the addition ofexogenous cholesterol, indicating that cholesterol is requiredfor Hh pathway activation resulting from Ptch1 deletion. Incontrast, treatment of cells harboring an activated Smo genewith simvastatin or triparanol treatment failed to reduce Gli1expression, indicating that cholesterol is dispensable for Hhpathway signaling resulting from constitutively active Smo.Consistent with the results described above, we found thatincreased levels of Ptch1 caused by overexpression of Gli1 werenot altered by treatment with simvastatin or triparanol (Fig.3E). Taken together, our data demonstrate that cholesterol isrequired for Smo function in the Hh signaling pathway(Fig. 3F).

Antagonists of cholesterol synthesis inhibit medulloblastomacell proliferation

Because cholesterol is necessary for Hh signaling, we hypoth-esized that inhibition of cholesterol synthesis might suppressmedulloblastoma cell proliferation. To test this idea, we exam-ined medulloblastoma cell proliferation in the presence orabsence of simvastatin or triparanol. Medulloblastoma cells iso-lated from Ptch1þ/� mice were briefly cultured in the presence ofcholesterol inhibitors or vehicle for 48 hours, and examined forproliferation by immunocytochemistry. As shown in Fig. 4A,about 60% of medulloblastoma cells were proliferating (Ki67positive) in control cultures, but the percentage of proliferatingcells declined significantly in a dose-dependent manner as theconcentration of simvastatin was increased (Fig. 4B–D). Reducedproliferation of medulloblastoma cells was also observed aftertreatment with triparanol (Fig. 4E). As shown in Fig. 4G–J,addition of exogenous cholesterol rescued the proliferationrepressed by simvastatin or triparanol treatment (Fig. 4K),

Figure 4.

Cholesterol biosynthesis inhibitors suppress proliferation of Ptch1þ/� medulloblastoma cells. A–K, Medulloblastoma cells isolated from Ptch1þ/� mouse werecultured in thepresence of vehicle (A) or simvastatin (B–D), triparanol (E), or togetherwithWSC (F–J) for 48hours, before cultured cellswere immunostained againstKi67 (red) and counterstained with DAPI. Scale bar, 10 mm. K, Percentage of Ki67-positive cells in cultured Ptch1þ/� medulloblastoma cells in the presenceof simvastatin, triparanol, or with addition of WSC. Significance asterisk key: P < 0.05 (�), P < 0.01 (��), P < 0.001 (���), P < 0.0001 (����).

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suggesting that reduced proliferation of medulloblastoma cellstreated with simvastatin or triparanol was indeed due to choles-terol deficiency.Wealsonoted that proliferation of vehicle-treatedcells increased upon exposure to exogenous cholesterol, indicat-ing that the endogenous levels are rate limiting under the exper-imental conditions used (Fig. 4F). These data indicate that antago-nists of cholesterol biosynthesis significantly inhibit medullo-blastoma cell proliferation. We next tested whether pharmaceu-tical inhibition of cholesterol synthesis could be utilized to treatmedulloblastoma. For this purpose, we generated amedulloblas-toma model by subcutaneous injection of CB17/SCID mice withmedulloblastoma cells isolated from Ptch1þ/� mice, as describedpreviously (19). This model was validated during the develop-ment of vismodegib and shown to accurately predict clinicalresponses in humans (37, 38). In contrast, human tumor celllines fail to sustain Hh pathway activity; therefore, they cannot beused to test the efficacy of inhibitors of Hh pathway activity (19).After the allograft tumor volume reached 200–400 mm3, micewere treated by intraperitoneal injection of 20 mg/kg/day simva-statin, 40 mg/kg/day simvastatin, or vehicle. No significant dif-ference in tumor growthwas observed between the control and 20mg/kg/day simvastatin treatment (P¼ 0.32; Fig. 5A). The volumeof tumors from the group of mice treated with 20 mg/kg/daysimvastatin was comparable with that from the control groupafter 21 days of treatment (Fig. 5B). However, tumor volume wasdramatically reduced after treatment with 40 mg/kg/day simva-statin, comparedwith either vehicle (P <0.001) orwith 20mg/kg/day (P <0.001), suggesting that simvastatin at a dose of 40mg/kg/day effectively suppressed medulloblastoma growth in vivo. Todetermine the mechanism responsible for suppressed tumorgrowth by simvastatin, we examined the proliferation and sur-vival of tumor cells following simvastatin treatment by IHC.Proliferation of medulloblastoma cells (Ki67 positive) wasmarkedly reduced after simvastatin treatment (Fig. 5C–C00) com-pared with vehicle. Moreover, simvastatin treatment promotedsignificant differentiation, evidenced by upregulation of NeuN inmedulloblastoma tissue (Fig. 5D–D00). In addition, an increasednumber of apoptotic cells (CC3 positive) was observed in tumortissues after simvastatin treatment, indicating increased apoptosis(Fig. 5E–E00). On the basis of qPCR and Western blot analyses,Gli1 mRNA and protein expression were significantly downregu-lated in tumors treated with 40 mg/kg/day simvastatin comparedwith the vehicle control (Fig. 5F–H), suggesting simvastatincompromised Hh signal transduction in medulloblastoma cells.In addition, Ptch1þ/� mice with established medulloblastomawere treatedwith 40mg/kg/day simvastatin for 2weeks. As shownin Supplementary Fig. S3A–S3D, simvastatin markedly inhibitedtumor cell proliferation and promoted tumor cell differentiationin medulloblastoma tissue. Moreover, activation of Hh pathwayin medulloblastoma tissue was repressed following simvastatintreatment. Together, these data demonstrate that simvastatintreatment can reduce medulloblastoma growth in vivo.

The SmoM2 mutation results in an amino acid substitution(W535L) in the seventh transmembrane domain, creating aconstitutively active form of Smo by disrupting G-proteincoupling (39). The SmoM2 mutation has been observed inhuman medulloblastoma and in basal cell carcinoma (39, 40).Hh signaling in MEF cells carrying a SmoM2 mutation was notaffected by simvastatin treatment (Fig. 3D), suggesting thatactivation of Hh pathway by SmoM2 is not dependent oncholesterol. To test the effect of simvastatin on the growth of

medulloblastoma resulting from Smo activation, we generatedmedulloblastoma using Math1-Cre/SmoM2 mice as describedpreviously (41). As shown in Supplementary Fig. S4A–S4C,proliferation of medulloblastoma cells in vitro was not alteredafter simvastatin treatment. In the medulloblastoma allograftmouse model generated using medulloblastoma cells fromMath1-Cre/SmoM2 mice, no obvious inhibition of medullo-blastoma growth was observed after simvastatin treatment(Supplementary Fig. S4D–S4E). These data indicate thatmedulloblastoma cells carrying the SmoM2 mutation are resis-tant to simvastatin treatment and that the SmoM2 mutationuncouples Smo from cholesterol dependency.

Statins synergize with Smo antagonist in medulloblastomatreatment

Previous studies reported that oxysterols and cholesterol bindto Smo at CRD-binding site, which is distinct from the bindingpocket of the conventional Smo antagonist, vismodegib (42).This led us to investigate the possible synergistic effects of statinsand vismodegib in the treatment of medulloblastoma. For thispurpose, mice bearing subcutaneous allograft tumors fromPtch1þ/� mice were treated with vismodegib, simvastatin, or thetwo in combination. Consistent with previous reports (43),treatment with vismodegib at 5 mg/kg/day reduced Hh-MBgrowth (Fig. 6A and B). However, combined treatment withvismodegib and simvastatin resulted in a complete inhibition ofHh-MB progression. Although 20 mg/kg/day simvastatin exhib-ited no inhibitory effect on medulloblastoma growth (P¼ 0.32),5 mg/kg/day vismodegib in combination with 20 mg/kg/daysimvastatin resulted in an additional reduction in tumor growthcompared with vismodegib alone (P < 0.001). These data suggestthat simvastatin and vismodegib act synergistically to prohibittumor growth. The synergistic effect of simvastatin and vismode-gib in medulloblastoma treatment was confirmed statistically asexplained in the Materials and Methods section.

CB17/SCID mice carrying allografts of Ptch1þ/� medulloblas-toma were also treated with another HMG-CoA inhibitor, ator-vastatin, alone or combined with vismodegib. Atorvastatin treat-ment at 10 mg/kg/day (P < 0.001) or 40 mg/kg/day (P < 0.001)significantly repressed tumor growth compared with vehicletreatment, confirming inhibitory effects of statins on medullo-blastoma growth (Fig. 6C and D). Moreover, 10 mg/kg/dayatorvastatin dramatically enhanced the inhibitory effects of vis-modegib on medulloblastoma growth, when compared withvismodegib treatment alone (P < 0.001), further confirming thesynergistic effects of statins and vismodegib in inhibiting medul-loblastoma growth.

Having observed the synergistic effects of statins and vismo-degib in medulloblastoma treatment, we next investigatedwhether cholesterol could affect the interaction between Smoand vismodegib. For this purpose, we pretreated NIH 3T3 cellsand Ptch1þ/� medulloblastoma cells with WSC and then exam-ined the inhibitory effects of vismodegib on Hh signaling basedon Gli1 protein expression. As expected, vismodegib signifi-cantly inhibited Gli1 expression in Hh-treated 3T3 cells (Fig.6E) and Ptch1þ/� medulloblastoma cells (Fig. 6F). However,pretreatment with cholesterol abrogated vismodegib-inducedinhibition of Hh signaling in a dose-dependent manner. Thesedata suggest that at high concentrations, binding of cholesterolto Smo may prevent its interaction with vismodegib, perhapsthrough an allosteric effect, providing a potential basis for the

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synergistic effect of statins and vismodegib in medulloblastomatreatment.

DiscussionCholesterol is an essential component of plasma mem-

branes, and it plays an important role in cell membrane

organization, dynamics, and function. During normal devel-opment, cholesterol homeostasis is tightly regulated by a com-plex protein network involving importation, biosynthesis,exportation, metabolism, and esterification. Altered expressionlevels, and mutations of genes involved in the cholesterolhomeostasis pathways, have been identified in many humanmalignancies, including glioblastoma, pancreatic cancer, and

Figure 5.

Simvastatin inhibits growth of Ptch1þ/�

medulloblastoma (MB). A, Growthrate of subcutaneous Ptch1þ/�

medulloblastoma following treatmentwith simvastatin or vehicle control.B, Tumor size of subcutaneousPtch1þ/� medulloblastomafollowing drug treatment. C–E00,Sections of subcutaneous Ptch1þ/�

medulloblastoma after treatmentwith vehicle (C–E) or 40 mg/kg/daysimvastatin (C0–E0) wereimmunostained for Ki67 (C–C00), NeuN(D–D00), or CC3 (E–E00). Percentage ofKi67þ cells (C00), NeuNþ cells (D00),andCC3þ cells (E00) inmedulloblastomatissue after designated drug treatment.Medulloblastoma tissues werecounterstained with DAPI. Scale bar, 10mm. F–H, mRNA (F) and protein (G)expression of Gli1 in Ptch1þ/�

medulloblastoma tissues after drugtreatment were analyzed byqPCR and Western blotting,respectively. H, Quantification of Gli1protein expression. Significanceasterisk key: P < 0.05 (�), P < 0.01 (��),P < 0.001 (���).

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melanoma (44). Impaired cholesterol homeostasis in tumorcells influences tumor progression and dictates the sensitivity oftumor cells to chemotherapy (45), although it is still debatedwhether cellular cholesterol levels influence tumor initiation.Our studies reveal enhanced expression of genes associatedwith cholesterol biosynthesis in Hh-MB, suggesting that theintracellular cholesterol levels in tumor cells are elevated.Inactivating mutations of Ptch1, the most common mutationin Hh-MB, may also contribute to increased cholesterol levelsin tumor cells, in that Ptch1 is involved in cholesterol effluxfrom cells (46).

Extensive evidence indicates that some exogenous oxysterols,such as 20 (S)-hydroxycholesterol and 25-hydroxycholesterolgenerated by cholesterol oxidation, activate Smo by binding toa site located in the extracellular CRD, demonstrating thatcholesterol derivatives are capable of triggering Hh pathway

activity (8–11). However, it is unclear whether these oxysterolsare physiologic activators of Smo, given that the endogenouslevels are significantly lower than the EC50 for Hh pathwayactivation. In this study, we found that, upon inhibition ofcholesterol biosynthesis, using proximal and distal inhibitors(simvastatin and triparanol, respectively), Hh pathway activityin medulloblastoma cells and fibroblasts was significantlyrepressed. Proliferation of medulloblastoma cells and tumorgrowth in vivo was repressed after pharmacologic or geneticinhibition of cholesterol biosynthesis. These data suggest thatendogenous cholesterol biosynthesis is important for Hh path-way activity. Compromised Hh pathway activity was observedin cholesterol-deficient fibroblasts after loss of Ptch1 but notfollowing constitutive activation of Smo or after overexpressionof Gli1. These data indicate that cholesterol biosynthesis isrequired for Smo to function in Hh signaling. Recent findings

Figure 6.

Statins synergize with vismodegib in inhibiting Ptch1þ/� medulloblastoma (MB) growth. A and B, Growth rate (A) and size (B) of subcutaneous Ptch1þ/�

medulloblastoma treated with 20 mg/kg/day simvastatin, 5 mg/kg/day vismodegib, or in combination. C and D, Growth rate (C) and size (D) of subcutaneousPtch1þ/� medulloblastoma treated with 10 mg/kg/day, 40 mg/kg/day atorvastatin, 5 mg/kg/day vismodegib, or in combination. E, Western blot analysis of Gli1protein expression in NIH 3T3 cells treated with vismodegib for 48 hours following overnight treatment withWSC and Hh-CM. F,Western blot analysis of Gli1 proteinexpression in Ptch1þ/� medulloblastoma cells treated with vismodegib for 48 hours after overnight treatment with WSC.

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that cholesterol synergizes with Hh to activate Smo throughbinding to the extracellular CRD (12, 13) are consistent withthe conclusion that cholesterol, but not oxysterols, functionsas the endogenous ligand responsible for Smo activationduring Hh pathway signaling, and that Ptch1 inhibits Smoby counteracting its activation by cholesterol. Thus, cholesterolplays a unique role as a second messenger in vertebrate Hhsignaling, by mediating the functional interaction betweenPtch1 and Smo. These findings resolve a longlasting questionof how Ptch1 suppresses Smo activity, and they identifycholesterol as a critical component for Smo activation byPtch1-mediated Hh signaling. Simvastatin also inhibits pro-liferation of group 3 medulloblastoma, which are associatedwith activating mutations in c-Myc and dysfunctional p53 (47).These findings imply that cholesterol may regulate many othersignaling pathways in medulloblastoma in addition to Hhsignaling.

The blood–brain barrier efficiently blocks transport of choles-terol from the bloodstream into the brain. Therefore, nearlyall brain cholesterol is produced via de novo synthesis (1). Recent-ly, it has been demonstrated that tumor cells in glioblastomamultiforme, the most malignant brain tumor in adults, exclusive-ly acquire cholesterol from astrocytes (45). In our studies, con-ditional inhibition of cholesterol biosynthesis in tumor cellsdramatically inhibited medulloblastoma cell proliferationand promoted the differentiation of tumor cells, whereas disrup-tion of cholesterol synthesis in astrocytes had no effects inmedulloblastoma cell proliferation and survival. These observa-tions suggest thatmedulloblastoma cells predominately rely on denovo synthesis of cholesterol. In our studies, cholesterol was notcompletely depleted after deletion of the Nsdhl gene in medul-loblastoma cells in Math1-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice fol-lowing tamoxifen treatment. On the basis of filipin staining,some regions of the tumor tissue were enriched with choles-terol. Such incomplete depletion of cholesterol in tumor tissuecould be due to insufficient Cre recombination and/or inade-quate tamoxifen penetration. However, we cannot completelyexclude the possibility that tumor cells at least partially acquirecholesterol from their microenvironment. Nevertheless, prolif-erating medulloblastoma cells predominantly accumulated incholesterol-enriched tumor regions of Math1-CreERT2/Nsdhlfl/fl/Ptch1þ/� mice, further confirming that cholesterol is critical formedulloblastoma growth.

The observation of Hh pathway dysregulation in tumors hascaused it to be an attractive pharmacologic target for cancertreatment formany years. All the available Hh pathway inhibitorsbeing studied in clinical trials target Smo. Our studies demon-strate that cholesterol biosynthesis is essential for medulloblas-toma progression. Statins effectively inhibited medulloblastomacell proliferation and suppressed tumor growth. The excellentsafety profile, together with low cost and ready availability ofstatins, makes cholesterol biosynthesis a promising target formedulloblastoma treatment and perhaps other malignanciesinvolving Hh signaling.

Smo inhibitors cause developmental bone abnormalities inyoungmice (43). Therefore, they are not recommended for use inyoung children. Potentially, the combined use of vismodegib andstatins could permit the use of lower doses of Smo inhibitors,potentially reducing the extent of bone toxicity in the pediatricHh-MB patient population. Currently, statins are being used tomanage dyslipidemia in some children and are among the drugs

approved for use in children (48, 49). So far, there have been noreports of bone toxicities or other phenotypes that could beascribed to Hh pathway inhibition. Nevertheless, further studiesare warranted to test the potential for combined toxicities of Smoinhibitors and statins in developing animal models prior to theiruse in children.

The potential efficacy of statins in Hh-MB therapy dependson which underlying mutation the tumor harbors in the Hhpathway. Among Hh-MB patients, approximately 80% of adulttogether with 50% of infant and childhood patients harbormutations in Hh, Ptch1, or Smo, who are most likely torespond to Smo antagonists including statins (40). However,medulloblastoma carrying the SmoM2 mutation are predictedto be resistant to Smo inhibitors as well as cholesterol inhibi-tors. Similarly, around 45% of infant and childhood Hh-MBpatients frequently harbor mutations downstream of Smo, suchas Sufu or Gli2 (40), rendering these tumors intrinsicallyresistant to any drugs targeting Smo.

Our studies reveal the synergistic effects of statin andvismodegib in treating medulloblastoma. It has been con-firmed that cholesterol activates Smo through interaction withthe CRD region, which is distinct from the binding site ofvismodegib. Recent structural studies found that binding withvismodegib precludes the interaction of Smo with cholesterol(14). In our studies, pretreatment with cholesterol overcamethe capacity of vismodegib to inhibit Hh signaling in NIH 3T3cells and medulloblastoma cells, implying that cholesterolcompromises the interaction between vismodegib and Smo.These data suggest an indirect competition between cholesteroland vismodegib, possibly through allosteric effects, in bindingto Smo.

Treatment with Smo antagonists results in dramatic regressionof tumor burden and reduction of symptoms, but there is a rapidemergence of drug resistance (50). The resistance often arises as aconsequence ofmutations in the transmembrane domain of Smothat prevent binding of antagonists. Therefore, cholesterol inhi-bitors could not only synergize with vismodegib in treatingmedulloblastoma, but also potentially be effective in treatingsome drug-resistant Hh-MB.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed by the authors.

Authors' ContributionsConception and design: R.E. Gordon, J.M.Y. Ng, A.J. Andrews, I. Astsaturov,T. Curran, Z.-J. YangDevelopment of methodology: R.E. Gordon, F. Du, J.M.Y. Ng, Z.-J. YangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):R.E. Gordon, L. Zhang, Y.-M. Kuo, J.M.Y. Ng, Z.-J. YangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.E. Gordon, S. Peri, Y.-M. Kuo, B.L. Egleston,J.M.Y. Ng, T. Curran, Z.-J. YangWriting, review, and/or revision of the manuscript: R.E. Gordon, L. Zhang,Y.-M. Kuo, B.L. Egleston, J.M.Y. Ng, T. Curran, Z.-J. YangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.E. Gordon, F. Du, J.M.Y. Ng, T. Curran,Z.-J. YangStudy supervision: R.E. Gordon, J.M.Y. Ng, T. Curran, Z.-J. Yang

AcknowledgmentsThe authors would like to thankDrs. G.E. Herman andD. Cunningham (The

Ohio State University, Columbus, OH) for providing Nsdhlfl/fl mice, Drs. T. YenandE.Golemis for thoughtful comments, andA. Efimov, E.Nicolas, F. Jin, and J.

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Oesterling for technical assistance. This work was also supported by NCI(CA178380, CA185504), ACS RSG (RSG1605301NEC), PA CURE HealthResearch Fund, Bucks County Chapter, and National Natural Science Founda-tion of China (81572724, 81573449). This research was supported by thesubsidy of the Russian Government to support the Program of competitivegrowth of Kazan Federal University to Renata E. Gordon.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 5, 2017; revised December 1, 2017; accepted January 9,2018; published OnlineFirst February 6, 2018.

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Gordon et al.

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2018;24:1375-1388. Published OnlineFirst February 6, 2018.Clin Cancer Res   Renata E. Gordon, Li Zhang, Suraj Peri, et al.   of MedulloblastomaStatins Synergize with Hedgehog Pathway Inhibitors for Treatment

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