PAK4PhosphorylatesFumaraseandBlocksTGF Induced Cell … · followed by immunoprecipitation and...

Metabolism and Chemical Biology

PAK4PhosphorylatesFumaraseandBlocksTGFb-Induced Cell Growth Arrest in Lung Cancer CellsTao Chen1, Ting Wang2,3,Wenhua Liang1, Qin Zhao2, Qiujing Yu2, Chun-Min Ma2,Lingang Zhuo2, Dong Guo2, Ke Zheng2, Chengzhi Zhou1, Shupei Wei1,WenhuaHuang1, Juhong Jiang1, Jing Liu1, Shiyue Li1, JianxingHe1,Yuhui Jiang2, andNanshan Zhong1

Abstract

Themetabolic activity of fumarase (FH) participates in genetranscription linking to tumor cell growth. However, whetherthis effect is implicated in lung cancer remains unclear. Here,we show TGFb induces p38-mediated FH phosphorylation atThr 90, which leads to a FH/CSL (also known as RBP-Jk)/p53complex formation and FH accumulation at p21 promoterunder concomitant activation of Notch signaling; in turn,FH inhibits histone H3 Lys 36 demethylation and therebypromotes p21 transcription and cell growth arrest. In addition,FH is massively phosphorylated at the Ser 46 by PAK4 innon–small cell lung cancer (NSCLC) cells, and PAK4-phosphorylated FH binds to 14-3-3, resulting in cytosolicdetention of FH and prohibition of FH/CSL/p53 complex

formation. Physiologically, FH Ser 46 phosphorylation pro-motes tumorigenesis through its suppressive effect on FH Thr90 phosphorylation–mediated cell growth arrest in NSCLCcells and correlates with poor prognosis in patients with lungcancer. Our findings uncover an uncharacterized mechanismunderlying the local effect of FH on TGFb-induced genetranscription, on which the inhibitory effect from PAK4 pro-motes tumorigenesis in lung cancer.

Significance: Fumarase counteracts CSL via its meta-bolic activity to facilitate TGFb-induced cell growtharrest, an effect largely blocked by PAK4-mediated phos-phorylation of fumarase.

IntroductionTGFb is a key regulator of cell fate during embryogenesis

and has also been implicated in tumor metastasis as a potentdriver (1–3). Canonically, TGFb stimulation leads to activation ofsignal transduction through transmembrane type I and type IIserine/threonine kinase receptors (TbRI and TbRII, respectively),which phosphorylates Smad2/Smad3 and promotes their com-plex formation with Smad4 and launches their transcriptionalactivity (4, 5). In addition, TGFb is also able to initiate anumber ofnon-Smad signaling pathways including p38 pathway, PI3Kpathway, and the Ras–ERK–MAPK pathway (6–8). It has beenknown that the axis of ubiquitin ligase TNF-receptor-associated

factor 6 (TRAF6)-TGFb–associated kinase 1 (TAK1) is critical forp38 activation in response to TGFb (6, 9). Functionally, previousfindings demonstrated p38 activation is essential for either TGFb-induced cellular apoptosis or epithelial-to-mesenchymal transi-tion (EMT; refs. 10, 11), although how its multifaceted effects arecoordinated remains elusive.

Crosstalk between Notch and TGFb signaling pathway hasbeen implicated in various physiologic events. TGFb stimula-tion is able to induce expression of Jagged1, which promotesEMT (12), and Notch activation is found to be indispensablefor TGFb-mediated EMT during cardiac development andtumorigenesis (13). HDAC6 has been reported to be requiredfor activation of Notch signaling by TGFb1 during EMT in lungcancer (14). Referring to downstream transcriptional factor,NICD and Smad3 can form physical complex and cooperate todrive the activation of synthetic promoters containing multi-merized CSL- or Smad3-binding sites (15). In terms of p21,p53 plays a positive role in TGFb-induced p21 expression andcell growth arrest at normal condition (16). A recent studyshows that CSL, which is the crucial transcriptional factorunder Notch signaling, blocks cell senescence through itsinhibitory effects on p53-mediated p21 transcription at theepigenetic level (17); this observation implies the regulatoryeffect of CSL on p53 is a potential linkage between TGFb andNotch signaling.

Emerging evidence in recent years indicate that metabolicenzymes, in addition to the basic role in metabolic pathways,also vitally participate in the epigenetic regulation through relatedmetabolic activity (18). Fumarase (FH), which separately distri-butes in mitochondria and cytosol, is responsible for the revers-ible hydration and dehydration of fumarate to malate in the

1State Key Laboratory of Respiratory Diseases; National Clinical Research Centerof Respiratory Diseases; Guangzhou Institute of Respiratory Health; First Affil-iated Hospital of Guangzhou Medical University, Guangzhou Medical University,Guangzhou, Guangdong, P.R. China. 2The Institute of Cell Metabolism andDisease, Shanghai Key Laboratory of Pancreatic Disease, Shanghai GeneralHospital, School ofMedicine, Shanghai JiaotongUniversity, Shanghai, P.R. China.3Department of Pharmacology, College of Basic Medical Sciences, TianjinMedical University, Tianjin, China.

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

T. Chen, T. Wang, and W. Liang contributed equally to this article.

Corresponding Authors: Yuhui Jiang, The Institute of Cell Metabolism andDisease, Shanghai Key Laboratory of Pancreatic Disease, Shanghai 200080,China. Phone: 8602-1377-97983; E-mail: [email protected]; and JianxingHe, [email protected]

doi: 10.1158/0008-5472.CAN-18-2575

�2019 American Association for Cancer Research.

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tricarboxylic acid cycle (19). Fumarate has been known to com-pete with a-ketoglutarate to bind to a-ketoglutarate–dependent dioxygenases that are involved in histone and DNAdemethylation, and thus exerts inhibitory effects (20). FH defi-ciency causes elevation of cellular fumarate level, which has beenimplicated in thedevelopment of renal cancer due to its inhibitoryeffect on TET-regulated DNA demethylation (21). FH is alsoreported to be importantly involved in DNA repair (22, 23) orgene transcription in glucose-deficient conditions (24), in whichchromatin-associated FH precisely regulates H3K36me2 at theregion of DNA breaks or gene promoter by antagonizing a-KG–dependent demethylase KDM2A/B. Given the tight relationshipbetween the functional status of FH and epigenetic regulation, itwould be meaningful to extensively understand the concrete roleof FH in this regard under distinct genetic contexts.

In this study, we first found that TGFb induces the interactionbetween FH and CSL in a manner dependent on Notch signalingactivation and p38-mediated phosphorylation of FH. This inter-action results in the formation of FH–CSL–p53 complex, andfacilitates the recruitment of FH at the promoter of p53-targetedgenep21. Through inhibition ofKDM2Aactivity, fumarate locallyproduced from promoter-associated FH leads to the increase ofH3K36me2 and hence p21 transcription, which eventually relivesthe negative effects of CSL on p53-mediated cell growth arrest.Moreover, we found that FH is largely phosphorylated by PAK4 atSer 46 in lung cancer cells, and 14-3-3 binds to PAK4 phosphor-ylated-FH, thus retaining FH in the cytosol and blocking its effecton CSL–p53 axis.

Materials and MethodsCell culture

Human normal bronchial epithelial cell line HBE and humanNSCLC cell lines A549, NCI-H520, HCC-827, and NCI-H358,were obtained from Shanghai Cell Bank (Shanghai BiologicalSciences, Chinese Academy of Sciences, Shanghai, China) thatwere originally obtained from the ATCC, authenticated by shorttandem repeat profiling within 6 months. Cells were maintainedin RPMI1640 supplemented with 10% FBS.

DNA constructs and mutagenesisThe sequence of Flag-tag of FH in the pcDNA6 vector is located

at the C-terminus of FH. Flag-FH (N) with deletion of 21N-terminal amino acids was constructed by PCR reactions andthe DNA constructs and mutagenesis were described previous-ly (23, 25). pGIPZ human FH shRNA was generated withthe oligonucleotides 50-GGAATTTAGTGGTTATGTT-30 and 50-AAATTGATATAAGCATCCA -30. pGIPZ human CSL shRNA wasgenerated with the oligonucleotide 50-ATAATTTCGCATAG-CTTCC-30 and 50-TCTGATTCATCATCATCCA-30. pGIPZ humanp53 shRNAswere generatedwith the oligonucleotides 50-GAAAC-CACTGGATGGAGAA-30 and 50-TTATCAGCAGTATGATAGC-30.pGIPZ human 14-3-3 zeta shRNAs were generated withthe oligonucleotides 50-ACGGTTCACATTCCATTAT-30 and 50-AGAACACAGAGAAGTTAAG-30. Human p21 siRNAs weregenerated with the oligonucleotides sense 50-GAUGGAACUUC-GACUUUGUUU-30; antisense: 50-ACAAAGUCGAAGUUCCA-UCUU-30. The pGIPZ controls were generated with controloligonucleotide 50-GCTTCTAACACCGGAGGTCTT-30 or 50-GCCCGAAAGGGTTCCAGCTTA-30.

MaterialsAntibodies that recognize FH (ab110286), PAK4 (ab62509),

CSL (ab25949), KDM2A (ab31739), H3 (ab1791), H3K36me2(ab9049), Lamin B1 (ab65986), Smad3 (ab28379), 14-3-3 zetawere purchased from Abcam. Antibodies that recognize p53(sc-126) were obtained from Santa Cruz Biotechnology. Antibo-dies against FH (#32975), Flag (#35535), His (#T505), and GST(#T509) were obtained from Signalway Antibody. Antibodiesagainst Flag (F3165), GST (G1160), and His (SAB4301134) werepurchased from Sigma. Antibodies against Phospho-MAPKAPK-2(#3044), Phospho-JunB (D3C6, #8053), p38 (D13E1, #8690),Phospho-p38 (Thr180/Tyr182; D3F9, #4511), Phospho-Akt(Ser473; D9E, #4060), Phospho-SMAD2 (Ser465/Ser467; E8F3R,#18338), Cleaved Notch1 (D3B8, #4147), SMRT (D8D2L,#62370), b-actin (#4970) were purchased from Cell SignalingTechnology. FITC-labeled antibody against BrdU (11-5071) waspurchased from eBioscience.

Rabbit polyclonal FH pThr-90 and FH pSer-46 antibodies wasgenerated by Signalway Antibody. A peptide containing pThr-90or FH pSer-46 was injected into rabbits. The rabbit serum wascollected and purified using an affinity column with nonpho-sphorylated FH Thr-90 and Ser-46 peptide to exclude the anti-bodies for nonphosphorylated FH, followed by an affinity col-umn with phosphorylated FH Thr-90 and Ser-46 peptide to bindto and purify the FH pThr-90 and FH pSer-46 antibody. The FHpThr-90 and FH pSer-46 antibodies were then eluted andconcentrated.

SP600125 and SB203580 were purchased from Cell Signal-ing Technology. AKT inhibitor and U0126 were purchased fromEMD Biosciences. PAK4 inhibitor PF-3758309 was obtainedfrom Selleck Chemicals. Anti-FLAG M2 beads, alkaline phos-phatase, and purified His-PAK4 were purchased from Abcam.TGFb was obtained from R&D Systems. Compound C, mono-ethyl fumarate, diethyl-malate, BrdU, EDTA-free ProteaseInhibitor Cocktail, and PhosSTOP were purchased from Sigma.Hygromycin, puromycin, DNase-free RNase A, and propidiumiodide were purchased from EMD Biosciences. Other materialswere described previously (23).

Immunoprecipitation and immunoblotting analysisProteins were extracted from cultured cells using a modifi-

ed buffer (50 mmol/L Tris-HCl (pH 7.5), 1% Triton X-100,150 mmol/L NaCl, 1 mmol/L DTT, 0.5 mmol/L EDTA, andprotease inhibitor cocktail or phosphatase inhibitor cocktail),followed by immunoprecipitation and immunoblotting withthe corresponding antibodies, as described previously (26). Forimmunoprecipitation of endogenous FH, FH antibody fromAbcam (ab110286) was used for immunoprecipitation (IP) andantibody fromSignalway Antibody (#32975)was used for immu-noblotting. Nuclear extract was collected using NE-PER Nuclearand Cytoplasmic Extraction Reagents (Thermo Fisher Scientific)according to the manufacturer's instructions. The protein concen-tration was determined using Bradford assay. Proteins from celllysates or nuclear extracts were separated by SDS-PAGE, trans-ferred onto polyvinylidene difluoride membrane (Millipore Cor-poration) and probed with the indicated antibodies.

Recombinant protein purificationWT and mutant GST-FH were expressed in bacteria and puri-

fied, as described previously (27).

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Gene expression analysisWe isolated total RNA using RNAzol RT (Molecular Research

Center) following the manufacturer's instructions. cDNA wassynthesized from 1 mg total RNA using iScript cDNA SynthesisKit (Bio-Rad) and quantified mRNA levels by real-time qRT-PCRusing SYBR Green (Bio-Rad). We ran samples in technical tripli-cates and calculated relative mRNA levels normalized to actinmRNA levels in the same samples. The qPCR primer sequenceswere listed as follows: p21-50-CGACTGTGATGCGCTAATGG (for-ward), 50-GGCGTTTGGAGTGGTAGAAATC (reverse); GAPDH-50-AGGTGAAGGTCGGAGTCAAC (forward), 50-GACAAGCTT-CCCGTTCTCAG (reverse).

p38 treatmentPurified WT and mutant GST-FH were incubated with p38 in

kinase assay buffer supplementedwith 0.2mmol/L AMP and coldATP in the presence or absence of 0.2 mCi/mL hot ATP (ICNBiochemicals) for 20minutes at 30�C. After the reaction, p38 wasremoved by extensive washing with RIPA buffer and kinase assaybuffer. The GST-FH bound beads were recovered by centrifuga-tion. For kinase phosphorylation analyses, the GST-FH–boundbeads were subjected to SDS-PAGE and then autoradiographyafter incubation with EN3HANCE (PerkinElmer). The GST-FH–

bound beads after p38 treatment in absence of hot ATP werefurther incubated with his-CSL for protein interaction assay asindicated.

Chromatin Immunoprecipitation assayA chromatin immunoprecipitation (ChIP) assay was per-

formed using an Upstate Biotechnology kit as described pre-viously (23). Quantitative real-time PCR was used to measurethe amount of bound DNA, and the value of enrichmentwas calculated according to the relative amount of input andthe ratio to IgG. The primers covering p53 DNA-bindingsites (p53RE) of human p21 gene promoter region [50-GTGGCTCTGATTGGCTTTCTG-30 (forward), 50-CTGAAAA-CAGGCAGCCCAAG-30 (reverse)] were used for the real-timePCR (28).

The primers covering proximal promoter region of human p21gene [50-GAAGTGCCCTCCTGCAGCACGCGAG-30 (forward) and50-CACCTCCTCTGAGTGCCTCGGTG-30 (reverse)] were used forthe real-time PCR. The primers covering promoter region ofhuman Smad7 gene [50-CCTCTGCTCGGCTGGTTCCACTGC-30

(forward) and50-TAGAAACCCGATCTGTTGTTTGCG-30 (reverse)]were used for the real-time PCR.

Mass spectrometry analysisFH phosphorylation was analyzed by LC/MS-MS as

described previously (23). Briefly, Flag-FH–associated proteinsfrom the IP assay were acetone-precipitated in vitro at �20�Covernight and resuspended in 50 mmol/L ammonium bicar-bonate buffer containing Rapigest (Waters Corp). The samplewas heated to 95�C for 10 minutes and allowed to cool downbefore 100 ng of sequencing-grade modified trypsin (Promega)was added. The digestion proceeded overnight at 37�C and wasanalyzed by LC/MS-MS using an Orbitrap-Elite mass spectrom-eter (Thermo Fisher Scientific). Proteins were identified bycomparing the fragment spectra against those in the SwissProtprotein database (EBI) using Mascot v.2.3 (Matrix Science) andSequest v.1.20 via Proteome Discoverer v.1.3 (Thermo FisherScientific) software.

Chromatin treatment in vitroIsolation of chromatin extract from cultured cells with recon-

stituted expression of WT or mutant rFH was performed asdescribed previously (23). Briefly, cells were washed with PBSand resuspended in solution A [10 mmol/L HEPES (pH 7.9),10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.34 M sucrose, 10% glyc-erol, 1 mmol/L dithiothreitol, 10 mmol/L NaF, 1 mmol/LNa2VO3, and protease inhibitors]. Triton X-100 was added to afinal concentration of 0.1%, and the cells were incubated for 5minutes on ice, followed by low-speed centrifugation (4 minutesat 1,300 � g at 4�C) to separate the cytoplasmic proteins fromthe nuclei. The isolated nuclei were then lysed in solution B(3 mmol/L EDTA, 0.2 mmol/L EGTA, 1 mmol/L DTT, andprotease inhibitors). Insoluble chromatin was collected by cen-trifugation. The resuspended chromatin extracts were incubatedwith the various concentration of malate for 30 minutes,followed by incubation with purified KDM2A in reaction buffer(20 mmol/L Tris-HCl at pH 7.5, 150 mmol/L NaCl, 50 mmol/L[NH4]2Fe[SO4]2, 100mmol/La-ketoglutarate, 2mmol/L Vc, and10 mmol/L phenylmethylsulfonylfluoride) for 3 hours. Afterreaction, the chromatin extracts were fixed and sonicated. TheCHIP analysis with H3K36me2 antibody was performed using anUpstate Biotechnology kit.

TransfectionHBE, A549, and NCI-H520 cells were transfected with various

plasmids and siRNA using Lipofectamine 3000 (Invitrogen)according to the vendor's instructions.

Cell proliferation assayAfter treatment, cells were incubatedwith 50mmol/L BrdU for 1

hour. Cells were collected by tryptase and centrifugation and fixedin 70% ethanol at 4�C for 1 hour and subsequently incubatedwith 2NHCl/0.5%Triton X-100 for 30minutes, 0.1mol/L boratesodium for 2 minutes. After anti-BrdU-FITC (eBioscience) incu-bation and washing, BrdU incorporation rate was analyzed byflow cytometry. Cell-cycle profile was measured by propidiumiodide staining and analyzed by flow cytometry.

Enzyme activity assayMeasurement of FH activity was performed as described pre-

viously (23). Briefly, purified WT or mutant FH proteinswere added to an enzyme assay buffer (50 mmol/L malate and10 mmol/L potassium phosphate at pH 7.3) and the absorbanceat OD 240 nm was recorded.

Fumarate measurementFumarate concentrations in cells were measured using the

Fumarate Detection Kit purchased from Abcam.

IHCIHC was performed on paraffin-embedded sections of human

lung cancer (611 lung cancer tissues). Formalin-fixed, paraffin-embedded consecutive human lung cancer tissue sections(3–5 mm) were deparaffinized and rehydrated. Antigen retrievalwas performed by boiling tissue sections in 10 mmol/L citratebuffer (pH 6.0) in amicrowave oven for 5minutes. The activity ofendogenous peroxidase was blocked with 3% hydrogen peroxidein methanol for 10 minutes at room temperature. After washing,nonspecific binding sites were blocked by incubating the slideswith 10% FBS/PBS for 30 minutes at room temperature. Sections

PAK4 Blocks TGFb-Induced Cell Growth Arrest

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were subsequently incubated with antibodies against PAK4(ab62509, 1:50) and FH pSer-46 (Signalway Antibody, 1:50) at4�C overnight. After incubation with the primary antibodies, thesections were washed and incubated with secondary antibodiesand DAB staining reagent with GTVision Detection System/Mo&Rb Kit according to the manufacturer's instructions. Aftercounterstain with hematoxylin and dehydration, the sectionswere mounted and imaged using the Leica microscope. Clinicalinformation of 681 consecutive resected NSCLC cases between2009 and 2010 in the first affiliated hospital of GuangzhouMedical University (Guangzhou, China) were collected. To illus-trate the natural prognosis of patients without confoundingeffects from postrecurrent treatments, only resected NSCLCs(stage I–III) were eligible and disease-free survival (DFS) wasselected as endpoint. The corresponding tissue array (with 681cases and a total of 23 holes as normal control) was subjected toIHC staining for FH pS46 expression. Immunoreactivity wasqualitatively evaluated according to the area of staining despitethe intensity to avoid artificial cut-off effect: the area of stainedcancer cellswas recorded as 0%(negative staining), >0%(positivestaining) of all cancer cells. Statistical analyses of the survival timeas well as the illustration of survival curves were performed usingthe Cox multivariate regression model in the SPSS 19 Software.Statistical significance was set at P < 0.05.

MouseSix-week-oldmale nu/numicewere injectedwith 2� 106 A549

cells in a volume of 150 mL of PBS/Matrigel [1/1 (v/v)]. Injectionsweremade subcutaneously in the left and right flanks, respectivelyat day 0. Tumor volume was measured using Vernier calipersweekly. All animal experiments were performed in accordancewith the regulations draftedby theAssociation forAssessment andAccreditation of Laboratory Animal Care in Shanghai and wereapproved by the East China Normal University Center for AnimalResearch.

Ethics approval and consent to participateAll animal experiments were approved by the East China

Normal University Center for Animal Research. The usage ofhuman tissues was approved by the Institutional Ethics Boardof the First Affiliated Hospital of Guangzhou Medical Universityand conforms to the Helsinki Declaration and to local legislation.Written informed consent was obtained from the patients.

Statistical analysisStatistical analysis was conducted with the two-tailed unpaired

Student t test. All data represent the mean � SEM of threeindependent experiments.

Resultsp38 mediates FH–CSL interaction induced by TGFb

To determine whether FH is involved in the regulation of TGFbsignaling under Notch activation, the potential relation betweenFH and CSLwas examined in human bronchial epithelial cell lineHBE cells. The reciprocal immunoprecipitation analysis indicatedTGFb stimulation dramatically increased the interaction betweenFH and CSL in HBE cells expressed with Notch intracellulardomain (NICD),while TGFb treatment orNICD expression alonecould not (Fig. 1A). FH was not associated with SMRT (Fig. 1A,left), a known transcriptional corepressor of CSL (29), which

basally binds to CSL, and their interactionwas decreased byNICDexpression (Fig. 1A, right). These data suggest that both TGFb andNotch signaling are required for FH–CSL complex formation,and the replacement of corepressor such as SMRT by NICD inCSL-related complex would facilitate FH–CSL interaction. Flowcytometry analyses showed that TGFb notably induced G0–G1

arrest and suppressed proliferation both in parent HBE (Supple-mentary Fig. S1A) and NICD-overexpressed HBE (NI-HBE) cells(Fig. 1B; Supplementary Fig. S1B). However, we found that TGFb-induced cell growth arrest was inhibited by FH depletion only inNI-HBE (Fig. 1B; Supplementary Fig. S1B), and this effect couldbe further reversed by CSL depletion (Fig. 1B; SupplementaryFig. S1B). These results reveal that FH is required for TGFb-induced growth arrest upon Notch activation and at this condi-tion exerts opposite effect to CSL.

TGFb-induced phosphorylation of Smad2, p38, and AKTin HBE (Supplementary Fig. S1C, left) or NI-HBE cells(Supplementary Fig. 1C, right) was not affected by FH depletion.Coimmunoprecipitation analyses showed CIP treatment in pre-cipitates from Flag-FH–disrupted FH-CSL complex formation(Fig. 1C), revealing FH interacts with CSL in a phosphoryla-tion-dependent manner. In addition, NI-HBE cells were pre-treated with AMPK inhibitor Compound C, JNK inhibitorSP600125, and p38 inhibitor SB203580, respectively (Supple-mentary Fig. S1D), and TGFb-induced FH–CSL interaction wasfound to be exclusively blocked by SB203580 (Fig. 1D), suggest-ing TGFb-induced FH–CSL interaction is dependent on p38activity and raising the possibility that FH could be the substrateof p38. This assumption was verified by an in vitro proteinphosphorylation assay, which showed purified activated p38could phosphorylate FH (Fig. 1E). Analysis of human FH aminoacid sequence indicated Thr 90 is the potential p38-phosphory-lated residue as S/TP site, and further in vitro protein phosphor-ylation assay indicated FH T90A mutant was resistant to p38-mediated FH phosphorylation (Fig. 1F).

Subsequently, coimmunoprecipitation analyses showed FHT90A mutant lost CSL-binding ability in NI-HBE cells underTGFb treatment (Fig. 1G). Consistently, a GST pull-down assayshowed that purified CSL can interact with WT FH, but not FHT90A, in the presence of p38 (Fig. 1H) and a phosphor-mimicform FH T90D (Supplementary Fig. S1E). These results suggestp38-mediated FH T90 phosphorylation is required for FHbinding to CSL. To investigate whether cellular location of FHaffects its interaction with CSL, Flag-FH was immunoprecipi-tated separately from nuclear and cytosolic fractions. As aresult, CSL was largely enriched in precipitates in the nucleusof NI-HBE cells stimulated with TGFb (Fig. 1I, left), whereasFH-90 phosphorylation was detected in either the nuclear orcytosolic lysates (Fig. 1I). In addition, the limited effect of CSLdepletion on the amount of nuclear FH-90 phosphorylation(Supplementary Fig. S1F) suggests that the nuclear transloca-tion of p38-phosphorylated FH is independent of CSL. Fur-thermore, we depleted endogenous FH in NI-HBE cells andreconstituted the expression of RNAi-resistant rFH(N), whichlost the mitochondrial localization, as 21 amino-terminalamino acid residues were deleted (23). As a result, FH(N)showed comparable FH-T90 phosphorylation to FH of full-length under TGFb stimulation (Supplementary Fig. S1G), andFH-T90 phosphorylation was not detected in the mitochondriaextracts (Supplementary Fig. S1H), suggesting mitochondrialocalization is dispensable for FH-T90 phosphorylation.

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Figure 1.

p38 mediates FH–CSL interaction induced by TGFb.A, HBE cells overexpressing Flag-FH and with or without NICDwere treated or untreated with TGFb(10 ng/mL) for 2 hours. Whole cellular extracts were subjected to immunoprecipitationwith an anti-FH antibody (left) or anti-CSL antibody (right). B, HBE cellsoverexpressing NICD (NI-HBE cells) were transfected with or without FH shRNA or/and CSL shRNA. Cells were treated or untreated with TGFb (10 ng/mL) for48 hours. The proportion of cells in cell cycle was examined by FACS analyses. The values are presented as mean� SEM (n¼ 3 independent experiments).�� , P < 0.01 (Student t test) between indicated groups. C,NI-HBE cells and Flag–FH were treated or untreated with TGFb (10 ng/mL) for 1 hour. Theimmunoprecipitates were treated with CIP (10 units) and analyzed by immunoblotting. D,NI-HBE cells overexpressing Flag-FH were pretreated with CompoundC (10 mmol/L), SP600125 (20 mmol/L), or SB203580 (10 mmol/L) for 1 hour (left), before being treated or untreated with TGFb (10 ng/mL) for 2 hours. E, In vitrophosphorylation analyses were performed bymixing the purified p38 with GST-FH proteins in the presence of [g-32P]ATP. F, The indicated purified GST-FHprotein was mixed with or without purified p38.G, NI-HBE cells expressed with indicated Flag–FH were treated or untreated with TGFb (10 ng/mL) for 2 hours.H, The indicated purified GST-FH protein was mixed with purified His-CSL protein with or without purified p38. GST pull-down analyses were performed. I,NI-HBE cells overexpressing Flag-FH were treated or untreated with TGFb (10 ng/mL) for 2 hours; nuclear (left) or cytosolic (right) extracts were subjected toimmunoprecipitation. In A–I, immunoblotting analyses were performed using the indicated antibodies; data represent one of three experiments.






FH forms a complex with CSL-p53 and promotes p21transcription through local fumarate production

Next, FH was depleted in NI-HBE cells and reconstituted withexpression of shRNA-resistant WT rFH(N) and rFH(N) T90A(Supplementary Fig. S2A). Consequently, rFH(N) T90A expres-sion significantly inhibited TGFb-induced G0–G1 arrest (Fig. 2A)and suppressed proliferation (Supplementary Fig. S2B) comparedwith its WT counterpart, indicating FH-T90 phosphorylation

promotes TGFb-induced growth arrest. p53 has been known tobe critical for TGFb-induced growth arrest (16). Coimmuno-precipitation analyses showed NICD overexpression inducedCSL–p53 complex formation (Supplementary Fig. S2C, left),which was not affected by TGFb treatment or FH depletion(Supplementary Fig. S2C, right). In contrast, TGFb largely inducedFH–p53 interaction in NICD-expressed HBE cells (Supplemen-tary Fig. S2D) in a CSL (Fig. 2B, left) and FH-T90 phosphorylation

Figure 2.

FH forms complex with CSL-p53 and promotes p21 transcription through local fumarate production.A, NI-HBE cells overexpressing indicated Flag-FH weretreated or untreated with TGFb (10 ng/mL) for 48 hours. Cell-cycle profile was examined by FACS analyses. B, NI-HBE cells with or without depleted CSL (left) oroverexpressing indicated Flag–FH (right) were treated or untreated with TGFb (10 ng/mL) for 2 hours. Whole cellular extracts were subjected toimmunoprecipitation with indicated antibodies. C,NI-HBE cells with or without p53 depletion, and with or without CSL and/or p53 depletion were treated oruntreated with TGFb (10 ng/mL) for 48 hours. Immunoblotting analyses were performed (left). Cell-cycle profile was examined by FACS analyses (middle andright). D, HBE or NI-HBE cells with or without FH depletion were treated or untreated with TGFb (10 ng/mL) for 12 hours. The mRNA level of p21was analyzed byreal-time PCR. E,NI-HBE cells with or without FH depletion and/or CSL depletion were treated or untreated with TGFb (10 ng/mL) for 12 hours. ThemRNA levelof p21was analyzed by real-time PCR. F,NI-HBE cells overexpressing indicated rFH(N) or with or without p53 depletion were treated or untreated with TGFb (10ng/mL) for 12 hours. The mRNA level of p21was analyzed by real-time PCR. G, NI-HBE cells with or without p53 depletion, and with or without CSL and/or p53depletion were overexpressed with indicated Flag-FH; cells were treated or untreated with TGFb (10 ng/mL) for 6 hours. ChIP analyses were performed by usingantibodies of anti-Flag. The y-axis shows the value normalized to the input. H,NI-HBE cells with FH depletion and reconstituted expression of indicated Flag-rFH(N) were treated or untreated with TGFb (10 ng/mL). ChIP analyses were performed with an anti-Flag antibody. The y-axis shows the value normalized to theinput. I, NI-HBE cells with FH depletion and reconstituted expression of indicated Flag–rFH(N) were treated or untreated with TGFb (10 ng/mL). Cells weretreated with indicated concentration of monoethyl fumarate. The mRNA level of p21was analyzed by real-time PCR. J, NI-HBE cells with FH depletion andreconstituted expression of indicated Flag-rFH(N) were treated or untreated with TGFb (10 ng/mL). Cells were treated with indicated concentration ofmonoethyl fumarate. Cell-cycle profile was examined by FACS analyses. In B and C, immunoblotting analyses were performed using the indicated antibodies. InA–J, data represent one of three experiments. In A and C–J, the values are presented as mean� SEM (n¼ 3 independent experiments). �� , P < 0.01 (Student ttest) between the indicated groups.

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(Fig. 2B, right) dependent manner. Functional analyses indicatedthat p53 depletion largely inhibited TGFb-induced growth arrestin NI-HBE cells, and this effect was not changed by CSL depletion(Fig. 2C; Supplementary Fig. S2E), demonstrating the dominativerole of p53 here. Along with aforementioned results, these datadelineate a proposed model in terms of the functional relation-ship among FH, CSL, and p53 under TGFb and Notch signaling(Supplementary Fig. S2F). Depletion of p21, a primary p53 targetgene relevant to growth arrest, apparently inhibited TGFb-induced growth arrest in either HBE cells (SupplementaryFig. S2G) or NI-HBE cells (Supplementary Fig. S2H). In line withresults shown in Fig. 1B, FH depletion notably decreased TGFb-induced p21 transcription in NI-HBE cells, but not in parentalHBE cells (Fig. 2D), and CSL depletion reversed these effects inNI-HBE cells (Fig. 2E). Meanwhile, TGFb dramatically promotedp21 transcription in WT FH-, but not FH T90A-expressed NI-HBEcells (Fig. 2F). Moreover, ChIP analysis showed WT FH-, but notFH T90A, was efficiently accumulated at p21 promoter region inTGFb-treated NI-HBE cells, which was blocked by either CSL orp53 depletion (Fig. 2G). These results reveal FH is recruited topromoter by CSL-p53 and thereby promotes p21 transcriptionand growth arrest by counteracting the suppressive effects of CSLon p53.

FH is able to regulate nonmetabolic events through its meta-bolic function (22, 23). Expression of the catalytically inactivemutant Flag-tagged FH(N) R233H showed TGFb induced com-parable FH-90 phosphorylation and FH-CSL complex to that inWT FH-expressed NI-HBE cells (Supplementary Fig. S2I). In linewith this, TGFb readily induced accumulation of FH(N) R233Haswell as WT FH(N) at p21 promoter regions (Fig. 2H). However,either reconstituted expression of rFH(N) R233Hor rFH(N) T90Anotably inhibited TGFb-induced p21 expression (Fig. 2I) andgrowth arrest (Fig. 2J) in NI-HBE cells. Of note, FH(N) T90A,which lost the enrichment at p21 promoter (Fig. 2H), showed acomparable catalytic activity to WT FH(N) and FH(N) T90D(Supplementary Fig. S2J). These data suggest both FH activityand promoter location are required for FH-CSL-p53–mediateddownstream events. To further determine the effect of FH activityon TGFb-induced growth arrest, exogenous monoethyl fumaratewas added into the cell-cultured medium with various amounts,which accordingly resulted in elevation of intracellular fumarateconcentration (Supplementary Fig. S2K). Consequently, FH (N)R233H or FH(N) T90A-repressed p21 transcription (Fig. 2I) andgrowth arrest (Fig. 2J; Supplementary Fig. S2L) in TGFb-treatedNI-HBE cells were partially reversed by exogenous fumarate in adose-dependent manner, while these rescue effects were blockedby p53 depletion (Supplementary Fig. S2M and S2N). On theother hand, addition of exogenous malate that led to a moderateincrease of fumarate levels (Supplementary Fig. S2O) did notsoundly affect TGFb-induced p21 transcription (SupplementaryFig. S2P) and growth arrest (Supplementary Fig. S2Q) in rFHR233H- or rFH T90A-expressed NI-HBE cells. Together, these dataindirectly demonstrate promoter-associated fumarate productionactivity of FH is indispensable for p53-p21–mediated down-stream event under TGFb signaling.

PAK4 phosphorylates FH and blocks FH–CSL interactionThe negative effect of TGFb signaling on cell growth is always

impeded during tumor development (30–32). In contrast withthe observation in HBE cells (Fig. 1A), TGFb stimulation onlyweakly promotes FH–CSL interaction in NICD-expressed A549

andNCI-H520 cells (Supplementary Fig. S3A). Accordingly, A549(Supplementary Fig. S3B) and NCI-H520 (Supplementary Fig.S3C) cells did not show significant enhancement of growth arrestafter TGFb treatment, unless with CSL depletion. Given thisdifference, we set forth to look for the upstream component thatwould be specific for the regulation of FH–CSL interaction incancer cells. A panel of inhibitors was collected and used to blockactivities of relevant kinases that are involved in tumorigenesis.Intriguingly, we found that the treatment of the p21 protein(Cdc42/Rac)-activated kinase 4, PAK4 inhibitor PF-3758309largely enhanced FH–CSL interaction in A549 (Fig. 3A) andNCI-H520 (Supplementary Fig. S3D) cells under TGFb stimulus.PAK4 belongs to serine/threonine protein kinase and its dysre-gulation has been implicated in lung cancer (33). Further coim-munoprecipitation analysis showed that PAK4 interactedwith FHinstead of CSL (Supplementary Fig. S3E), implying that FHwouldalso be a substrate of PAK4. As revealed in the protein phosphor-ylation assay, purified activated PAK4 phosphorylated FH and thephosphorylation was recognized by an antibody against Serphosphorylation (Fig. 3B). Mass spectrometric analysis indicatedthat FH Ser46 underwent phosphorylation inA549 cells (Fig. 3C).Mutagenesis analyses showed that FH S46A mutant, which didnot affect p38-mediated FH T90 phosphorylation (Supplemen-tary Fig. S3F), robustly abolished PAK4-mediated FH phosphor-ylation, as demonstrated by autography and immunoblottinganalysis by using a specific FH-S46 phosphorylation antibody(Fig. 3D).

Subsequently, immunoblotting analysis showed that PAK4-mediated FH-S46 phosphorylation is insensitive to TGFbstimulation in A549 cells (Supplementary Fig. S3G, left), andhardly detected in NI-HBE cells (Supplementary Fig. S3G,right). Meanwhile, PF-3758309 treatment, which blockedFH-S46 phosphorylation, did not affect TGFb-induced FHT90 phosphorylation in A549 cells (Supplementary Fig.S3H). Immunoprecipitation analyses indicated that TGFb stim-ulation failed to significantly promote the binding of phos-phor-mimic mutant FH S46D to CSL in NI-HBE cells (Supple-mentary Fig. S3I). In contrast, FH S46A displayed an increasedbinding to CSL and p53 in TGFb-treated A549 and NCI-520cells (Fig. 3E; Supplementary Fig. S3J). Functionally, TGFbtreatment largely promoted cell growth arrest in A549 cellswith PAK4 inhibitor treatment (Supplementary Fig. S3K), orwith reconstituted expression of rFH(N) S46A, but not rFH(N)S46A/T90A (Fig. 3F; Supplementary Figs. S3L and S3M). Thesedata strongly suggest FH-S46 phosphorylation by PAK4impedes FH–CSL interaction, and thus releases the repressiveeffects of CSL on TGFb-induced growth arrest. In addition,TGFb-induced growth arrest was inhibited by p53 depletionin A549 cells with FH S46A expression (Fig. 3G; SupplementaryFig. S3N), and TGFb treatment displayed limited effects on cellgrowth in p53-null H1299 cells, which was not changed by CSLdepletion (Supplementary Fig. S3O), supporting the pivotalrole of p53 in TGFb-induced cell growth arrest. Consistently,TGFb readily stimulated p21 transcription (Fig. 3H, left) as wellas FH promoter accumulation (Fig. 3I) in FH S46A-expressed,but not WT FH- or FH S46A/T90A-expressed A549 cells, whichwere repressed by p53 depletion [Fig. 3H (right) and I]. In termsof the effect of metabolic activity of FH, we found exogenousfumarate (Supplementary Fig. S3P) partially promoted p21transcription (Fig. 3J) and growth arrest (Fig. 3K) in A549 cellstreated with TGFb.






PAK4-phosphorylated FH binds to 14-3-3Sequential in vitro kinase assays indicated p38- and PAK4-

mediated FH phosphorylation were not mutually affected (Sup-plementary Fig. S4A). In A549 cells, TGFb induced FH-T90phosphorylation in a p38 activity–dependent manner (Supple-mentary Fig. S4B), which was not significantly changed underFH-S46A expression (Supplementary Fig. S4C). Given the impor-

tance of FH-T90 phosphorylation for FH binding to CSL, thesedata are in contrast with the result that FH S46A–CSL interactionwas largely enhanced in TGFb-treated NSCLC cells (Fig. 3E), andimplies that the inhibitory effect of PAK4 on FH–CSL interactionis attributed to an additional mechanism. Cellular fraction anal-ysis indicated that FH-S46 phosphorylation showed an exclusivelocalization in the cytosol of A549 cells (Fig. 4A) and expressionof

Figure 3.

PAK4 phosphorylates FH and blocks FH–CSL interaction. A,A549 cells overexpressing NICD (NI-A549 cells) were transfected with Flag-FH and were pretreatedwith PF-3758309 (10 mmol/L), U0126 (20 mmol/L), or LY294002 (10 mmol/L) for 1 hour (left), before being treated or untreated with TGFb (10 ng/mL) for 2 hours.Immunoprecipitation analysis was performed using anti-Flag antibody. B, In vitro phosphorylation analyses were performed by mixing the purified PAK4 withGST-FH proteins in the presence of [g-32P]ATP. C, NI-A549 cells were overexpressed with Flag-FH. Immunoprecipitation analysis was performed using anti-Flagantibody, and the extracts were analyzed by mass spectrometry. The results of a mass spectrometric analysis of a tryptic fragment at m/z 510.70 (mass error,1.13 ppm)matched those of the doubly charged peptide 111-2-120, suggesting that S46 was phosphorylated. The Sequest score for this match was Xcorr¼ 2.38.The Mascot score was 288.46. D, The indicated purified GST-FH proteins were mixed with or without purified PAK4. E,NI-A549 cells expressed with indicatedFlag-FH were treated or untreated with TGFb (10 ng/mL) for 2 hours. Immunoprecipitation analysis was performed using anti-Flag antibody. F, NI-A549 cellsoverexpressing indicated Flag-FH were treated or untreated with TGFb (10 ng/mL) for 48 hours. Cell-cycle profile was examined by FACS analyses. G, NI-A549cells with or without p53 depletion were overexpressed with indicated Flag-FH. Cells were treated or untreated with TGFb (10 ng/mL) for 48 hours. Cell-cycleprofile was examined by FACS analyses. H, NI-A549 cells with or without CSL depletion (left) or p53 depletion (right) were expressed with indicated Flag-FH.Cells were treated or untreated with TGFb (10 ng/mL) for 48 hours. The mRNA level of p21was analyzed by real-time PCR. I, NI-A549 cells with or without CSLdepletion (left) or p53 depletion (right) were overexpressed with indicated Flag-FH. Cells were treated or untreated with TGFb (10 ng/mL) for 6 hours. ChIPanalyses were performed with an anti-Flag antibody. The y-axis shows the value normalized to the input. J, A549 cells incubated with the indicatedconcentrations of monoethyl-fumarate were treated or untreated with TGFb (10 ng/mL) for 12 hours. The mRNA level of p21was analyzed by real-time PCR.K, A549 cells incubated with the indicated concentrations of monoethyl-fumarate were treated or untreated with TGFb (10 ng/mL) for 48 hours. Cell-cycleprofile was examined by FACS analyses. In A, B,D, and E, immunoblotting analyses were performed using the indicated antibodies. InA, B and D–K, datarepresent one of three experiments. In F–K, the values are presented as mean� SEM (n¼ 3 independent experiments). � , P < 0.05; �� , P < 0.01 (Student t test)between the indicated groups.

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FH S46A led to a dramatic increase of FH–CSL interaction in thenucleus, but not cytosol, under TGFb stimulation. In contrast, thenuclear level of FH-T90 phosphorylation was significantlyincreased in FH S46A–expressed A549 cells with TGFb stimulus,with a concomitant drop in the cytosol (Fig. 4A). Further cellularfraction analysis in rFH (N) S46A-expressed A549 cells indicatedthat FH with FH-T90 phosphorylation accounts for around 20%of total nuclear FH (Supplementary Fig. S4D, left), a large appor-tion of which, interacted with CSL (Supplementary Fig. S4D,right). In addition, rFH(N) R233H and rFH T90A showed acomparable nuclear accumulation toWT FH (Supplementary Fig.S4E, left), while rFH(N) S46A and rFH S46A/T90A with nuclearlocation increased by around 23% compared with that of WT FH(Supplementary Fig. S4E, right) in A549 cells treated with TGFb.These data suggest that FH T90 or S46 phosphorylation accountsfor a small proportion of total FH, and FH pS46 exerts negativeeffects on the nuclear translocation of T90-phosphorylated FHand the subsequent FH–CSL interaction. Of note, the limitedeffect of FHpS46 on nuclear translocation of total FH implies thatthere remain unidentified mechanisms involved in FH nucleartranslocation.

14-3-3 proteins are critically implicated in signal transductionduring various biological processes through the specific binding

to protein phospho-Ser/Thr–containing sequence motifs (34).Scansite analysis showed that FH Ser46 is located in the sequenceas a potential binding site motif for 14-3-3 zeta. We wonderwhether 14-3-3 zeta is responsible for the cytosolic arrest of FH.Ectopic and endogenous coimmunoprecipitation analysis show-ed that 14-3-3 zeta was associated with FH in the cytosol (Fig. 4B,left) but not nucleus (Fig. 4B, right) of A549 cells. In contrast, FHS46A failed to interact with 14-3-3 zeta (Fig. 4B, left). Consistentwith the limited FH-S46 phosphorylation levels, NI-HBE cellsshowed a limited complex formation of FH-14-3-3 zeta (Supple-mentary Fig. S4F). The pull-down assay showed purified GST-14-3-3 zeta associated with WT FH, but not FH S46A, in A549 cells(Fig. 4C). Consistent with results shown in SupplementaryFig. S4E, immunoblotting of nuclear extracts indicated expressionof 14-3-3 zeta shRNA (Supplementary Fig. S4G) only partiallyenhanced the nuclear translocation of total FH, while leading to asignificant increase of nuclear FH-T90 phosphorylation in A549cells treatedwith TGFb (Fig. 4D). These data indicate that FHpS46exerts critical effects on nuclear translocation of FH-T90 phos-phorylation and the subsequent FH–CSL interaction (Fig. 4E). Inaddition, 14-3-3 zeta has been reported to destabilize p53 (30);this implies that TGFb-inducedp53-FHS46A interactionmight beattributed to the potential change of p53 protein levels resulting

Figure 4.

PAK4-phosphorylated FH binds to 14-3-3. A, NI-A549 cells overexpressing indicated Flag-FH were treated or untreated with TGFb (10 ng/mL) for 2 hours;nuclear (left) or cytosolic (right) extracts were subjected to immunoprecipitation with an anti-Flag antibody. B, NI-A549 cells overexpressing indicated Flag-FHwere treated or untreated with TGFb (10 ng/mL) for 2 hours; nuclear (left) or cytosolic (right) were subjected to immunoprecipitation with an anti-Flag antibody.C,NI-A549 cells overexpressing indicated Flag-FH. Cellular extracts were collected and incubated with or without GST–14-3-3. D, NI-A549 cells were transfectedwith 14-3-3 zeta shRNA and were treated or untreated with TGFb (10 ng/mL) for 2 hours. Nuclear extracts were collected. E, A cartoon showing 14-3-3 binds toSer46-phosphorylated FH and leads to its sequestration in the cytosol. In A–D, immunoblotting analyses were performed using the indicated antibodies. Datarepresent one of three experiments.






from FH S46A expression. However, immunoblotting analysisindicated that reconstituted expression of rFH S46A in A549 cellsdid not result in significant change of p53 protein levels (Sup-plementary Fig. S4H).

Local fumarate maintains H3K36Me2 through inhibition ofKDM2 activity

Fumarate can regulate histone methylation via its antagonizedeffect on a-ketoglutarate (aKG)-dependent demethylases (22).TGFb stimulation in NI-HBE cells increased H3K36me2 andH3K4me3, but not H3K27me2 or H3K9me2 levels at the p21promoter region covering p53RE (Supplementary Fig. S5A),whileonly H3K36me2 accumulation, which is associated with tran-scriptional activation (35), was apparently blocked by rFH T90A-or rFH R233H expression in TGFb-treated NI-HBE cells (Supple-mentary Fig. S5B). We further expressed H3K36R in NI-HBE cellsand found H3K36R was readily accumulated at p21 promoterregion (Supplementary Fig. S5C). p53, CSL, and p38 proteinlevels remained at steady levels under H3K36R expression (Sup-plementary Fig. S5D) and H3K36R expression largely repressedTGFb-induced p21 transcription in NI-HBE cells (SupplementaryFig. S5E) or rFH(N) S46A-expressed A549 cells (SupplementaryFig. S5F). In addition, rFH(N) T90A expression did not show asignificant effect on TGFb-induced transcription of smad7, ofwhich the gene promoter region contains binding site of SBE(Smad-binding element), but not p53RE, compared with WTcounterpart (Supplementary Fig. S5G). Meanwhile, ChIP analysisindicated FH accumulation was hardly to be detected at thepromoter region of smad7, andH3K36me2 deposition on smad7promoter was not changed by TGFb treatment (SupplementaryFig. S5H), suggesting FH-regulated H3K36me2 accumulation atpromoter would be prone to promoter of genes that cover p53RE.In A549 cells, rFH(N) S46A expression largely promotedH3K36me2 accumulation at p21 promoter (Fig. 5A) and p21transcription (Supplementary Fig. S5I) induced by TGFb com-paredwithWT rFH(N),while this failed to be recapitulated in cellsexpressing rFH(N) S46A/R233H or S46A/T90A (Fig. 5B). Inaddition, expression of rFH(N) S46A/R233H or S46A/T90A hadno additional effects on H3K36R-repressed p21 transcription(Fig. 5C). These data indicate promoter-associated FH activity isrequired for H3K36me2 regulation at promoter region.

KDM2 is the primary histone demethylase responsible forH3K36me2 demethylation (22). ChIP analysis showed thatKDM2A was constantly detected at the p21 promoter region inNI-HBE cells or rFH(N) S46A-expressed A549 cells (Supplemen-tary Fig. S5J). KDM2A depletion notably inhibited rFH(N)S46A/T90A- and rFH(N) S46A/R233H-repressed H3K36me2accumulation at p21 promoter (Fig. 5B), p21 transcription(Fig. 5D), and growth arrest (Fig. 5E; Supplementary Fig. S5K)in TGFb-treated A549 cells. These data indicate that FH promo-tes TGFb-induced growth arrest through inhibition of KDM2A-mediated demethylation of H3K36me2. Furthermore, exogenousmonoethyl fumarate was added into A549 cells with KDM2Aoverexpression that led to a decrease in overall H3K36me2 levels(Supplementary Fig. S5L). Ectopic expression of KDM2A robustlysuppressed H3K36me2 at p21 promoter, which was blocked byexogenous fumarate in a dosage-dependent manner (Fig. 5F).However, if exogenous a-KG was concomitantly added intocells with fumarate, the inhibitory effect from KDM2A over-expression was significantly reversed (Fig. 5F). Moreover, exoge-nous fumarate partially reversed FH(N) S46A/T90A or FH(N)

S46A/R233H-suppressed H3K36me2 at p21 promoter (Fig. 5G)induced by TGFb, in which FH S46 phosphorylation did notaffect FH metabolic activity (Supplementary Fig. S5M). At thesame condition, addition of high dosage of exogenousmalate didnot exert significant effect on H3K36me2 accumulation at p21promoter (Supplementary Fig. S5N), p21 transcription (Supple-mentary Fig. S5O), or cell growth (Supplementary Fig. S5P).Measurement of fumarate indicated high dosage of exogenousmalate only led to moderate increase of intracellular fumaratelevels (Supplementary Fig. S5Q). To determine the direct effectsof fumarate on KDM2A activity, chromatin was collected fromA549 cells expressing rFH(N) S46A or rFH(N) S46A/R233H.In vitro assay showed that KDM2A abolished TGFb-inducedH3K36me2 on the p21 promoter region, which was partiallyinhibited by the malate incubation (Fig. 5H, 1st–4th lanes). Incontrast, malate failed to affect H3K36me2 from FH R233H-expressed A549 cells (Fig. 5H, 5th–7th lanes). These data indicateFH catalytic activity for fumarate production is critical for TGFb-induced growth arrest through inhibition of KDM2A-mediateddemethylation at p21 promoter.

FH S46 phosphorylation is required for tumorigenesisSeahorse analysis indicated only A549 cells expressed with rFH

R233H, but not rFH T90A, rFH S46A, or rFH S46A/T90A, dis-played a lower mitochondrial oxygen consumption rate (OCR),compared with WT counterpart (Supplementary Fig. S6A),excluding that FH-S46 or FH-T90 phosphorylation might affectcell growth through its potential impact on mitochondrial activ-ity. To further analyze the physiologic relevance of FH Ser46phosphorylation, four lung cancer cell lines NCI-H520, A549,HCC-827, and NCI-H358 were collected. As a result, each cancercell line exhibited distinct FH Ser46 phosphorylation levels,which was in line with the PAK4 protein levels and inverselyrelated to the amount of FH–CSL complex (Fig. 6A). Functionalanalysis indicated that FH-S46 phosphorylation and PAK4 levelsare positively correlated with cell growth in these cell lines underTGFb stimulation (Fig. 6B). As shown in Supplementary Fig. S6B,expression of rFH S46A, but not rFH S46A/T90A, largely blockedcell growth in NCI-H520 cells under TGFb treatment. To furtherdetermine the functional impact of PAK4-mediated FH-S46 phos-phorylation on tumorigenesis, A549 cells with reconstitutedexpression of WT rFH, rFH T90A, rFH S46A, or rFH S46A/T90Awere subcutaneously injected into athymic nudemice. Comparedwith WT counterpart, A549 cells expressing rFH T90A did notshow remarkable change of tumor growth (Supplementary Fig.S6C), while expression rFH S46A, but not rFH S46A/T90A,notably inhibited tumor growth (Fig. 6C), revealing the criticaleffect of FH S46 phosphorylation. Consistently, 14-3-3 depletionor PF-3758309 treatment in A549 cells significantly repressedtumor growth (Supplementary Fig. S6D). High activity of PAK4correlates with poor prognosis for patients with lung cancer (33).To further determine the clinical relevance of our finding thatFH-S46 phosphorylation promotes tumor cell growth, we per-formed IHC analyses to examine the levels of PAK4 and FH-S46phosphorylation in serial sections of human lung tumor speci-mens (Fig. 6D, left and middle). Staining quantification showedthat FH-S46 phosphorylation levels were positively correlatedwithPAK4 expression levels (Fig. 6D, right).No case in thenormalcontrols showed significant FH pS46 signals (Supplementary Fig.S6E). As illustrated in Table 1, the expression rate of FH pS46 washigher in female than in male, and was increased as the N stage

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and TNM stage increased. However, the proportions were similarbetween different age, smoking history, histology, and tumor size.In survival analyses, the censored rate was 37.6% with a medianfollow-up timeof 5.06 (range 0.05–6.99) years. After adjusting forsex, age, histology, and stage, we found that the expression statusof FH pS46 did not stratify prognosis in overall population (HR

1.040; 95% CI, 0.772–1.400, P¼ 0.797). However, patients withFH pS46 expression were potentially associated with inferior DFSin patients with squamous cell carcinoma (HR 1.668; 95% CI,0.863–3.222; P ¼ 0.128; Fig. 6E, left). and in those with lymphnode metastases (HR 1.435; 95% CI, 0.956–2.154; P ¼0.082; Fig. 6E, right).

Figure 5.

Local fumarate maintains H3K36me2 through inhibition of KDM2 activity. A,NI-A549 cells with FH depletion and reconstituted expression of indicated Flag-rFH(N) were treated or untreated with TGFb (10 ng/mL) for 6 hours. B, NI-A549 cells with FH depletion and reconstituted expression of indicated Flag–rFH(N) and with or without KDM2A depletion were treated or untreated with TGFb (10 ng/mL) for 6 hours. C, NI-A549 cells with FH depletion andreconstituted expression of indicated Flag-rFH(N) and indicated H3. Cells were treated or untreated with TGFb (10 ng/mL) for 12 hours. Immunoblottinganalyses were performed using the indicated antibodies (left). The mRNA level of p21 was analyzed by real-time PCR (right). D, NI-A549 cells with FHdepletion and reconstituted expression of indicated Flag-rFH(N) were transfected with or without KDM2A shRNA. Cells were treated or untreated withTGFb (10 ng/mL) for 12 hours. The mRNA level of p21 was analyzed by real-time PCR. E, NI-A549 cells with FH depletion and reconstituted expression ofindicated Flag-rFH(N) were transfected with or without KDM2A shRNA. Cells were treated or untreated with TGFb (10 ng/mL) for 48 hours. Cell-cycleprofile was examined by FACS analyses. F, NI-A549 cells with FH depletion and reconstituted rFH(N) S46A were overexpressed with or without KDM2A.Cells added with indicated concentration of monoethyl fumarate and octyl-a�KG were treated or untreated with TGFb (10 ng/mL) for 12 hours. ThemRNA level of p21 was analyzed by real-time PCR. G, NI-A549 cells with FH depletion and reconstituted expression of indicated Flag-rFH(N) weretreated with indicated concentration of monoethyl fumarate. Cells were treated or untreated with TGFb (10 ng/mL) for 6 hours. H, NI-A549 cells with FHdepletion and reconstituted expression of indicated Flag-rFH(N) were treated with TGFb (10 ng/mL) for 6 hours. Chromatin extracts were collected andmixed with or without the indicated concentration of malate for 30 minutes and followed by incubation with KDM2A, and then chromatin extracts werefixed and used for ChIP analyses (left). Chromatin extracts subjected to immunoblotting analysis with an indicated antibody (right). In A, B, G, and H,ChIP analyses with indicated antibodies were performed. The y-axis shows the value normalized to the input. The values represent mean � SEM(n ¼ 3 independent experiments). �� , P < 0.01 (Student t test) between indicated groups. In A–H, data represent one of three experiments.






Figure 6.

FH S46 phosphorylation is required for tumorigenesis. A, NCI-H520 A549, NCI-H520, HCC-827, and NCI-H358 cells overexpressing NICD were treated with TGFb(10 ng/mL) for 48 hours. Whole cellular extracts were subjected to immunoprecipitation with an anti-FH antibody. B,NCI-H520 A549, NCI-H520, HCC-827, andNCI-H358 cells overexpressing NICDwere treated or untreated with TGFb (10 ng/mL) for 48 hours. Cell-cycle profile was examined by FACS analyses (left).Cellular proliferation rate was examined by BrdU incorporation assay and FACS analyses (right). The values are presented as mean� SEM (n¼ 3 independentexperiments). � , P < 0.05; �� , P < 0.01 (Student t test) between indicated groups. C,A total of 2� 106 A549 cells with overexpression of NICD and FH depletionand reconstituted expression of indicated rFH(N) were subcutaneously injected into the athymic nude mice. Representative tumor xenografts are shown (left).Tumor volumes were measured using length (a) and width (b) and calculated using the following equation: V¼ ab2/2. Data represent the means� SEM (n¼ 7,right). D, IHC staining with anti-PAK4 and anti-FH pSer46 antibodies was performed on 611 human lung tumor specimens. Representative photos of staining insquamous cell carcinoma are shown. Scale bar, 20 mm (left and right). Relation between categorized variables was examined by x2 test (right). E, The survivaltimes for patients with negative (black curve) versus positive (red curve) FH pS46 were compared. The Kaplan–Meier method and log-rank tests indicate thesignificance level of the association of FH pS46 (P¼ 0.01182) with inferior DFS in patients with squamous cell carcinoma (HR, 1.668; 95% CI, 0.863–3.222; P¼0.128) and in patients with lymph nodemetastases (HR, 1.435; 95% CI, 0.956–2.154; P¼ 0.082). F, A schematic model representing integration of TGFb-p38-FHand NICD-CSL signaling and its negative regulation in tumor cells. TGFb induces p38 activation, which can phosphorylate FH at T90. Upon Notch activation,nuclear NICD promotes the interaction between CSL and p38-phosphorylated FH; this in turn promotes FH/CSL/p53/Smad complex formation and theenrichment of FH on p21 promoter region. Eventually, the local production of fumarate by FH inhibits KDM2A-mediated histone H3 demethylation and promotesp21 expression responsible for growth arrest. On the other hand, PAK4 is able to phosphorylate FH and impedes FH–CSL interaction. In normal cells, cells areprone to growth arrest under TGFb treatment, while upregulated PAK4 activity prevents growth arrest induced by TGFb treatment in cancer cells.

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DiscussionThe physiologic consequence of TGFb signaling is compli-

cated by the concrete context and its crosstalk with othersignaling pathways. A recent study showed CSL, the centraleffector under Notch signaling pathway, can interact with p53and thereby block p53-mediated cell senescence (17). Giventhe critical role of p53 in the TGFb-induced cell growth arrest,this finding provides a potential regulatory linkage betweenTGFb and Notch signaling, and prompts us to further investi-gate the underlying mechanism. In this study, we found thatTGFb induces FH Thr 90 phosphorylation by p38. Upon Notchactivation, nuclear NICD promotes the interaction betweenCSL and p38-phosphorylated FH and thus FH/CSL/p53/Smadcomplex formation; this facilitates FH recruitment to p53-targed p21 promoter, where FH inhibits KDM2A-mediateddemethylation of H3K36me2 through local production offumarate; accumulation of H3K36me2 relieves the inhibitoryeffect of CSL on p53 and promotes TGFb-induced cell growtharrest (Fig. 6F). These results elucidate a novel mechanism ofH3K36me2 regulation by p38/FH signaling axis, and demon-strate that the metabolic effect of FH on transcriptional regu-lation is importantly implicated in cellular response to TGFbsignaling.

Previous studies indicated that FH localized in the nucleusdisplayed a local effect on histonemethylation (23). Consistentlyhere, we found that promoter-associated FH facilitates p53-medi-ated transcription through inhibition of H3K36me2 demethyla-tion (Fig. 6F), in which FH-catalyzed metabolic reaction in thedirection from fumarate to malate is shown to be crucial. How-ever, the supporting evidence in this regard remains indirect in this

study and it has become critical to develop the novel experimentalmethod in future to precisely detect the amount of local meta-bolites and to better clarify the localmetabolic effects of FH underthe specific context. Our results indicate that CSL upon Notchactivation is shown to block the transcriptional activity p53 underTGFb signaling, which is in line with the previous finding indi-cating the inhibitory effects of CSL on p53-mediated transcrip-tion (17). The interaction between CSL and T90-phoshorylatedFH promotes the formation of the protein complex containingFH/CSL/p53, which is the prerequisite for FH accumulation andlocal effect at the promoter of targeted gene; Of note, it has beenknown that Notch activation is accompanied with the alterationofCSL-associated components (36), thus, thismight be critical forthe accessibility of CSL binding for T90-phoshorylated FH. Con-sistently, the essential role of Notch–CSL activation for FHinvolvement in TGFb-p53–p21 signaling is supported by resultsshowing that exclusive FH–CSL interaction in the nucleus appearswhen both signaling pathways are triggered. Hence, it seems thatthe additively negative control of CSL by FH is able to make genetranscription regulated in amore precise manner on the signalingcrosstalk. However, it remains to be further investigated whetherthis effect is also dependent on Notch activation under othersignaling context.

Besides the inhibitory effects on cell growth, TGFb is known toparadoxically drive tumor metastasis during cancer develop-ment (2). It has been a hot issue to decipher how the functionalrole of TGFb is converted. In contrast with observations in HBEcells, we found that TGFb-induced FH–CSL interaction and cellgrowth arrest are notably suppressed in lung cancer cells, whichwas reversed by the PAK4 inhibition. Overexpression or hyper-activation of PAK4 is recorded in human lung cancer (33). In

Table 1. Summary of patient characteristics and their correlation with FH pS46 expression

FH pS46 expressionCharacteristics N (%) Negative (%) Positive (%) P

Total 681 (100) 33.9 66.1Sex 0.025Male 397 (58.4) 37.6 62.4Female 283 (41.6) 28.8 71.2

Age 0.144<70 551 (80.9) 32.5 67.5�70 130 (19.1) 40.2 59.8

Smoking history 0.231No 405 (63.4) 31.8 68.2Yes 234 (36.6) 36.9 63.1

Histology 0.651Squamous 150 (22.1) 37.5 62.5Adenocarcinoma 479 (70.5) 33 67Others 50 (7.4) 34 66

Tumor size (cm) 0.243<3 307 (45.7) 36.3 63.73–5 212 (31.5) 33 675–7 101 (15.0) 31.3 68.7�7 52 (7.7) 29.8 70.2

N stage 0.0730 410 (63.1) 36.2 63.81 90 (13.8) 30.5 69.52 150 (23.1) 28.1 71.9

TNM stage 0.020Ia 143 (22.0) 39.2 60.8Ib 202 (31.1) 36.8 63.2IIa 105 (16.2) 31.9 68.1IIb 36 (5.5) 18.8 81.3IIIa 161 (24.8) 29.5 70.5IIIb 3 (0.5) 0 100






accordance, our study indicates that PAK4 is highly expressed inlung cancer cells and clinical samples. Further analyses show thatPAK4 can phosphorylate FH at Ser 46; and this phosphorylationsequesters FH at cytosol through its binding to 14-3-3; in turn, FHSer 46 phosphorylation blocks nuclear function of FH relevant toTGFb-induced cell growth arrest (Fig. 6F). Collectively, theseresults suggest that the aberrant levels of PAK4 would be animportant strategy undertook by cancer cells to avoid negativeeffects of TGFb on cell growth, and hence to indirectly potentiatetumor metastasis under TGFb signaling. Given the importantphysiologic effects of FH phosphorylation by PAK4, it could bepredicted that FH expression levels and activity would be tightlyrelated to tumorigenesis in lung cancer cells with high expressionlevels of PAK.

The regulation of metabolic enzymes is fundamentallylinked to various physiologic events in addition to the directeffect on metabolism (37, 38). The local effect of FH on TGFb-induced gene transcription exemplifies the subtle interactionbetween metabolic pathway and cellular growth signaling.Meanwhile, the inhibitory effect of PAK4 on p38/FH axisuncovers a novel mechanism of spatial regulation of FH activ-ity. With respect to tumorigenesis, the dysregulation of PAK4activity is identified here as a tumor-specific way to escape fromthe negative effects of TGFb, which potentially provides amolecular basis for improving the clinical treatment againsttumors with upregulated PAK4 activity.

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

Authors' ContributionsConception and design: Y. Jiang, T. Chen, W. Liang, J. He, N. ZhongDevelopment of methodology: T. Chen, T. Wang, W. Liang, Q. Yu, C. Zhou,W. Huang, J. He, N. Zhong

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Chen, T. Wang, W. Liang, Q. Zhao, K. Zheng,C. Zhou, S. Wei, J. Jiang, S. Li, J. HeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Chen, W. Liang, Q. Zhao, D. Guo, C. Zhou, J. HeWriting, review, and/or revision of themanuscript: Y. Jiang, T. Chen, T. Wang,W. Liang, Q. YuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q. Zhao, Q. Yu, C.-M. Ma, L. Zhuo, K. Zheng,C. Zhou, S. Wei, J. Liu, S. Li, J. HeStudy supervision: Y. Jiang, J. He, N. Zhong

AcknowledgmentsWe thank D.-L. Li at the East China Normal University for mice study

assistance, and L.-J. Liao at the East China Normal University for assistanceof mass spectrometry analysis. This work was supported by National NatureScience Foundation of China 81773006 (to Y. Jiang), Shanghai Committeeof Science and Technology 16QA1403200 (to Y. Jiang), Shanghai MunicipalEducation Commission-Gaofeng Clinical Medicine Grant 20161319(to Y. Jiang), Program for Eastern Young Scholar at Shanghai Institutionsof Higher Learning (to Y. Jiang), The First Affiliated Hospital of GuangzhouMedical University, the Open Project of State Key Lab of Respiratory DiseaseGrant SKLRD2016OP006 (to Y. Jiang), SKLRDQN201702 (to T. Chen),National Natural Science Foundation of China 81490534 (to N. Zhong),81871893 (to W. Liang), 81501996 (to W. Liang), Training of ExcellentYouth in Shanghai Health System 2017YQ056 (to T. Wang), GuangdongProvincial Science and Technology Planning Project 2018A030313525 (to T.Chen), the First Affiliated Hospital of Guangzhou Medical University, the"Young Elite Talents Program," and the "Science and Technology SupportProjects 201520-gyfyy" (to T. Chen).

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 indicatethis fact.

Received August 17, 2018; revised December 6, 2018; accepted January 23,2019; published first January 25, 2019.

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2019;79:1383-1397. Published OnlineFirst January 25, 2019.Cancer Res Tao Chen, Ting Wang, Wenhua Liang, et al. Growth Arrest in Lung Cancer Cells

-Induced CellβPAK4 Phosphorylates Fumarase and Blocks TGF

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