menin epigenetically represses hedgehog signaling in...

Tumor and Stem Cell Biology

Menin Epigenetically Represses Hedgehog Signaling inMEN1 Tumor Syndrome

Buddha Gurung1, Zijie Feng1,3, Daniel V. Iwamoto1, Austin Thiel1, Guanghui Jin3, Chen-Min Fan4,Jessica M.Y. Ng2, Tom Curran2, and Xianxin Hua1

AbstractMultiple endocrine neoplasia type 1 (MEN1) is an inherited tumor syndrome that includes susceptibility to

pancreatic islet tumors. This syndrome results from mutations in the MEN1 gene, encoding menin. Althoughmenin acts as an oncogenic cofactor for mixed lineage leukemia (MLL) fusion protein–mediated histone H3lysine 4 methylation, the precise basis for how menin suppresses gene expression and proliferation ofpancreatic beta cells remains poorly understood. Here, we show that menin ablation enhances Hedgehogsignaling, a proproliferative and oncogenic pathway, in murine pancreatic islets. Menin directly interacts withprotein arginine methyltransferase 5 (PRMT5), a negative regulator of gene transcription. Menin recruitsPRMT5 to the promoter of the Gas1 gene, a crucial factor for binding of Sonic Hedgehog (Shh) ligand to itsreceptor PTCH1 and subsequent activation of the Hedgehog signaling pathway, increases repressive histonearginine symmetric dimethylation (H4R3m2s), and suppresses Gas1 expression. Notably, MEN1 disease-related menin mutants have reduced binding to PRMT5, and fail to impart the repressive H4R3m2s mark atthe Gas1 promoter, resulting in its elevated expression. Pharmacologic inhibition of Hedgehog signalingsignificantly reduces proliferation of insulinoma cells, and expression of Hedgehog signaling targetsincluding Ptch1, in MEN1 tumors of mice. These findings uncover a novel link between menin and Hedgehogsignaling whereby menin/PRMT5 epigenetically suppresses Hedgehog signaling, revealing it as a target fortreating MEN1 tumors. Cancer Res; 73(8); 2650–8. �2013 AACR.

IntroductionMultiple endocrine neoplasia type 1 (MEN1) is an inherited

tumor syndrome, with development of tumors in severalendocrine organs including pancreatic islets (1–4). The genemutated in this syndrome, MEN1, encodes a nuclear protein,menin (5, 6).Menin interactswith diverse proteins to regulate avariety of cellular functions including control of gene tran-scription (7, 8). Target-based therapy against MEN1 syndromeis currently lacking, yet highly desirable.

Epigenetic regulation of gene expression via histonemethylation is crucial for the regulation of onset andmaintenance of various cancers (9). Menin can positively

or negatively affect gene expression. It upregulates expres-sion of antiproliferative genes, such as cyclin-dependentkinase inhibitors p18 and p27, partly via upregulating mixedlineage leukemia (MLL)-mediated histone H3 lysine 4(H3K4) methylation (10, 11). However, it is not yet wellunderstood how menin represses gene transcription andsignaling pathways.

The Hedgehog signaling pathway regulates diverse bio-logic processes ranging from embryonic development to cellcycle and tumorigenesis (12). With assistance from acces-sory proteins, such as GPI-anchored cell surface proteinsGAS1, BOC, or CDO (13–15), Hedgehog ligands bind to thecell surface receptor Patched (PTCH1), resulting in therelease of PTCH1-mediated repression of the cell membraneprotein Smoothened (SMO; ref. 12). Activated SMO entersthe primary cilia and promotes the dissociation of GLIproteins from SuFu, Fused, and Cos2 complex (16), resultingin nuclear translocation of transcription factors, GLI1 andGLI2, and transcription of target genes including proproli-ferative genes (12). Concomitantly, GLI3 proteolysis togenerate the repressive form, GLI3R, is inhibited (17). Con-stitutively activated Hedgehog signaling triggers the devel-opment of tumors, such as medulloblastomas and basal cellcarcinoma (BCC; refs. 18, 19). It is not yet clear whethermenin influences Hedgehog signaling during the develop-ment of the MEN1 tumor. Here, we show that meninpotently suppresses Hedgehog signaling at least partly viaprotein arginine methyltransferase 5 (PRMT5)-mediated

Authors' Affiliations: 1Department of Cancer Biology, Abramson FamilyCancer Research Institute, Abramson Cancer Center, University of Penn-sylvania Perelman School of Medicine; 2Department of Pathology andLaboratoryMedicine, TheChildren'sHospital of Philadelphia, Philadelphia,Pennsylvania; 3Department of Basic Medical Sciences, Medical College,XiamenUniversity, Xiamen, Fujian, China; and 4Department of Embryology,Carnegie Institution for Science, Baltimore, Maryland

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Xianxin Hua, Department of Cancer Biology,Abramson Cancer Center, Abramson Family Cancer Research Institute,University of Pennsylvania Perelman School of Medicine, 421 Curie Blvd.,Philadelphia, PA19104.Phone: 215-746-5567; Fax: 215-746-5525; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-3158

�2013 American Association for Cancer Research.

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suppression of GAS1, and Men1 mutations lead to enhancedHedgehog signaling.

Materials and MethodsPlasmids and cell cultureLentiviral constructs expressing Prmt5 short hairpin RNA

(shRNA) were obtained from Open Biosystems. Lentiviralpackaging plasmids, pMD2G and pAX2G, were purchased fromAddgene. Retroviral plasmids expressing Flag-tagged ormutant menin have been described elsewhere (20). GST-PRMT5 fragments were generated using the Site-DirectedMutagenesis Kit (Stratagene). Men1-null mouse embryonicfibroblasts (MEF) cells complemented with wild-type ormutant menin (20), and 293 cells were cultured in Dulbecco'sModified Eagle'sMedium (supplementedwith 10%FBS and 1%Pen/Strep).

Immunoprecipitation and immunoblottingNuclear extract was isolated as described previously (21).

Immunoprecipitation and immunoblotting were conductedusing standard techniques. Details can be found in Supple-mentary Materials and Methods. Antibodies used were anti-menin (Bethyl, A300-105A), anti-PRMT5 (Abcam, ab31751and ab109451), anti-MEP50 (Bethyl, A301-562A), anti-PRMT7 (Abcam, ab126965), anti-FLAG (Sigma, F3165), andanti-b-Actin (Sigma, A5441). Rabbit antibodies to GAS1 aredescribed elsewhere (22).

RNA extraction and quantitative real-time PCRTotal RNAwas extracted from cultured cells with Trizol and

an RNeasy Extraction Kit from Qiagen. Details and primersequences can be found in Supplementary Materials andMethods.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assay was con-

ducted as previously described using a Quick ChIP Kit fromImgenex (23). Details and primer sequences can be found inSupplementary Materials and Methods. Antibodies used forChIP were anti-menin (Bethyl, A300-105A), anti-PRMT5(Abcam, ab31751), anti-Histone H4 Symmetric Di-MethylR3 (Abcam, ab5823), and anti-Histone H3 (ab1791).

MiceAll laboratorymiceweremaintained on a 12-hour light–dark

cycle in the animal facility at the University of Pennsylvania(Philadelphia, PA). All experiments on mice in our researchprotocol were approved by the Institutional Animal Care andUse Committee of the University of Pennsylvania and werecarried out in accordance with relevant Institutional andNational guidelines and regulations. Men1l/l;CreER, Men1l/l;RipCre, and Men1l/l;Pdx1CreER mice were generated asdescribed previously (24). Genotyping of mice was conductedby PCR on mouse-tail DNA.

Excision of the floxed Men1 gene using tamoxifenMen1l/l;Cre-ER and Men1l/l;pdx1Cre-ER and their littermate

controls were fed tamoxifen (TAM; MP Biomedicals) at

200 mg/kg of body weight per day for 2 consecutive days,followed by 1 day off and then for another 2 consecutive days asdescribed previously (20).

Physiologic measurementsGDC-0449 was obtained from ChemieTek. Blood glucose

levels were assayed from tail vein blood by a glucose meter(OneTouch, Lifescan). Blood serum insulin levels were mea-sured by ELISA using a Mouse Insulin Kit (Crystal Chem).

Detection of mRNA levels in islets of mice pancreasIslets were individually isolated from mouse pancreas

by collagenase digestion and separated using a Ficollgradient as previously described (11). The islets weredigested in TRIzol, and RNA was extracted using a RNeasyMini Kit (Invitrogen). A total of 50 ng RNA was reversetranscribed into cDNA using the Omniscript RT Kit fromQiagen.

ImmunofluorescenceImages were captured using a Nikon Eclipse E800 fluoreses-

cence microscope equipped with a CCD digital camera. Totalinsulin staining area was quantified using Metamorph soft-ware (Molecular Devices Corporation). Antibodies used forimmunostaining were insulin (Abcam, ab7842) and bromo-deoxyuridine (BrdUrd; Accurate Chemical and Scientific,OBT0030G). Secondary antibodies used were fluorescein iso-thiocynate (Abcam, ab6904) and Alexa fluor 546 (Invitrogen,A11035).

PRMT5 and menin-binding assayHis-Menin (in pET28a vector) andGST-PRMT5 (frompGEX-

4T1 vector) were expressed in BL21 Codon Plus (Stratagene).Details and antibodies used can be found in SupplementaryMaterials and Methods.

Histone methyltransferase assayCells were lysed in Tween-20 buffer [50 mmol/L Hepes-

KOH, pH 8, 150 mmol/L NaCl, 2.5 mmol/L EGTA, 1 mmol/LEDTA, 0.1% Tween 20, 1 mmol/L phenylmethylsulfonyl-fluoride (PMSF), and 1 mmol/L dithiothreitol ) supplemen-ted with a cocktail of protease inhibitors, and menin com-plexes were collected using anti-Flag M2 beads (Sigma).Beads were washed in Tween-20 buffer, and then incubatedwith 2.5 mCi S-adenosyl-L-(methyl-3H) methionine (SAM;Amersham Pharmacia) and 1 mg recombinant Histone H4(NEB) in a total volume of 25 mL of methyltransferasebuffer [50 mmol/L Tris-HCl (pH 8.0), 50 mmol/L NaCl, and1 mmol/L PMSF] for 2 hours at 30�C. The reaction mixturewas resolved on SDS-PAGE, and the gel was soaked inAmersham Amplify solution (GE Healthcare) for 30 min-utes, and subsequently exposed to film at �80�C forautoradiography.

Statistical analysesStatistical analyses were conducted by using Graphpad

Prism (version 5.0; Graphpad Software). The data are presentedas the mean� SD of n determinations unless noted otherwise.

Epigenetic Repression of Hedgehog Signaling by Menin

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A 2-tailed Student t test was used for measuring statisticaldifferences.

ResultsMenin represses expression of Gas1, a crucial cofactorfor Hedgehog signaling

Our previous microarray analysis showed that Men1 exci-sion in MEFs upregulated expression of Gas1 (21), and recentreports show that GAS1 plays a crucial role in enhancingHedgehog signaling in cultured cells and during embryonicdevelopment (13–15).We found that ectopicmenin expressionin menin-null MEFs reduced the mRNA and protein levels ofGAS1 (Fig. 1A). Quantitative real-time PCR (qRT-PCR) showedthat complementing the menin-null cells with menin sup-pressed Sonic Hedgehog ligand (Shh)-induced expression ofGli1 (Fig. 1B), an effector of Hedgehog signaling (17). Ourresults suggest that menin suppresses Hedgehog signaling.

Menin directly interacts with PRMT5, a repressivehistone H4 arginine 3 methyltransferase, and coeluteswith the PRMT5 complex

As menin interacts with proteins like MLL to upregulategene expression (11, 25), we sought to identify transcriptionalrepressive partner(s) of menin that may downregulate theexpression of Gas1. Ion-exchange chromatography showedthat the majority of menin from nuclear extracts eluted in therange of 200 to 300mmol/L NaCl (Fig. 2A). Themenin-contain-ing fractions were subjected to affinity purification with anti-Flag M2-conjugated beads. Menin-interacting proteins wereeluted and separated by gel electrophoresis, followed by silverstaining (Fig. 2B). Mass spectrometry analysis of the purifiedproteins showed that PRMT5 and its associated factor MEP50were among the major eluted proteins (Fig. 2B). PRMT5symmetrically dimethylates protein arginine residues, such ashistone H4 arginine 3 (H4R3) and histone H3 arginine 8,resulting in repression of gene expression (26, 27).

Reciprocal coimmunoprecipitation using lysates fromHEK293 cells ectopically expressing Flag-tagged menin, fol-lowed byWestern blotting, confirmed the interaction between

menin and PRMT5 (Fig. 2C). Furthermore, MEP50 was alsodetected in the anti-menin immunoprecipitate (Fig. 2C).Importantly, in HEK293 cells expressing only endogenousmenin and PRMT5, immunoprecipitation with 2 independentmenin antibodies effectively pulled down PRMT5 (Fig. 2D).Similarly, immunoprecipitation with anti-PRMT5 antibodypulled down menin (Fig. 2E), showing interaction of theendogenous proteins. Gel filtration chromatography showedthat although menin eluted at several peaks, the fractionseluting at approximately 500 kDa contained PRMT5 andMEP50 (Fig. 2F, Lanes 5–6), suggesting that menin/PRMT5/MEP50 coexist in a complex. Glutathione S-transferase (GST)pulldown with proteins expressed and purified from Escher-ichia coli showed that GST-PRMT5 directly interacted withHis-tagged menin (Fig. 2G). Further analysis of GST-PRMT5fragments of various lengths (Supplementary Fig. S1A) showedthat amino acid residues 1-210 bound to menin (Supplemen-tary Fig. S1B, lane 3). Deletional analysis of PRMT5 fragment 1-210 indicated that amino acid 1-93 was sufficient for binding tomenin (Supplementary Fig. S1C, lane 4). A biotinylated PRMT5peptide (amino acid 23-51 of PRMT5) conjugated to avidinbeads was sufficient for pulling down menin (Fig. 2H), indi-cating that menin binds to the N-terminus of PRMT5.

Menin pulls down PRMT5-associated histone H4methyltransferase activity

To test whether immunoprecipitated menin pulls downhistone methylating activity mediated by PRMT5, 293T cellswere transfected withMEN1 and/or PRMT5 cDNA, followed byimmunoprecipitation with an anti-menin antibody. Histonemethyltransferase (HMT) assay with the immunoprecipitatedcomplex, 3H-SAM, and recombinant histone H4 showed thatmenin pulled down a histone H4 methylating activity, and thisactivity was enhanced further when both menin and PRMT5were ectopically expressed (Fig. 2I, lane 4 vs. 3), thus indicatingthat menin-associating PRMT5 is enzymatically functional.As controls, menin and PRMT5 were expressed as expected(Fig. 2J).

Microarray analysis reveals common targets of meninand PRMT5 including Gas1, and shows correlation withthe Hedgehog pathway signature

We conducted cDNA microarray analysis with primary pan-creatic islets from control Men1l/l or Men1l/l mice expressing aTAM-inducible Cre under control of the ubiquitous Ubc9 pro-moter (Men1l/l;Ubc9CreER) 14 days after Men1 excision (24).Excision of Men1 in pancreatic islet cells was confirmed bycoimmunostaining for menin and insulin (Supplementary Fig.S2). Gene set enrichment analysis (GSEA; ref. 28) of genesenriched uponMen1 excision in pancreatic islets, in an unbiasedsearch, matched a group of genes upregulated in the Prmt5-knockdown NIH3T3 subset including Gas1 ref. (27; Supplemen-tary Fig. S3A). Moreover, the leading-edge subset of the associ-ated significant gene sets included Gas1 (Supplementary Fig.S3A). Because GAS1 is actively involved in promoting propro-liferative Hedgehog signaling, these findings suggest that meninand PRMT5 functionally interact to suppress a common set ofgenes including Gas1 to modulate Hedgehog signaling.

Figure 1. Menin regulates GAS1 and Hedgehog signaling. A, qRT-PCRand immunoblotting showing expression of Gas1 mRNA and protein inmenin-null MEFs complemented with either vector or wild-type menin.Ponceau S is included as a loading control. B, qRT-PCR showingexpression of Gli1 mRNA in menin-null MEFs complemented with eithervector or wild-type menin cultured in varying concentrations of Shh-conditioned medium (Shh-CM). Error bars indicate � SD.

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Ablation of all 3 Hedgehog coreceptors, GAS1, CDO, andBOC abrogates Shh-dependant neural progenitors (14), and acomplete loss of Hedgehog-dependant cerebellar granule neu-ron precursors (CGNP) proliferation (13). Knockdown of Gas1in MEFs inhibits Shh-induced gene expression (15). From ourmicroarray analysis of isolated islets from control andMen1D/D

mice, GSEA indicated that there was a modest increase in theHedgehog signaling gene set (Supplementary Fig. S3B). Thesefindings suggest that menin may effectively dampen, but notcompletely block Hedgehog signaling.

PRMT5 is required for suppressing Gas1 expression, andfor optimal histone H4 arginine 3 symmetricdimethylation at the Gas1 promoterWe knocked down Prmt5 in MEFs using short hairpin

RNAs (shRNA), and qRT-PCR showed that 2 independentshRNA clones reduced expression of Prmt5 (Fig. 3A), andmoderately increased the mRNA levels of Gas1 (Fig. 3B).Consistently, reduced PRMT5 expression correlated with

increased expression of GAS1 at the protein level, as shownby Western blotting (Fig. 3C). To determine the impact ofPrmt5 knockdown on histone H4 arginine 3 symmetricdimethylation (H4R3m2s), catalyzed by PRMT5 (27), at theGas1 promoter, we conducted ChIP assay and found thatPRMT5 bound to the Gas1 promoter, and PRMT5 knock-down moderately reduced PRMT5 binding (Fig. 3D). Con-sistently, PRMT5 knockdown also reduced H4R3m2s levelsat the Gas1 promoter (Fig. 3D and Supplementary Fig. S4A).As a control, no difference in binding was observed at theactin promoter (Supplementary Fig. S4B). We cultured bothcontrol and PRMT5 knockdown cells in the presence orabsence of Shh ligand and found that PRMT5 knockdownmarkedly increased Shh-induced expression of Gli1 andPtch1 (Fig. 3E and F), both targets of Hedgehog signaling(17). Together, these results suggest that PRMT5 plays acrucial role in suppressing Hedgehog signaling, at least inpart by repressing GAS1 and increasing the repressiveH4R3m2s mark at the promoter.

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Figure2. Menin interacts directlywithPRMT5 tomethylatehistoneH4.A, immunoblottingofmenin innuclear extract fromHEK293cells expressingFlag-meninfractionated by anion exchange chromatography using a Q-Sepharose column eluted with varying concentrations of NaCl. B, silver stainingofmenin-containing fractions after affinity purification using anti-FlagM2beads. Visible bandswere excised for identificationbymass spectrometry. �, PRMT5peptide fragments identified by mass spectroscopy; 77DWNTLIVGK, 228AAILPTSIFLTNKK, 241KGFPVLSK, 334YSQYQQAIYK, 369GPLVNASLR. C,immunoblotting showingmenin (top), PRMT5, andMEP50 (bottom) in nuclear extract of HEK293 cells ectopically expressing Flag-menin immunoprecipitatedwith indicated antibodies. D and E, endogenous interaction between menin and PRMT5 in HEK293 cells immunoprecipitated with 2 independent anti-menin(D) or anti-PRMT5 antibodies (E). F, immunoblotting for menin, PRMT5, andMEP50 in gel filtration chromatography fractions of nuclear extract from HEK293cells ectopically expressing Flag-menin. G, immunoblotting ofHis-menin expressed inE. coli immunoprecipitated byGST-PRMT5 immobilized to glutathionebeads. H, biotinylated PRMT5 peptides of various lengths were immobilized on streptavidin beads, and the binding of His-menin was identified byimmunoblotting. I, HMT assay using anti-menin immunoprecipitates from 293T cells transfected with Flag-menin and/or Myc-PRMT5 incubated with3H-AdoMet andhistoneH4.Autoradiography for 3H-methylated histoneH4 (middle); Coomassie stain ofH4 is includedasa loading control. J, immunoblottingof menin and PRMT5 in cells used for HMT assay in I. Ponceau S is included as a loading control. IgG, immunoglobulin G.






Menin binds to the promoter ofGas1 and recruits PRMT5to increase H4R3m2s

To determine whether menin directly regulates expressionof Gas1, we conducted ChIP assay with menin-expressing ormenin-null cells, and showed that menin bound to the Gas1promoter (Fig. 4A). Consistently, PRMT5 binding andH4R3m2s at the Gas1 promoter were also markedly reducedin Men1-null cells (Fig. 4A), indicating that menin recruitsPRMT5 to methylate H4R3 at this target gene.

To determine whether MEN1 disease-related menin pointmutations affect Gas1 expression, we complemented menin-null MEFs with either wild type or mutant forms of menin, andshowed that wild type and mutant menin were expressed atcomparable levels based on Western blotting (Fig. 4B). How-ever, although wild-type menin expression reduced Gas1mRNA levels, mutant menin lost or partially lost their abilityto repress Gas1 expression (Fig. 4B). ChIP assay showed thatthe menin mutants largely retained their ability to bind to theGas1 promoter, but failed to impart the H4R3m2s mark at thepromoter (Fig. 4C and Supplementary Fig. S4C). As a control,no changes in menin binding or histone H4R3 methylationwere observed at the actin promoter (Supplementary Fig. S4D).

To determine whether the mutations affect the ability ofmenin to interactwith PRMT5,we ectopically expressedmeninand PRMT5 in 293 cells (Supplementary Fig. S5), and observedthat the interaction between menin and PRMT5 was reducedconsiderably in themeninmutants upon comparisonwithwildtype (Fig. 4D, top). Furthermore, HMT assay with the immu-noprecipitated menin complex, 3H-SAM, and recombinant

histone H4 showed that the histone H4 methylating activitywas dramatically reduced in the mutant menin immune com-plexes compared with wild-type menin (Fig. 4D, third). Col-lectively, these results suggest that menin-mediated repressiveH4R3m2s at the Gas1 promoter plays a role in suppressingMEN1 tumorigenesis.

Men1 excision in primary islets results in increasedHedgehog signaling

To determine whether Men1 is also crucial for regulatingGas1 expression and Hedgehog signaling, we isolated primaryislets from controlMen1l/l andMen1l/l; Rip-cremice (8-months-old), and qRT-PCR showed that Men1 excision resulted inundetectableMen1mRNA levels (Fig. 5A), but increasedmRNAlevels of Gas1 (Fig. 5B), Gli1 (Fig. 5C), and Ptch1 (Fig. 5D),indicative of enhanced Hedgehog signaling. These resultssuggest that menin plays a crucial role in vivo in pancreaticislets in repressing the expressions of Gas1, Gli1, and Ptch1,thereby suppressing Hedgehog signaling.

Treatment ofMen1-excised mice harboring insulinomasby a pharmacologic SMO inhibitor reduces proliferationof insulinoma cells and inhibits Hedgehog signaling

Effective treatment for MEN1 tumors is not yet available.A Hedgehog signaling inhibitor, GDC-0449, is proven safeand effective for treating human BCC and medulloblastomas(29). Because the mouse MEN1 tumor model largely phe-nocopies the human MEN1 tumor syndrome (30, 31), wetreated 8-month-old Men1l/l; Rip-cre mice, which developed

Figure 3. Loss of PRMT5 results in elevated Gas1 levels and enhanced Hedgehog signaling. A, qRT-PCR showing reduction of Prmt5 mRNA in MEFsexpressing 2 independent Prmt5-targeting shRNA clones compared with MEFs expressing scrambled control shRNA. B, qRT-PCR showingexpression of Gas1 mRNA levels in MEFs expressing shRNA targeting Prmt5. C, immunoblotting for PRMT5 and GAS1 in MEFs expressing eithercontrol or Prmt5-targeting shRNAs. Ponceau S is included as a loading control. D, ChIP with antibodies against PRMT5 and H4R3m2s at the Gas1promoter. ChIP amplicon,Gas1 (�780 bp/�609 bp). E and F,MEFs expressing control or 2 independent shRNA clones targetingPrmt5were cultured in eithercontrol or Shh-conditioned medium (Shh-CM) and Gli1 (E) and Ptch1 (F) mRNA levels were quantitated by qRT-PCR. Error bars indicate � SD. IgG,immunoglobulin G.

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insulinomas, with either control vehicle or GDC-0449 for4 weeks (Fig. 6A), and found that GDC-0449 treatmentsignificantly reduced the number of BrdUrd-positivecells in insulinomas (Fig. 6B and C), by approximately60% (Fig. 6D). The marked impact of inhibiting Hedgehogsignaling on suppressing neuroendocrine tumors is under-scored by the common practice of using the mitotic index(proliferation) as a common parameter for grading andprognosis of human neuroendocrine tumors (32). GDC-0449 treatment did not affect the area of the insulinoma(Supplementary Fig. S6A), likely because of the limitedduration of the treatment and/or relative slow growth ofneuroendocrine tumors. However, the treatment reducedthe level of blood insulin, an indicator of secreted insulinfrom insulinomas (Supplementary Fig. S6B). This is consis-tent with the reported observation that mutation of SMO inadult mice reduces the production of insulin in islets (33).Moreover, qRT-PCR showed that expression of Ptch1, a

Hedgehog signaling target, was reduced in isolated isletsfromMen1l/l; pdxCreERmice treated with GDC-0449 (Fig. 6E)compared with vehicle-treated mice, indicating that GDC-0449 was effective in suppressing Hedgehog signaling. Wecannot rule out that the SMO inhibitor also, at least, partiallyinhibits Hedgehog signaling from stromal cells surroundingthe Men1-deleted tumor cells that act in a paracrine fashion.Nevertheless, our findings clearly indicate that GDC-0449effectively suppresses proliferation of MEN1 tumor cells invivo, link menin to suppression of Hedgehog, and uncoversthe Hedgehog signaling pathway as a promising target forimproving therapy against neuroendocrine tumors.

DiscussionAlthoughmenin regulates cell-cycle genes and represses cell

proliferation (34), it has been underexplored as to what sig-naling pathways are dysregulated by menin mutations. Ourfindings suggest that normally menin "gates" the Hedgehog

Figure 4. Menin recruits PRMT5 and its associated histone modification mark, H4R3m2s, to the Gas1 promoter. A, ChIP with antibodies against menin,PRMT5, and H4R3m2s at the Gas1 promoter in Men1-null MEFs complemented with either empty vector or wild-type (WT) menin. ChIP amplicon,Gas1 (�780 bp/�609 bp). B, qRT-PCR showing expression of Gas1 mRNA in menin-null MEFs complemented with empty vector, WT menin,or MEN1 disease-related mutants L22R, and A242V. Levels of ectopic WT and mutant menin expression are shown, and immunoblotting for b actin isincluded as a loading control. C, ChIP with antibodies against menin and H4R3m2s at the Gas1 promoter in Men1-null MEFs complemented with vector,WT menin, or MEN1 disease-related mutants L22R and A242V. ChIP amplicon, Gas1 (�780 bp/�609 bp). D, coimmunoprecipitation of PRMT5in menin immunoprecipitates from 293T cells ectopically expressing PRMT5 and either WT or MEN1 disease-related menin mutants (top). Anti-meninimmunoprecipitates from cells above were incubated with 3H-AdoMet and histone H4 for HMT assay, and 3H-methylated histone H4 was detected byautoradiography (third). Coomassie stain of H4 is included as a loading control (bottom). Error bars indicate � SD. IgG, immunoglobulin G.






signaling threshold, at least partly, by repressing Gas1 toprevent overt activation of Hedgehog signaling in islet cells.Men1 mutations in some islet cells may stochastically triggerovert Hedgehog signaling, which can induce expression ofproproliferative genes such as cyclin D1 (35), thereby increas-ing cell proliferation. Consistently, expression of Gli1 andPtch1, an indicator of activated Hedgehog signaling, wasincreased in primary islets from Men1-excised mice. The link

between menin and Hedgehog signaling represents a newmechanism for menin-mediated tumor suppression (Fig. 6F).

We found that menin directly interacts with PRMT5, whichcatalyzes repressive H4R3m2s (27). Both menin and PRMT5directly bound to the promoter of Gas1, a component of theHedgehog signaling pathway. Menin is crucial for recruitmentof PRMT5 to and for effective methylation of H4R3 at the Gas1promoter (Fig. 4A), a novel means for menin-mediated repres-sion of gene expression. However, it must be noted that itremains unclear howmenin is recruited to the Gas1 promoter,and whether menin directly binds the Gas1 promoter DNA torepress Gas1 expression. In addition to PRMT5, the repressivehistone H4R3m2s mark is catalyzed by another type II PRMTenzyme, PRMT7 (36, 37). However, we found that only PRMT5was detected in the menin immunoprecipitates, whereasPRMT7 did not coimmunoprecipitate with menin (Supple-mentary Fig. S7). This clearly indicates that the menin-depen-dant histone H4R3m2s mark at the Gas1 promoter can beattributed to PRMT5, and not PRMT7.

Our data show that menin immunoprecipitates possessHMT activity toward recombinant histone H4, and that thisactivity is lost in certain disease-related Men1 mutationsresulting from compromised interaction with PRMT5 (Fig.4D). It has previously been reported that menin immunopre-cipitates have HMT activity toward histone H3 but not histoneH4 (25), whereas HMT activity toward recombinant histoneH4was clearly observed in our studies. It is possible that theimmunoprecipitate conditions used by Hughes and colleaguespreferentially retain MLL activity that methylates H3K4. Alter-natively, the MLL enzymatic activity toward histone H3 mightbe more robust than the PRMT5 activity toward histone H4 inthe menin immunoprecipitates. This would result in prefer-ential methylation of histone H3 when a mixture of corehistones is used. In contrast, purified recombinant histoneH4 alone was used in our study, thus favoring observation ofhistone H4 methylation. Nevertheless, our data clearly show

Figure 5. Loss of Men1 correlates with activated Hedgehog signaling inMen1-null mice. A–D, pancreatic islets were isolated from 8-month-oldMen1l/l;RipCre and control Men1l/l mice (n ¼ 4 mice) and qRT-PCR wasused to quantitate the mRNA levels of Men1, P < 0.0001 (A), Gas1, P <0.0044 (B), Gli1, P ¼ 0.0538 (C), and Ptch1, P ¼ 0.0258 (D). Error barsindicate � SD.

Figure 6. Inhibition of Hedgehogsignaling in Men1-excised miceresults in decreased islet cellproliferation. A, scheme forinhibition of the Hedgehogpathway with GDC-0449 inMen1-excised mice. B and C,immunofluorescence for BrdUrdand insulin in pancreas of Men1-excised mice gavaged with eithervehicle (B) or GDC-0449 (C) for 4weeks at a doseof 100mg/kg twicedaily. D, quantitation of BrdUrdincorporation in islets of miceabove (same as B and C). E,qRT-PCR for Ptch1mRNA in isletsofMen1l/l;pdxCreERmice gavagedfor 10 days with either vehicle orGDC-0449 at a dose of 100 mg/kgtwice daily, P ¼ 0.0010. F, a modelfor menin-PRMT5–mediatedinhibition of Hedgehog signalingthrough epigenetic regulation ofGAS1. Error bars indicate � SD.

Gurung et al.





that menin interacts with PRMT5, and the associated histonearginine methylation activity is necessary for repressing Gas1and the canonical Hedgehog signaling pathway.Although multiple factors have been previously reported to

affect Hedgehog signaling, such as PKA-mediated phosphor-ylation of GLI proteins and proteolysis of GLI proteins (16), ithas been poorly understood as to whether and how epigeneticfactors affect Hedgehog signaling. We found that both meninand PRMT5 are crucial for PRMT5-mediated H4R3m2s at theGas1 promoter. PRMT5-mediated H4R3m2s suppresses geneexpression of b-globin genes in hematopoietic cells partlythrough recruiting DNMT3A via increasing repressive markH4R3m2s at the promoter to silence the gene by promoterDNAmethylation (38). Our results reveal the PRMT5-mediatedepigenetic mechanism in repressing Hedgehog signaling andsuppression of tumorigenesis.Our studies involving MEN1 disease-related menin mutants

showed that certain point mutants failed to suppress theexpression of Gas1 (Figs. 4B and 4C), suggesting that menin-mediated suppression of Gas1 and canonical Hedgehog signal-ing may be relevant to its function in suppressing MEN1 tumorsyndrome. Furthermore, inhibition of Hedgehog signaling withthe SMO inhibitor GDC-0449 markedly reduces tumor cellproliferation in a MEN1 mouse model (Fig. 6D), indicating thatcanonical Hedgehog signaling plays a significant role in prolif-eration of Men1-excised islet cells. It is noteworthy that GDC-0449 inhibition of SMO may have additional effects besidesrestoring the role of menin in repressing Gas1, as inhibition ofHedgehog signaling in a transgenic mouse model in which theartificial Rip-driven SV40 T antigen promotes development ofinsulinoma, represses growth of the transgene-induced islettumors (39). However, we found, for the first time, that Men1mutation inb cells in vivo leads toenhancedHedgehog signaling,which may contribute to increasing b-cell proliferation.The normal role of Hedgehog signaling in regulating pan-

creatic islets andb cells is complex, influenced by temporal andspatial factors (33). Hedgehog ligand stimulates production ofinsulin in cultured b cells (40), whereas knockout of SMO inpancreatic epithelial cells initially reduces the size of isletsduring embryonic development and decreases total insulinproduction in adult mice (33). Hedgehog ligand expression orHedgehog signaling is also increased in human gastrointestinalneuroendocrine tumors or in mouse small cell lung cancer (41,42). Our results show that Men1 ablation leads to increase inexpression of Hedgehog target genes in pancreatic islets, andthe SMO inhibitor suppresses proliferation of MEN1 tumors.However, MEN1 deletion in mice does not result in the typicalHedgehog pathway-related tumors such as medulloblastoma.

These findings are consistent with the notion that menin/PRMT5 modulates (but does not completely block) Hedgehogsignaling partly through reducing Gas1 expression. It is likelythatmenin does so in endocrine cells likeb cells becausemeninis more abundantly expressed in b cells (24). Similarly, it hasbeen shown that Men1 excision in the liver has no affect onproliferation (43), thus highlighting the tissue-specific effect ofMen1 ablation. Although PRMT5 can promote tumorigenesisin multiple tissues via regulating various partners such as p53(44), cyclin D1 (45), and Janus-activated kinase-2 (46), it maypartner with menin to dampen Hedgehog signaling in certainendocrine cells. In aggregate, our findings, for the first time,reveal that MEN1 mutation leads to enhanced Hedgehogsignaling via a previously unknown epigenetic mechanism,and pharmacologic inhibition of this pathway significantlyreduces proliferation of Men1-mutated pancreatic tumors inmice. As more than 40% of pancreatic neuroendocrine tumorsharbor somatic mutations in the MEN1 gene (47), Hedgehogantagonists may be valuable to improve therapy for neuroen-docrine tumors with MEN1 mutation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: B. Gurung, C.-M. Fan, J.M.Y. Ng, X. HuaDevelopment ofmethodology: B. Gurung, Z. Feng, J.M.Y. Ng, T. Curran, X. HuaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):B.Gurung, D.V. Iwamoto, A. Thiel, J.M.Y. Ng, T. CurranAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):B. Gurung, Z. Feng, D.V. Iwamoto, A. Thiel, G. Jin, J.M.Y. Ng, T. CurranWriting, review, and/or revision of the manuscript: B. Gurung, D.V.Iwamoto, C.-M. Fan, J.M.Y. Ng, T. Curran, X. HuaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Z. Feng, C.-M. FanStudy supervision: T. Curran, X. Hua

AcknowledgmentsThe authors thank Dr. J.A. Diehl for myc-PRMT5 plasmid construct and

stimulating discussions, Dr. Chaoxing Yuan at Penn Proteomics facility for massspectrometry analysis, Dr. Allen Bale and their colleagues for critically readingthe manuscript, and Shivani Sethi for editing the manuscript.

Grant SupportThis work was supported in part by grants from the NIH (R01-CA-113962 and

R01-DK085121 to X. Hua, R01 DK084963 to C.-M. Fan) and Caring for CarcinoidFoundation-AACR Grant Care for Carcinoid Foundation (11-60-33; to X. Hua).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received August 13, 2012; revised January 17, 2013; accepted January 29, 2013;published OnlineFirst April 10, 2013.

References1. Libe R, Bertherat J. Molecular genetics of adrenocortical tumours,

from familial to sporadic diseases. Eur J Endocrinol 2005;153:477–87.2. BurgessJR,NordB,DavidR,GreenawayTM,ParameswaranV,Larsson

C, et al. Phenotype and phenocopy: the relationship between genotypeand clinical phenotype in a single large family with multiple endocrineneoplasia type 1 (MEN 1). Clin Endocrinol (Oxf) 2000;53:205–11.

3. Farrell WE, Azevedo MF, Batista DL, Smith A, Bourdeau I, Horvath A,et al. Unique gene expression profile associated with an early-onset

multiple endocrine neoplasia (MEN1)-associated pituitary adenoma. JClin Endocrinol Metab 2011;96:E1905–14.

4. Marx SJ. Molecular genetics of multiple endocrine neoplasia types 1and 2. Nat Rev Cancer 2005;5:367–75.

5. Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE,Collins FS, Emmert-Buck MR, et al. Positional cloning of the genefor multiple endocrine neoplasia-type 1. Science 1997;276:404–7.






6. Lemmens I, Van de Ven WJ, Kas K, Zhang CX, Giraud S, Wautot V,et al. Identification of the multiple endocrine neoplasia type 1(MEN1) gene. The European Consortium on MEN1. Hum Mol Genet1997;6:1177–83.

7. Balogh K, Racz K, Patocs A, Hunyady L. Menin and its interactingproteins: elucidation of menin function. Trends Endocrinol Metab2006;17:357–64.

8. Yang Y, Hua X. In search of tumor suppressing functions ofmenin. MolCell Endocrinol 2007;265–266:34–41.

9. Baylin SB, Jones PA. A decade of exploring the cancer epigenome -biological and translational implications. Nat Rev Cancer 2011;11:726–34.

10. Milne TA, Hughes CM, Lloyd R, Yang Z, Rozenblatt-Rosen O, Dou Y,et al.Menin andMLLcooperatively regulate expressionof cyclin-depen-dent kinase inhibitors. Proc Natl Acad Sci U S A 2005;102:749–54.

11. Karnik SK, Hughes CM, Gu X, Rozenblatt-Rosen O, McLean GW,Xiong Y, et al. Menin regulates pancreatic islet growth by promotinghistone methylation and expression of genes encoding p27Kip1 andp18INK4c. Proc Natl Acad Sci U S A 2005;102:14659–64.

12. Jiang J, Hui CC. Hedgehog signaling in development and cancer. DevCell 2008;15:801–12.

13. Izzi Luisa CF. Boc and Gas1 each form distinct Shh receptor com-plexes with Ptch1 and are required for Shh-mediated cell proliferation.Dev Cell 2011;20:788–801.

14. Allen BL, Song JY, Izzi L, Althaus IW, Kang JS, Charron F, et al.Overlapping roles and collective requirement for the coreceptorsGAS1, CDO, and BOC in SHH pathway function. Dev Cell 2011;20:775–87.

15. Martinelli DC, Fan CM.Gas1 extends the range of Hedgehog action byfacilitating its signaling. Genes Dev 2007;21:1231–43.

16. Wang Y, McMahon AP, Allen BL. Shifting paradigms in Hedgehogsignaling. Curr Opin Cell Biol 2007;19:159–65.

17. Katoh Y, Katoh M. Hedgehog target genes: mechanisms of carcino-genesis induced by aberrant hedgehog signaling activation. Curr MolMed 2009;9:873–86.

18. Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley S,Chidambaram A, et al. Mutations of the human homolog ofDrosophilapatched in the nevoid basal cell carcinoma syndrome. Cell 1996;85:841–51.

19. Taipale J, Chen JK, CooperMK,WangB,Mann RK,Milenkovic L, et al.Effects of oncogenic mutations in Smoothened and Patched can bereversed by cyclopamine. Nature 2000;406:1005–9.

20. Schnepp RW, Chen YX, Wang H, Cash T, Silva A, Diehl JA, et al.Mutation of tumor suppressor gene Men1 acutely enhances prolifer-ation of pancreatic islet cells. Cancer Res 2006;66:5707–15.

21. La P, Schnepp RW, C DP, A CS, Hua X. Tumor suppressor meninregulates expression of insulin-like growth factor binding protein 2.Endocrinology 2004;145:3443–50.

22. Lee CS, Buttitta L, Fan CM. Evidence that the WNT-induciblegrowth arrest-specific gene 1 encodes an antagonist of sonichedgehog signaling in the somite. Proc Natl Acad Sci U S A2001;98:11347–52.

23. Chen YX, Yan J, Keeshan K, Tubbs AT, Wang H, Silva A, et al. Thetumor suppressor menin regulates hematopoiesis and myeloid trans-formation by influencing Hox gene expression. Proc Natl Acad SciU S A 2006;103:1018–23.

24. Yang Y, Gurung B, Wu T, Wang H, Stoffers DA, Hua X. Reversalof preexisting hyperglycemia in diabetic mice by acute deletionof the Men1 gene. Proc Natl Acad Sci U S A 2010;107:20358–63.

25. Hughes CM, Rozenblatt-Rosen O, Milne TA, Copeland TD, Levine SS,Lee JC, et al. Menin associates with a trithorax family histone methyl-transferase complex and with the hoxc8 locus. Mol Cell 2004;13:587–97.

26. FriesenWJ,Wyce A, Paushkin S, Abel L, Rappsilber J, MannM, et al. Anovel WD repeat protein component of the methylosome binds Smproteins. J Biol Chem 2002;277:8243–7.

27. Pal S, Vishwanath SN, Erdjument-BromageH, Tempst P, Sif S. HumanSWI/SNF-associated PRMT5 methylates histone H3 arginine 8 andnegatively regulates expression of ST7 and NM23 tumor suppressorgenes. Mol Cell Biol 2004;24:9630–45.

28. SubramanianA, TamayoP,Mootha VK,Mukherjee S, Ebert BL,GilletteMA, et al. Gene set enrichment analysis: a knowledge-based approachfor interpreting genome-wide expression profiles. Proc Natl Acad SciU S A 2005;102:15545–50.

29. Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA, Fu L, et al.Treatment ofmedulloblastomawith hedgehog pathway inhibitorGDC-0449. N Engl J Med 2009;361:1173–8.

30. Bertolino P, Tong WM, Herrera PL, Casse H, Zhang CX, Wang ZQ.Pancreatic beta-cell-specific ablation of the multiple endocrine neo-plasia type 1 (MEN1) gene causes full penetrance of insulinomadevelopment in mice. Cancer Res 2003;63:4836–41.

31. Crabtree JS, Scacheri PC,WardJM,McNally SR,SwainGP,MontagnaC, et al. Of mice and MEN1: Insulinomas in a conditional mouseknockout. Mol Cell Biol 2003;23:6075–85.

32. Strosberg J, Nasir A, Coppola D,WickM, Kvols L. Correlation betweengrade and prognosis in metastatic gastroenteropancreatic neuroen-docrine tumors. Hum Pathol 2009;40:1262–8.

33. Lau J,HebrokM.Hedgehog signaling in pancreas epithelium regulatesembryonic organ formation and adult beta-cell function. Diabetes2010;59:1211–21.

34. Wu T, Zhang X, Huang X, Yang Y, Hua X. Regulation of cyclin B2expression and cell cycle G2/m transition by menin. J Biol Chem2010;285:18291–300.

35. Kenney AM, Rowitch DH. Sonic hedgehog promotes G(1) cyclinexpression and sustained cell cycle progression in mammalian neu-ronal precursors. Mol Cell Biol 2000;20:9055–67.

36. Lee JH, Cook JR, Yang ZH, Mirochnitchenko O, Gunderson SI, FelixAM, et al. PRMT7, a new protein arginine methyltransferase thatsynthesizes symmetric dimethylarginine. J Biol Chem 2005;280:3656–64.

37. Karkhanis V, Wang L, Tae S, Hu YJ, Imbalzano AN, Sif S. Proteinarginine methyltransferase 7 regulates cellular response to DNA dam-age by methylating promoter histones H2A and H4 of the polymerasedelta catalytic subunit gene, POLD1. J Biol Chem2012;287:29801–14.

38. Zhao Q, Rank G, Tan YT, Li H, Moritz RL, Simpson RJ, et al. PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, couplinghistone and DNA methylation in gene silencing. Nat Struct Mol Biol2009;16:304–11.

39. Fendrich V, Wiese D, Waldmann J, Lauth M, Heverhagen AE, RehmJ, et al. Hedgehog inhibition with the orally bioavailable Smoantagonist LDE225 represses tumor growth and prolongs survivalin a transgenic mouse model of islet cell neoplasms. Ann Surg2011;254:818–23.

40. Thomas MK, Rastalsky N, Lee JH, Habener JF. Hedgehog signalingregulation of insulin production by pancreatic beta-cells. Diabetes2000;49:2039–47.

41. Shida T, FuruyaM,Nikaido T, HasegawaM,KodaK,OdaK, et al. SonicHedgehog-Gli1 signaling pathway might become an effective thera-peutic target in gastrointestinal neuroendocrine carcinomas. CancerBiol Ther 2006;5:1530–8.

42. Fendrich V, Waldmann J, Esni F, Ramaswamy A, Mullendore M,Buchholz M, et al. Snail and Sonic Hedgehog activation in neuroen-docrine tumors of the ileum. Endocr Relat Cancer 2007;14:865–74.

43. Scacheri PC, Crabtree JS, Kennedy AL, Swain GP, Ward JM, MarxSJ, et al. Homozygous loss of menin is well tolerated in liver, a tissuenot affected in MEN1. Mamm Genome 2004;15:872–7.

44. Durant ST, ChoEC, LaThangueNB. p53methylation–theArg-ument isclear. Cell Cycle 2009;8:801–2.

45. Aggarwal P, Vaites LP, KimJK,Mellert H,GurungB,NakagawaH, et al.Nuclear cyclin D1/CDK4 kinase regulates CUL4 expression and trig-gers neoplastic growth via activation of the PRMT5methyltransferase.Cancer Cell 2010;18:329–40.

46. Liu F, Zhao X, Perna F, Wang L, Koppikar P, Abdel-Wahab O, et al.JAK2V617F-mediated phosphorylation of PRMT5 downregulates itsmethyltransferase activity and promotes myeloproliferation. CancerCell 2011;19:283–94.

47. Jiao Y, Shi C, Edil BH, de Wilde RF, Klimstra DS, Maitra A, et al.DAXX/ATRX, MEN1, and mTOR pathway genes are frequentlyaltered in pancreatic neuroendocrine tumors. Science 2011;331:1199–203.

Gurung et al.





2013;73:2650-2658. Published OnlineFirst April 15, 2013.Cancer Res Buddha Gurung, Zijie Feng, Daniel V. Iwamoto, et al. Tumor SyndromeMenin Epigenetically Represses Hedgehog Signaling in MEN1

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