Chromatin Remodeling Factor LSH Drives Cancer Progression ...Chromatin remodeling factor LSH is...

Molecular and Cellular Pathobiology

Chromatin Remodeling Factor LSH Drives CancerProgression by Suppressing the Activity ofFumarate HydrataseXiaozhen He1,2,3,4, Bin Yan1,2,3,4, Shuang Liu1, Jiantao Jia1,2,3,Weiwei Lai1,2,3, Xing Xin1,2,3,4,Can-e Tang1, Dixian Luo5, Tan Tan5, Yiqun Jiang1,2,3, Ying Shi1,2,3,4, Yating Liu1,2,3,4,DeshengXiao6, LingChen1,2,3,4, ShaoLiu7,ChaoMao1,2,3,GangYin8,YanCheng9, JiaFan10,11,Ya Cao1,2,3,4, Kathrin Muegge12, and Yongguang Tao1,2,3,4

Abstract

Chromatinmodification is pivotal to the epithelial–mesenchy-mal transition (EMT), which confers potent metastatic potentialto cancer cells. Here, we report a role for the chromatin remodel-ing factor lymphoid-specific helicase (LSH) in nasopharyngealcarcinoma (NPC), a prevalent cancer in China. LSH expressionwas increased inNPC,where it was controlled by the Epstein–Barrvirus-encoded protein LMP1. In NPC cells in vitro and in vivo, LSHpromoted cancer progression in part by regulating expression offumarate hydratase (FH), a core component of the tricarboxylicacid cycle. LSH bound to the FH promoter, recruiting the epige-

netic silencer factor G9a to repress FH transcription. Clinically, wefound that the concentration of TCA intermediates inNPCpatientsera was deregulated in the presence of LSH. RNAi-mediatedsilencing of FH mimicked LSH overexpression, establishingFH as downstream mediator of LSH effects. The TCA intermedi-ates a-KG and citrate potentiated the malignant character ofNPC cells, in part by altering IKKa-dependent EMT gene expres-sion. In this manner, LSH furthered malignant progressionof NPC by modifying cancer cell metabolism to support EMT.Cancer Res; 76(19); 5743–55. �2016 AACR.

IntroductionChromatin-modifying enzymes utilize cofactors and substrates

that are key components of core metabolic pathways. The cellular

concentration of intermediates fluctuates as a function of themetabolic status and thereby transduces homeostatic responsesvia gene expression (1–3). Strikingly, chromatin-modifyingenzymes sense intermediary metabolism products and processthis information into gene regulation, which modulates diseaseprogression, including cancer (4). Lymphoid-specific helicase(LSH), a protein belonging to the SNF2 family of chromatin-remodeling ATPases, is critical for normal development of plantsand mammals by establishing correct DNA methylation levelsand patterns (5–8). LSH maintains genome stability in mamma-lian somatic cells (9, 10). LSH serves as a target for DeltaNp63al-pha driving skin tumorigenesis in vivo and co-operates with theoncogenic function of E2F3 (11, 12). The role of LSH in metab-olism remains unknown.

Epithelial–mesenchymal transition (EMT) is a key mechanismof cancer progression including metastasis (13–17). The meta-bolic reprogramming that is associated with EMT demands fun-damental changes of regulatory networks (16). EMT is a dynamicand reversible process and is provoked by signals from themicroenvironments (17, 18). Whether and how metabolic inter-mediates are involved in EMT during cancer progression remainspoorly understood.

Altered cellular metabolism, in particular the Warburg effect, isa hallmark of cancer cells, with the tricarboxylic acid (TCA) cycle atthe center of oxidative metabolism serving as a robust source forintermediates required for anabolic reactions (19, 20). Oncome-tabolites are defined as metabolites whose abnormal expressioncauses metabolic and epigenetic dysregulation and transforma-tion to malignancy (21). The mitochondrial enzyme fumaratehydratase (FH), a key component of the TCA cycle, catalyzes thehydration of fumarate tomalate and is essential for cellular energy

1Center for Medicine Research, Xiangya Hospital, Central South Univer-sity, Changsha, Hunan, China. 2Cancer Research Institute, School ofBasic Medicine,Central South University, Changsha, Hunan,China. 3KeyLaboratory of Carcinogenesis and Cancer Invasion (Central South Uni-versity), Ministry of Education, Hunan, China. 4Key Laboratory of Car-cinogenesis (Central SouthUniversity),MinistryofHealth,Hunan,China.5National and Local Joint Engineering Laboratory of High-throughputMolecular Diagnosis Technology, Translational Medicine Institute, TheFirst People's Hospital of Chenzhou, University of South China, Chenz-hou,Hunan,China. 6DepartmentofPathology,XiangyaHospital,CentralSouth University, Changsha, Hunan, China. 7Department of Pharmacy,Xiangya Hospital, Central South University, Changsha, Hunan, China.8Department of Pathology, School of Basic Medicine, Central SouthUniversity, Hunan, China. 9Department of Pharmacology, School ofPharmaceutical Sciences, Central South University, Changsha, Hunan,China. 10Liver Surgery Department, Liver Cancer Institute, ZhongshanHospital,KeyLaboratoryofCarcinogenesis andCancer Invasion (FudanUniversity), Ministry of Education, Fudan University, Shanghai, China.11Institute of Biomedical Sciences, Fudan University, Shanghai, China.12Mouse Cancer Genetics Program, National Cancer Institute, BasicScience Program, Leidos Biomedical Research, Inc., Frederick NationalLaboratory for Cancer Research, Frederick, Maryland.

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

X. He, B. Yan, and S. Liu contributed equally to this article.

Corresponding Author: Yongguang Tao, Xiangya Hospital, Central South Uni-versity, 110 Xiangya Road, Changsha 410078, China. Phone: 86-731-84805448;Fax: 86-731-84470589; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-0268

�2016 American Association for Cancer Research.

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production andmacromolecular biosynthesis. FH has been iden-tified as a tumor suppressor, and its inactivation by geneticmutations alters the level of 2-oxoglutarate–dependent oxyge-nases and leads to epigenetic deregulation of oncogenes or tumorsuppressors (21, 22). The molecular mechanisms by which FHgene expression is controlled remain unclear.

Epstein–Barr virus (EBV) infects more than 90% of the globaladult population and contributes to several malignancies includ-ing nasopharyngeal carcinoma (NPC), a prevalent cancer in thesouthern region of China and South-East Asia (23, 24). Epigeneticchanges induced by EBV are key events in the viral process ofcarcinogenesis. Chromatin remodeling factors are crucial factorsof epigenetics and play a critical part in the development of severalmalignancies (25, 26), but their role in the progress of NPCremains unknown.

In this study, we examined the physiologic role of LSH in NPCby focusing on cancer progression. We found that LSH wasoverexpressed in NPC and its expression correlated with EBVinfection. We also demonstrate that LSH directly suppresses FHin complex with G9a. The downregulation of FH promotes cellmigration and invasion in vitro and tumor growth and metastasisin vivo. Moreover, LSH controls expression of TCA intermediatesthat promote cancer progression through activation of IKKa, achromatin modifier, and transcriptional activator. Our studyreveals a critical role of LSH in cancer progression that hasimportant implications for the development of novel therapeuticstrategies to treat NPC.

Materials and MethodsCell culture, antibodies, plasmids, siRNAs, and chemicals

Four latent membrane protein 1 (LMP1)–negative NPC celllines (CNE1,HNE2,HNE3, andHK1) and two LMP1-positive celllines (HNE2-LMP1 andCNE1-LMP1)were obtained fromCancerResearch Institute of Central South University. The EBV-positiveNPC cell line C666-1 was kindly provided by Professor S.W. Tsao,University of Hong Kong, Pokfulam, Hong Kong. The NPC celllines were cultured in RPMI-1640 (GIBCO, Life Technologies)with 10% heat-inactivated FBS (Hyclone; Invitrogen). All celllines were maintained at 37�C with 5% CO2. The cell lines testednegative for mycoplasma contamination. All cell lines were pas-saged less than 10 times after initial revival from frozen stocks. Allcell lines were authenticated by short tandem repeat profilingprior to use. The detail in antibodies, plasmids, siRNAs, andchemicals was listed into Supplementary Material and Methods.

Western blot analysis and coimmunoprecipitation assayDetails of the Western blot analysis and coimmunoprecipita-

tion (co-IP) assay were described previously (27). The detailprocedure was listed into Supplementary Material and Methods.The antibodies used forWestern blot detection were the LSH, G9aantibodies.

Immunohistochemistry analysis and in situ hybridization oftumor biopsies

NPC biopsies, validated by pathologist Dr. Desheng Xiao(Xiangya Hospital, Hunan, China), were obtained from theDepartment of Pathology of Xiangya Hospital. The NPC tissuearray was purchased from Pantomics. IHC analysis of paraffinsections from NPC patient or xenograft samples was describedpreviously (28). In situ hybridization was performed using the

EBV-encoded RNA (EBER) horseradish peroxidase–conjugatedprobe and DAB as substrate from the ISH Kit (Life Technologies),according to the instructions of the manufacturers.

Quantitative real-time PCRDetails of the procedures were described previously (27, 28).

The primer sequences used were used in Supplementary Table S1.The mean � SD of three independent experiments was shown.

Cell proliferation assay, migration and invasion assay, andplate-colony formation assay

Details of the procedures were described previously (27, 28).The detail procedure was listed into Supplementary Material andMethods.

Immunofluorescence assay and Operetta High ContentScreening and High Content Analysis

Details of the procedures were described previously (27). Thedetail procedure was listed into Supplementary Material andMethods.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assays were essential-

ly performed as previously described (27, 28). ChIP DNA wasanalyzed by qPCR with SYBR Green (Bio-Rad) in ABI-7500(Applied Biosystems) using the primers as listed in Supplemen-tary Table S2. The antibodies used are as indicated.

Targeted GC–MS, 2-HG, and TCA metabolite measurementsGC/MS assays were essentially performed as described (29).

The detail procedure was listed into Supplementary Material andMethods.

Nude mice and study approvalA xenograft tumor formation was essentially performed as

previously described (28). The detail procedure was listed intoSupplementary Material and Methods.

Statistical analysisThe experiments were repeated at least 3 times except the nude

mice experiments. Results are expressed as mean � SD or SEM asindicated. A two-tailed Student t test was used for intergroupcomparisons. A P value less than 0.05 was considered statisticallysignificant.

ResultsChromatin remodeling factor LSH is overexpressed in NPC

To determine the role of LSH in NPC, we performed immu-nohistochemical analysis in tissues derived from NPC patients.LSH protein was present in normal inflamed nasopharyngealtissues, and its expression greatly increased in NPC tissues (Fig.1A). Next, NPC tissues were grouped into EBV negative, EBVpositive (þ), and strongly positive (þþ) based on the expressionof EBER and EBV-encoded LMP1 using in situ hybridization andimmunohistochemical analysis, respectively (Fig. 1A). LSHexpression was elevated in EBV-positive NPCs compared withEBV-negative NPCs (Fig. 1B). Overall, the expression of the EBVmarker was positively correlated with LSH protein level in EBV-infected NPC.

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Cancer Res; 76(19) October 1, 2016 Cancer Research5744




Next, we addressed the question whether LSH is associated withthe progression of NPC (30). The expression of LSH was signifi-cantly increased in advanced clinical stage IV comparedwith earlierstages of (P < 0.05; Fig. 1C). LMP1 is expressed in many malig-nancies includingNPC(31).WescreenedLSHexpression inapanelof NPC cells using Western Blot analysis (Supplementary Fig. S1)and selected four NPC cell lines, CNE1, HK1, HNE3, and C666-1,for subsequent studies. We found low levels of LSH in HK1 cells, acell line that is LMP1 negative, whereas the cell line C666-1, whichexpresses theendogenousLMP1protein, had increasedLSHproteinlevels (Fig. 1D). Using two other LMP1-positive NPC cell lines, we

could further confirm the positive relationship between LMP1expression and LSH expression (Fig. 1E).

Overexpression of LSH promotes cancer progression in vitroand in vivo

To uncover the physiologic role of LSH in NPC, we stablyoverexpressed LSH in three NPC cell lines, CNE1-LSH, HK1-LSH,and HNE3-LSH (inlet of Fig. 2A and B; Supplementary Fig. S2A).LSH overexpression increased significantly the growth of all celllines in vitro (Fig. 2A and B; Supplementary Fig. S2A). Stableexpression of LSH in these cell lines enhanced colony formation

Figure 1.

LSH is highly expressed in NPC. A,immunohistochemical analysis was used todetermine LSH protein level (LSH) in an NPCtissue array from NPC patients (top). The level ofEBERwas analyzed by in situ hybridization inNPCtissues (bottom). The level of LMP1 was furtheranalyzed in NPC tissues. B, relative expression ofLSH in NP and NPC tissues with different levels ofEBER. n, number of analyzed samples; � , P < 0.05;�� , P < 0.01. C, elevated expression of LSH issignificantly associated with late stages of NPC.D,Western analysis for detectionof LSHprotein inHK1 and C666-1 cells. E, Western analysis fordetection of LSH protein in CNE1-LMP1 and HNE2-LMP1 cells and the parental cells.

LSH Promotes Cancer Progression by Suppressing FH

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(Fig. 2C and Supplementary Fig. S2B). Stable expression of LSH inCNE1 and HK1 cells increased migration and invasion in an invitro assay (Fig. 2D and E; Supplementary Fig. S2C) andmigrationactivity in a wound healing assay in CNE1 cells (Fig. 2F) andHNE3 cells (Supplementary Fig. S2D).

Furthermore, stable expression of LSH impaired the expressionlevel of epithelial markers (ZO-1 and E-cadherin) and increasedthe expression level of mesenchymal markers (vimentin) inCNE1, HK1, and HNE3 cells (Supplementary Fig. S2E–S2G),suggesting that LSH promotes the transition from the epithelial

Figure 2.

Overexpression of LSH promotes cancer progression in vitro and in vivo. MTT assay was applied to assess cell viability in CNE1 (A) and HK1 (B) NPCs withoverexpression of LSH expression. LSH protein levels are shown in the inset. C, plate colony formation assay was measured in cells as indicated. D and E, themigrating colony number is shown as bar graph (mean � SD from three separate experiments; D), and a representative experiment is shown for invasion (E).F, HK1 cells with a stable expression of LSH were analyzed for their ability to migrate in a wound healing assay. G, a representative experiment is shown forE-cadherin and vimentin in CNE1 cells stably expressing a control vector or LSH using High Content Screening and High Content Analysis. Relative intensity ofE-cadherin (H) and vimentin (I) is shown. Data from nude mice are shown after injection of HK1 cells stably expressing control vector or LSH expressionplasmids, and tumor volume was monitored at indicated time points (J), and tumor weight (K) was recorded. L, CNE1-MOCK and CNE1-LSH cells were injected intothe tail vein of SCID mice. Animals (n ¼ 6 for each group) were euthanized and the development of lung metastases was assessed macroscopically or bymicroscope in paraffin-embedded sections stained with hematoxylin and eosin (H&E). � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.

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stage to the mesenchymal stage. Using high content imagingsystem, stable expression of LSH decreased the staining level ofE-cadherin and increased the staining level of vimentin in CNE1(Fig. 2G), HK1, and HNE3 cells (Supplementary Fig. S2H andS2I). Relative intensity of E-cadherin staining decreased in thepresence of LSH inCNE1,HK1, andHNE3 cells (Fig. 2H),whereasrelative intensity of vimentin staining increased with overexpres-sing of LSH (Fig. 2I). Taken together, LSH can promote growth,migration, and invasion of NPC cancer cells in vitro.

To address the question whether LSH can also play a role inNPC in vivo, we applied a xenograft model. First, we testedxenograft tumor formation in nude mice. We found significantlylarger tumors after 2 months using HEN3-LSH cells as comparedwith HNE3 cells (Supplementary Fig. S3A), whereas the wholebodyweight remained unchanged (Supplementary Fig. S4A). Theinjection of HK1-LSH cells (1 � 107) showed that LSH over-expression significantly increased the tumor size (SupplementaryFig. S3B), tumor volume (Fig. 2J), and tumor weight (Fig. 2K),whereas the whole body weight remained unchanged (Supple-mentary Fig. S4B).

To further extend these observations, we examined EMT mar-kers in biopsies from xenograft tumors in mice. We found thatLSH decreased the expression of the epithelial marker ZO-1 andincreased vimentin (Supplementary Fig. S3D).Moreover, staining

of tumor sections showed that the level of LSH protein expressionwas associated with a decrease in E-cadherin and ZO-1 and anincrease in vimentin (Fig. 3E). Similar changes in EMT markerexpression caused by LSH overexpression were detected in anoth-er panel of tumor tissues (HNE3 and HNE3-LSH; SupplementaryFig. S4C).

To further confirm the role of LSH in vivo, we used an exper-imentalmetastasis model in which the tumor cells (2� 106) weredirectly injected into the tail vein of SCID mice. When animalswere euthanized after 2 months, we found that all CNE1-LSHrecipient mice (6/6) had increased numbers of metastases in thelung and some metastasis at other sites (chest and abdomen;Fig. 2L and Supplementary Fig. S3C). In contrast, control mice(CNE1) had just one animal with signs of lung metastases (1/6).This indicated that LSH promoted the colonization of lung, chest,and abdomenwith tumor cells. Together, our results demonstratethat LSH expression is linked to cell migration, invasion, andtumor growth and colonization in vivo, suggesting a criticalfunction of LSH in tumor growth and metastasis.

Knockdown of LSH inhibits cancer progression in vitro and invivo

To further validate the physiologic role of LSH in NPC carci-nogenesis, we generated stable LSH knockdown in C666-1 cancer

Figure 3.

Knockdown of LSH inhibits cancerprogression in vitro and in vivo. A, theMTT assay was performed to assess cellviability in C666-1 NPCs that were stablytransfected with two distinct LSHshRNAs expression vectors (siLSH#1 andsiLSH#2) and control cells (siCTRL). LSHprotein levels as detected by Westernanalysis are shown in the inset. B and C,a representative experiment is shownfor the migration and invasion assayafter stable knockdown of LSH in C666-1cells (B), and the colony number ofmigratory and invasive cells is shown(C). D, relative intensity of E-cadherinand vimentin is shown. E, a xenograftmodel of tumor growth was establishedin nude mice to evaluate the ability ofC666-1 cells with a stable knockdown ofLSH to form tumors within 27 days, andtumor volume was monitored. F,immunohistochemical analysis fordetection of E-cadherin and b-catenin intumor samples from nude mice.� , P < 0.05; �� , P < 0.01.






cells. The knockdown approach successfully reduced LSH proteinto less than10%(inlet of Fig. 3A). The knockdownof LSH resultedin significantly reduced cell growth (Fig. 3A) and impaired theformation of colonies (Supplementary Fig. S5A and S5B). Fur-thermore, stable knockdown of LSH decreased activity for migra-tion and invasion (Fig. 3B and C). Stable knockdown of LSHincreased relative intensity of E-cadherin staining and decreasedrelative intensity of vimentin staining (Fig. 3D). Next, we injected3 � 106 of C666-1 cells into nude mice and observed that LSHdepletion significantly impaired the tumor volume (Fig. 3E),tumor formation, and tumor weight (Supplementary Fig. S5Cand S5D),whereas the bodyweight didnot change significantly ineither groups (Supplementary Fig. S5E).

Finally, we found that reduction of LSH increased ZO-1 anddecreased vimentin expression in C666-1 cells (SupplementaryFig. S5F), and decreased ZO-1 and E-cadherin expression anddecreased vimentin expression in biopsies from tumors generatedin nudemice (Supplementary Fig. S5G). IHC confirmed that LSHknockdown enhanced E-cadherin and ZO-1 expression, anddiminished vimentin expression in tumors that formed afterinjection into nude mice (Fig. 3F). Taken together, these findingsindicate a physiologic role of LSH in the growth, migration, andinvasion characteristics of NPC cancer cells in vitro and in vivo.

LSH controls FH expression and LSH binds to the fh promoterand interacts with G9a

To understand more about the molecular mechanism and toidentify potential targetsmediating the TCA cycle, we performed aPCR array in C666-1, HK1, and HK1 cells with overexpression ofLSH.We noticed that the FHmRNAwas decreased in C666-1 cellscompared with the level in HK-1 cells (data not shown). More-over, LSH overexpression in HK1 cells repressed specificallymRNAof the FH gene, whereas genes encoding other componentsof the TCA cycle were not affected by LSH overexpression (Fig.4A). The inverse correlation between LSH and FH expression wasfurther corroborated inNPC cells after stable expression of LSH inHNE3, HK1, and CNE1 cells using Western analysis (Fig. 4B). Inaddition, after stable knockdown of LSH in C666-1 cells, FHexpression was increased (Supplementary Fig. S6A), suggesting arepressive regulatory role of LSH in FH expression.

Subsequently, we confirmed the inverse regulation of FH andLSH protein expression in transplanted tumors from nude mice(Supplementary Fig. S6B–S6E). Next, we examined the relation-ship between FH and LSH expression, in human tumors perform-ing IHC analysis in NPC biopsies. While FH was highly expressedin normal inflamed nasopharyngeal tissues (NP), FH proteinexpression was greatly decreased in NPC tumor samples, and FHhad the lowest expression levels in metastatic tissues of NPC(Supplementary Fig. S6F). The evaluation of LSH and FH proteinlevels of all 61 biopsies corroborated the reverse correlationbetween LSH and FH (P < 0.01; Supplementary Fig. S6G). Takentogether, these results indicate that LSH is amajor regulator of FHexpression and downregulates FH protein level in NPC.

Because LSH is localized in the nucleus and acts a chromatinmodifier in DNA methylation (32), we performed ChIP assay todetermine whether LSH could directly bind to the fh promoter.LSH was directly associated with the fh promoter (Fig. 4C).Bisulfite sequencing revealed no differences in CG methylationat the fh promoter in dependence of LSH (data not shown),suggesting other mechanisms than DNA methylation involvedin. G9a, also known as euchromatic histone-lysine N-methyl-

transferase 2, is an important epigenetic regulator, which monoand dimethylates Lysine-9 (33). It has been previously detected ina complex with LSH and implicated as mediator of LSH-inducedgene repression in mice (8). Because we noted colocalization ofG9a and LSH in C666-1 cells using immunofluorescence staining(Fig. 4D), we further evaluated a possible interaction betweenLSH and G9a in human cells. LSH coimmunoprecipitated withG9a and vice versa (Fig. 4E), and Flag-tagged LSH coprecipitatedwith G9a using a Flag pull-down assay (Fig. 4F).

To further address the role of G9a in FH regulation, we per-formed a ChIP assay to examine G9a binding to the fh promoter.G9a was associated with the fh promoter region, and its bindingwas enhanced in LSH overexpressing cells (Fig. 4G). Knockdownof LSH significantly reduced G9a binding to the fh promoter (Fig.4H), indicating that LSH controlled G9a association to the fhpromoter region.Moreover, the level ofH3K4Me3modification, achromatin mark that indicates promoter activity, was concomi-tantly deceased in the presence of LSH (Fig. 4I). Furthermore,sequential ChIP assay was performed to determine whether LSHand G9a were simultaneously present at the fh promoter regions.Successive precipitations of LSH followed by G9a precipitation(Fig. 4J) and vice versa (Fig. 4K) were equally successful, indicat-ing that both, LSH and G9a, were recruited to the fh promoter asan intact complex.

TCA cycle intermediates are dependent on LSH, and TCAintermediates promote cancer progression in NPC cells

To evaluate the efficiency of the TCA cycle in NPC, we first usedGC-MS to detect the intermediates of TCA cycles. We found thatlevels of citrate, a-KG, oxaloacetate (OAA), and cis-aconitate (CisAco) were significantly higher in the sera of patients with NPCthan normal controls (Fig. 5A). Themetabolite levels of succinate,fumarate, and malate were markedly lower in tumor patients ascompared with healthy controls (Fig. 5A).

To address the role of LSH in TCA cycle intermediates, weexamined the concentration of several intermediates in LSH over-expressingNPC.We observed that LSH increased citrate anda-KGconcentration in CNE1, HK1, and HNE3 cells (SupplementaryFig. S7A and S7B). LSH knockdown in C666-1 cells led to asignificant decrease in the concentration of a-KG and citrate(Supplementary Fig. S7C). Furthermore, we found that the ratioof a-KG/succinate and a-KG/fumarate increased significantlyafter stable expression of LSH in CNE1, HK1, and HNE3 cells(Supplementary Fig. S7D and S7E). In contrast, LSH knockdownin C666-1 significantly lowered the ratio of a-KG/succinate anda-KG/fumarate (Supplementary Fig. S7F). Taken together, theTCA cycle intermediates and the ratio of a-KG/succinate anda-KG/fumarate are regulated by LSH. There was no correlationbetween the EBV status and the intermediates of TCA cycles inNPC patients (Supplementary Fig. S8), suggesting that the EBVstatus does not affect TCA cycle–related intermediates in NPCpatients.

To address the role of TCA intermediates in NPC cells, wetreated NPC cells with citrate, cis-Aco, and a-KG. The addition ofcitrate and a-KG promoted plate colony formation in both HK1cells (Fig. 5B) and HNE3 cells (Fig. 5C). Next, we found that thetreatment of citrate and a-KG in CNE1 cells resulted in increasedmigration and invasion in an in vitro assay (Fig. 5D). Also, theaddition of citrate anda-KG inHK1 andHNE3 cells decreased theconcentration of succinate, fumarate, andmalate (SupplementaryFig. S9A and S9B). Furthermore, Western analysis demonstrated a

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decrease of E-cadherin and ZO-1 and an increase of vimentinprotein levels after addition of citrate anda-KG (Fig. 5E) inHNE3cells. Similar findings of E-cadherin, ZO-1, and vimentin altera-tions were detected in CNE1 cells after addition of citrate anda-KG (Supplementary Fig. S9C and S9D), suggesting that theseTCA metabolites can promote EMT. Interestingly, we alsoobserved a slight decrease in FH protein levels under these cultureconditions, suggesting a feedback loop between TCAmetabolitesand FH expression. Lastly, we examined the effect of a-KG onC666-1 tumor characteristics mediated by LSH. The addition of

a-KG increased migration and invasion of C666-1 cells that wasreduced after LSH knockdown (Fig. 5F), suggesting that thepromoting effect of a-KG on tumor characteristics of NPCsdepends in part on the presence of LSH.

TCA intermediates a-KG and citrate decrease the binding ofIKKa to the epithelial makers

Finally, we addressed the question how TCA intermediatesmay regulate gene expression in NPC. Abnormal levels of TCAintermediates might activate nuclear factor kappa-B (NF-kB)

Figure 4.

LSH represses FH expression through the recruitment of G9a to the fh promoter. A, RT-PCR analysis for detection of FH mRNA using total RNA derived fromHK1-MOCK and HK1-LSH cells. B,Western analysis to assess FH protein levels in cells that were stably transfected with a LSH expression plasmid. C, ChIP analysis fordetection of LSH binding to the fh promoter in CNE1 and HK1 cells. D, C666-1 cells were stained with colocalization of LSH and G9a. E, Co-IP of LSH and G9a inlysates derived from C666-1 cells, followed by immunoblotting for detection of LSH or G9a. F, equal amounts of protein from HK1-MOCK and HK1-LSH wereimmunoprecipitated (IP) with anti-Flag M2 agarose and were immunoblotted to detect LSH or G9a. G, ChIP analysis for detection of G9a binding to the fhpromoter in CNE1 and HK1 cells. H, ChIP analysis for detection of G9a binding to the fh promoter in C666-1 cells in the depletion of LSH. I, ChIP analysis for detectionof H3K4Me3 at the fh promoter in CNE1 and HK1 cells. J, ChIP analysis with anti-Flag M2 agarose detected the recruitment of LSH at the fh promoter. K,re-ChIP assay of Flag and G9a detected the binding of LSH at the fh promoter. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.






independent of inhibitor of nuclear factor kappa-B kinase alpha(IKKa; ref. 34). We first examined whether the TCA intermediatesa-KG and citratemay regulate IKKa in NPC, andwe observed thattreatment of CNE1 cells with either citrate or a-KG increasedexpression of IKKa (Fig. 6A). Likewise, IKKa was enhanced inHNE3 cells after addition of citrate and a-KG (Fig. 6B). Further-more, IKKa was increased in both HK1 and HNE3 cells afteraddition of succinate, fumarate, and malate, respectively; mean-while, E-cadherin also changed and FH expression was decreased(Supplementary Fig. S10A–S10F). We noted that LSH increasedIKKa expression inCNE1,HK1, andHNE3 cells (Fig. 6C),whereasLSHknockdown inC666-1 cells led to a significant decrease in theexpression of IKKa (Fig. 6D), consistent with the notion that LSHoverexpression leads to higher levels ofa-KG and citrate, which inturn upregulate IKKa levels. In addition, IHC analysis showedthat LSH increased IKKa expression in the nucleus in CNE1 cellsafter examining transplanted tumors from SCID mice (Fig. 6E).

Because IKKa can function as a chromatin modifier (35–37), weexamined association to IKKa putative target genes.

We observed that IKKawas enriched at the promoters for ZO-1and E-cadherin, and its association greatly reduced in CNE1 andHK1 cells with LSH overexpression (Fig. 6F and G). In contrast,LSH overexpression enhanced the association of IKKa to thevimentin promoter (Fig. 6H), consistent with a positive trans-criptional activity of IKKa (33). Finally, we examined directly therole of TCA intermediates on IKKa recruitment to the promoterregions of genes involved in EMT. Although the addition of eithera-KG or citrate for 3 days reduced the enrichment of IKKa at theZO-1 and E-cadherin promoter regions (Fig. 6I and J), addition ofthese compounds increased IKKa recruitment to the vimentinpromoter (Fig. 6K). Taken together, our data suggest that thechromatin regulator and transcriptional activator IKKa may beinvolved in the regulation of EMTmarkers mediating the effect ofLSH and TCA intermediates.

Figure 5.

TCA intermediates are regulated by LSH and increase cancer progression in NPC cells. A, GC-MS measured the indicated TCA metabolites in the serum ofNPC patients (Tumor) and healthy controls (Normal). Growth in plate colony formation was measured in HK1 (B) and HNE3 (C) cells after the cells were treatedwith TCA intermediates as indicated. D, the colony numbers of migration and invasion cells are shown in CNE1 cells after addition of citrate and a-KG. HNE3cells (E)were treatedwith citrate (left) anda-KG (right) as indicated andWestern analysis performed to assess FH andEMTproteins.F, the effect ofa-KGaddition onmigration and invasion ability was measured after depletion of LSH in C666-1 cells. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.

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Knockdown of FH promotes cancer progressionLastly, we detected FH expression in a panel of NPC cells, and

we selected HNE3 cells for the next study (Supplementary Fig.S11). We generated stable FH knockdown in HNE3 cells, andthe knockdown of FH resulted in the decrease of the epithelialmarker ZO-1, whereas LSH protein level did not change (Fig.7A). Furthermore, FH knockdown decreased significantly therelative intensity of E-cadherin staining and increased therelative intensity of vimentin staining in HNE3 cells (Fig.7B). These findings indicate that FH is directly involved in theregulation of EMT genes and suggest that LSH acts upstream ofthe FH pathway. FH knockdown also increased colony numbers(Fig. 7C) and resulted in an increased capacity for migration

and invasion (Fig. 7D). Lastly, we examined directly the role ofFH in regulation of TCA cycle intermediates. We observed adecreased in succinate, fumarate, and malate concentrationsafter knockdown of FH in HNE3 cells (Fig. 7E), whereasknockdown of FH had no effect on a-KG or citrate (Supple-mentary Fig. S12). However, the ratio of a-KG/succinate anda-KG/fumarate increased after depletion of FH (Fig. 7F). Insummary, LSH represses FH that in turn affects cancer progres-sion through deregulation of TCA intermediates.

Based on our findings, we propose a model for LSH-medi-ated signaling and enhancement of NPC tumorigenesis (Fig.7G). In this model, LSH acts as a driver of cancer progressioninvolving EMT, invasion, and migration. LSH directly targets

Figure 6.

IKKa is a critical regulator of LSH and oncometabolites a-KG and citrate induced cancer progression. CNE1 cells (A) and HNE3 cells (B) were treated with citrate(left) and a-KG (right), and the expression of IKKa and LSH proteins was analyzed. C,Western analysis was used to assess IKKa protein levels in cells as indicated.D, IKKa expression was analyzed in C666-1 cells after stable depletion of LSH. E, immunohistochemical analysis was used to analyze the IKKa expression intransplanted tumor tissues after injection of HK1-LSH cells. ChIP analysis for detection of IKKa binding to the ZO-1 (F), E-cadherin (G), and vimentin (H) promoters inCNE1 and HK1 cells in the presence of LSH. ChIP analysis for detection of IKKa binding to the ZO-1 (I), E-cadherin (J), and vimentin (K) promoters in CNE1cells after addition of a-KG and citrate, respectively. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.






the FH promoter, recruits G9a, and leads to FH repression.Reduction of FH leads to a reduction of succinate, fumarate,and malate. TCA intermediates promote cancer progressionthrough the decrease of epithelial markers and the increase ofmesenchymal marker expression. The changes of epithelialmarker gene expression are medicated by IKKa that directlybind to these promoters.

DiscussionIn this study, we provide evidence that LSH plays a

critical role in cancer progression. Our findings suggest thatLSH acts as a driver in NPC by promoting cell growth,migration, and invasion that are key characteristics of cancerprogression.

Figure 7.

Knockdown of FH promotes cancer progression and working model. A, HNE3 cells were stably transfected with two distinct LSH shRNA expression vectors(siFH#1 and siFH#2) and control cells (siCTRL), respectively.Western analysis detected the levels of FH, ZO-1, and LSHprotein.B,growth in the plate colony formationassay was measured in the depletion of FH. C, relative intensity of E-cadherin and vimentin is shown in the depletion of FH in HNE3 cells. D, a representativeexperiment is shown for the migration and invasion assay after stable knockdown of FH in HNE3 cells. E, GC-MS was used to measure the concentration of indicatedTCA metabolites after stable knockdown of FH in HNE3 cells. F, ratio of a-KG to succinate and ratio of a-KG to fumarate are shown in HNE3 cells that stablyknockdown FH. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001. G, the schematic model of LSH in cancer progression.

He et al.





Acquired epigenetic abnormalities participate together withchromatin alterations in the early stages of carcinogenesis.Because the EBV methylome is unchanged comparing NPC celllines from southern China and the primary NPCs from southernEurope (38), additional studies are necessary to address the role ofEBV and its products in epigenetics. Here, we report that LMP1,encoded by the EBV genome, upregulated the expression level ofLSH in NPCs.

LSH is critical for chromatin function and establishment ofDNA methylation (6, 7, 32, 39). Reports show that LSH con-tributes to the malignant progression of prostate cancer, mela-noma, head and neck cancer, etc. (12, 40, 41); however, themolecular mechanisms are not well understood. Here, we dem-onstrated that LSH was linked to cancer progression of NPC. Wefound that FHexpressionwas repressed in the presence of LSH. FHmay serve as a direct target of LSH function, becausewe foundLSHwas associated with the fh promoter. LSH may repress the fhpromoter independent of DNA methylation. LSH can increasenucleosome density (32), cause RNA polymerase II stalling (42,43), or promote gene silencing via a G9a/GLP complex duringdifferentiation and early development (8). Here, we provideevidence for an interaction of LSH with G9a, recruitment ofG9a to the fh promoter in a LSH-dependent manner, and subse-quent chromatin modification leading to FH promoter repres-sion, thus linking epigenetic regulation by LSH with suppressionof the emerging tumor suppressor gene FH (21, 44, 45).

FH is a key component of the TCA cycle and mediates thereversible conversion of fumarate to malate. Although FH wasdiscovered as a tumor suppressor (46), the regulators of thisimportant TCA cycle enzyme and the consequences of FH down-regulation remain unclear. We found that both fumarate andmalate were decreased in the serum of NPC patients, which isconsistent with TCA intermediate changes observed after inacti-vation of FH caused by genetic mutations (22, 44, 45). Moreover,loss of FH leads to cellular senescence due to formation ofsuccinicGSH, a covalent adduct between fumarate and glutathi-one (47).

Distinct cancer tissue types may affect the selective accumu-lation of TCA metabolite levels in different ways. Metaboliteconcentrations of colon and stomach tissues are superimposedon a metabolic pathway map including the TCA cycle. Forexample, while malate level decreases and citrate level increasesin cancer compared with normal tissue, the concentrations ofCis Aco remain unchanged, comparing caner with normaltissues (48). Also, organ-specific differences were observed inthe metabolite levels of the TCA cycle and other intermediates(48, 49). What causes a selective accumulation of initial meta-bolites of the TCA cycle during tumorigenesis while other TCAintermediates show extremely low concentrations is poorlyunderstood.

Oncometabolite 2-Hydroxyglutarate (2-HG) is a competitiveinhibitor ofa-KG–dependent dioxygenases in gliomas andhema-tological malignancies that carry mutations of isocitrate dehydro-genase genes (IDH1 and IDH2; refs. 50, 51). However, we did notfindmutations of IDH1 and IDH2 genes and any accumulation of2-HG in NPC (data not shown). We found the ratio of a-KG tosuccinate and a-KG to fumarate was increased in NPC patients.Interestingly, an elevated ratio of a-KG to succinate has beenshown to influence the pluripotency state in embryonic stem cellsvia the alterationofmultiple chromatinmodifications (52). Itwillbe interesting to determine which TCA intermediates alter chro-

matin, including histonemodifications andDNAmethylations inNPC and how these epigenetic changes contribute to cancerprogression.

EMT is not required formetastasis but induces chemoresistancein cancer, and the disturbance of the epithelial balance is causedby altering several layers of regulation including epigenetic reg-ulation (18, 53–55). The functional interactions between EMT-inducing transcription factors and the modulators of chromatinconfiguration give crucial insights into the underlyingmechanismof cancer progression (18). Metabolic competition can drivecancer progression (56), and this competition is due to thedisturbed balance of TCA intermediates that could trigger EMT.The reprogramming of gene expression during EMT is initiatedand controlled by signaling pathways that respond to extracellularcues and lead to metabolic reprogramming. Here, we demon-strated that overexpression of LSH is linked to EMT by increasingmigration and invasion ability in NPC. It also indicated that EMTinduction by LMP1 is mediated by LSH. We found that the othermanykey regulators, such as TWIST andSnail, that induce EMTareaffected by LMP1 (Supplementary Fig. S13). Furthermore, LSHoverexpression, as well as deregulation of TCA intermediates,leads to IKKa recruitment to the promoters of EMT-related genes.In this way, LSH induces a cascade of epigenetic and metabolicchanges that result in further epigenetic regulations via IKKa andEMT.

In summary, our study highlights the importance of LSH-mediated regulation of TCA intermediates in cancer progression.LSH, together with G9a, represses FH. Reduced FH level leads to areduction of succinate, fumarate, andmalate, also increases in theratio ofa-KG to fumarate. TCA intermediates including a-KG andcitrate decrease E-cadherin and ZO-1 expression and increasevimentin. The changes of EMT marker gene expression are con-trolled by IKKa that binds directly to these promoters. Thepathway leads to EMT, promotingmigration, invasion, and cancerprogression (Fig. 7G). Repression of LSH and its downstreameffects may serve as potential target for the development of noveltherapeutic approaches.

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

Authors' ContributionsConception and design: X. He, B. Yan, S. Liu, J. Fan, Y. TaoDevelopment of methodology: X. He, B. Yan, S. Liu, C.-e Tang, Y. Jiang, Y. Liu,L. Chen, C. Mao, Y. TaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. He, B. Yan, S. Liu, W. Lai, X. Xin, D. Luo, T. Tan,Y. TaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. He, B. Yan, S. Liu, J. Jia, X. Xin, Y. Jiang, L. Chen,S. Liu, Y. TaoWriting, review, and/or revision of the manuscript: X. He, B. Yan, S. Liu,Y. Cheng, Y. Cao, K. Muegge, Y. TaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. He, B. Yan, S. Liu, J. Jia, W. Lai, X. Xin, D. Luo,Y. Jiang, Y. Shi, D. Xiao, G. Yin, Y. TaoStudy supervision: J. Fan, Y. Tao

Grant SupportThis work was supported by the National Basic Research Program of China

[2011CB504300 (Y. Tao); 2015CB553903 (Y. Tao)]; the National NaturalScience Foundation of China [81171881, 81372427, and 81672787 (Y. Tao),81271763 (S. Liu), 81302354 (Y. Shi), 81201675 (G. Yin), 81300429 (T. Tan),






81422051 and 81472593 (Y. Cheng)]. This project has been funded in part withfederal funds from the Frederick National Laboratory for Cancer Research, NIH,under contract HHSN261200800001E (K. Muegge).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 1, 2016; revised May 26, 2016; accepted May 26, 2016;published OnlineFirst June 14, 2016.

References1. Katada S, Imhof A, Sassone-Corsi P. Connecting threads: Epigenetics and

metabolism. Cell 2012;148:24–8.2. Kaelin WGJr, McKnight SL. Influence of metabolism on epigenetics and

disease. Cell 2013;153:56–69.3. Lu C, Thompson CB. Metabolic regulation of epigenetics. Cell Metab

2012;16:9–17.4. Gut P, Verdin E. The nexus of chromatin regulation and intermediary

metabolism. Nature 2013;502:489–98.5. ZemachA, KimMY,HsiehPH,Coleman-DerrD, Eshed-Williams L, ThaoK,

et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyl-transferases to access H1-containing heterochromatin. Cell 2013;153:193–205.

6. Yu W, McIntosh C, Lister R, Zhu I, Han Y, Ren J, et al. Genome-wide DNAmethylation patterns in LSH mutant reveals de-repression of repeat ele-ments and redundant epigenetic silencing pathways. Genome Res2014;24:1613–23.

7. Tao Y, Xi S, Shan J, Maunakea A, Che A, Briones V, et al. Lsh, chromatinremodeling family member, modulates genome-wide cytosine methyla-tion patterns at nonrepeat sequences. Proc Natl Acad Sci U S A2011;108:5626–31.

8. Myant K, Termanis A, Sundaram AY, Boe T, Li C, Merusi C, et al. LSH andG9a/GLP complex are required for developmentally programmed DNAmethylation. Genome Res 2011;21:83–94.

9. Fan T, Yan Q, Huang J, Austin S, Cho E, Ferris D, et al. Lsh-deficient murineembryonal fibroblasts show reduced proliferation with signs of abnormalmitosis. Cancer Res 2003;63:4677–83.

10. Burrage J, Termanis A, Geissner A, Myant K, Gordon K, Stancheva I. TheSNF2 family ATPase LSHpromotes phosphorylation ofH2AX and efficientrepair of DNA double-strand breaks in mammalian cells. J Cell Sci2012;125(Pt 22):5524–34.

11. Keyes WM, Pecoraro M, Aranda V, Vernersson-Lindahl E, Li W, Vogel H,et al. DeltaNp63alpha is an oncogene that targets chromatin remodeler Lshto drive skin stem cell proliferation and tumorigenesis. Cell Stem Cell2011;8:164–76.

12. von Eyss B, Maaskola J, Memczak S, Mollmann K, Schuetz A, Lod-denkemper C, et al. The SNF2-like helicase HELLS mediates E2F3-dependent transcription and cellular transformation. EMBO J 2012;31:972–85.

13. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition.J Clin Invest 2009;119:1420–8.

14. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymaltransitions in development and disease. Cell 2009;139:871–90.

15. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. Theepithelial-mesenchymal transition generates cells with properties of stemcells. Cell 2008;133:704–15.

16. De Craene B, Berx G. Regulatory networks defining EMT during cancerinitiation and progression. Nat Rev Cancer 2013;13:97–110.

17. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mes-enchymal transition. Nat Rev Mol Cell Biol 2014;15:178–96.

18. Tam WL, Weinberg RA. The epigenetics of epithelial-mesenchymal plas-ticity in cancer. Nat Med 2013;19:1438–49.

19. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell2011;144:646–74.

20. Desideri E, Vegliante R, Ciriolo MR. Mitochondrial dysfunctions in cancer:Genetic defects and oncogenic signaling impinging on TCA cycle activity.Cancer Lett 2015;356(2 Pt A):217–23.

21. Thompson CB. Metabolic enzymes as oncogenes or tumor suppressors.N Engl J Med 2009;360:813–5.

22. Adam J, Yang M, Soga T, Pollard PJ. Rare insights into cancer biology.Oncogene 2014;33:2547–56.

23. Mesri EA, Feitelson MA, Munger K. Human viral oncogenesis: A cancerhallmarks analysis. Cell Host Microbe 2014;15:266–82.

24. Lieberman PM. Virology. Epstein-Barr virus turns 50. Science 2014;343:1323–5.

25. Suva ML, Riggi N, Bernstein BE. Epigenetic reprogramming in cancer.Science 2013;339:1567–70.

26. Chen T, Dent SY. Chromatin modifiers and remodellers: Regulators ofcellular differentiation. Nat Rev Genet 2014;15:93–106.

27. Shi Y, Tao Y, Jiang Y, Xu Y, Yan B, Chen X, et al. Nuclear epidermal growthfactor receptor interacts with transcriptional intermediary factor 2 toactivate cyclin D1 gene expression triggered by the oncoprotein latentmembrane protein 1. Carcinogenesis 2012;33:1468–78.

28. Jiang Y, Yan B, Lai W, Shi Y, Xiao D, Jia J, et al. Repression of Hox genes byLMP1 in nasopharyngeal carcinoma andmodulation of glycolytic pathwaygenes by HoxC8. Oncogene 2015;34:6079–91.

29. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, et al.Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature2009;462:739–44.

30. LoKW,ToKF,HuangDP. Focus onnasopharyngeal carcinoma.CancerCell2004;5:423–8.

31. Raab-Traub N. Epstein–Barr virus transforming proteins: Biologic proper-ties and contribution to oncogenesis. In:Damania B, Pipas JM, editors.DNA tumor viruses. New York, NY: Springer; 2009. p. 259–84.

32. Ren J, Briones V, Barbour S, Yu W, Han Y, Terashima M, et al. The ATPbinding site of the chromatin remodeling homolog Lsh is required fornucleosome density and de novo DNA methylation at repeat sequences.Nucleic Acids Res 2015;43:1444–55.

33. Shinkai Y, Tachibana M. H3K9 methyltransferase G9a and the relatedmolecule GLP. Genes Dev 2011;25:781–8.

34. Shanmugasundaram K, Nayak B, Shim EH, Livi CB, Block K, SudarshanS. The oncometabolite fumarate promotes pseudohypoxia throughnoncanonical activation of NF-kappaB signaling. J Biol Chem 2014;289:24691–9.

35. Anest V, Hanson JL, Cogswell PC, Steinbrecher KA, Strahl BD, Baldwin AS.A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-depen-dent gene expression. Nature 2003;423:659–63.

36. Yamamoto Y, Verma UN, Prajapati S, Kwak YT, Gaynor RB. Histone H3phosphorylation by IKK-alpha is critical for cytokine-induced gene expres-sion. Nature 2003;423:655–9.

37. Huang WC, Ju TK, Hung MC, Chen CC. Phosphorylation of CBP byIKKalpha promotes cell growth by switching the binding preference ofCBP from p53 to NF-kappaB. Mol Cell 2007;26:75–87.

38. Fernandez AF, Rosales C, Lopez-Nieva P, Grana O, Ballestar E, Ropero S,et al. The dynamic DNA methylomes of double-stranded DNA virusesassociated with human cancer. Genome Res 2009;19:438–51.

39. Yu W, Briones V, Lister R, McIntosh C, Han Y, Lee EY, et al. CG hypo-methylation in Lsh�/�mouse embryonic fibroblasts is associated with denovo H3K4me1 formation and altered cellular plasticity. Proc Natl AcadSci U S A 2014;111:5890–5.

40. Waseem A, Ali M, Odell EW, Fortune F, Teh MT. Downstream targets ofFOXM1: CEP55 and HELLS are cancer progression markers of head andneck squamous cell carcinoma. Oral Oncol 2010;46:536–42.

41. Ryu B, KimDS,Deluca AM, Alani RM. Comprehensive expression profilingof tumor cell lines identifies molecular signatures of melanoma progres-sion. PLoS One 2007;2:e594.

42. Tao Y, Liu S, Briones V, Geiman TM, Muegge K. Treatment of breast cancercells with DNA demethylating agents leads to a release of Pol II stalling atgenes with DNA-hypermethylated regions upstream of TSS. Nucleic AcidsRes 2011;39:9508–20.

43. Tao Y, Xi S, Briones V, Muegge K. Lsh mediated RNA polymerase II stallingatHoxC6andHoxC8 involvesDNAmethylation. PLoSOne2010;5:e9163.

44. Adam J, Yang M, Bauerschmidt C, Kitagawa M, O'Flaherty L, MaheswaranP, et al. A role for cytosolic fumarate hydratase in urea cycle metabolismand renal neoplasia. Cell Rep 2013;3:1440–8.

He et al.





45. TernetteN, YangM, LaroyiaM,KitagawaM,O'Flaherty L,Wolhulter K, et al.Inhibition of mitochondrial aconitase by succination in fumarate hydra-tase deficiency. Cell Rep 2013;3:689–700.

46. Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, et al.Germline mutations in FH predispose to dominantly inherited uterinefibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet2002;30:406–10.

47. Zheng L, Cardaci S, Jerby L, MacKenzie ED, Sciacovelli M, Johnson TI, et al.Fumarate induces redox-dependent senescence by modifying glutathionemetabolism. Nat Commun 2015;6:6001.

48. Hirayama A, Kami K, Sugimoto M, Sugawara M, Toki N, Onozuka H, et al.Quantitative metabolome profiling of colon and stomach cancer micro-environment by capillary electrophoresis time-of-flightmass spectrometry.Cancer Res 2009;69:4918–25.

49. Ho PC, Bihuniak JD, Macintyre AN, Staron M, Liu X, Amezquita R, et al.Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cellresponses. Cell 2015;162:1217–28.

50. Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH, et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-depen-dent dioxygenases. Cancer Cell 2011;19:17–30.

51. Turcan S, Rohle D, Goenka A, Walsh LA, Fang F, Yilmaz E, et al. IDH1mutation is sufficient to establish the glioma hypermethylator phenotype.Nature 2012;483:479–83.

52. Carey BW, Finley LW, Cross JR, Allis CD, Thompson CB. Intracellularalpha-ketoglutarate maintains the pluripotency of embryonic stem cells.Nature 2015;518:413–6.

53. Easwaran H, Tsai HC, Baylin SB. Cancer epigenetics: Tumor heterogeneity,plasticity of stem-like states, and drug resistance. Mol Cell 2014;54:716–27.

54. Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, et al.Epithelial-to-mesenchymal transition is dispensable for metastasis butinduces chemoresistance in pancreatic cancer. Nature 2015;527:525–30.

55. Fischer KR, Durrans A, Lee S, Sheng J, Li F, Wong ST, et al.Epithelial-to-mesenchymal transition is not required for lungmetastasis but contributes to chemoresistance. Nature 2015;527:472–6.

56. Chang CH, Qiu J, O'Sullivan D, Buck MD, Noguchi T, Curtis JD, et al.Metabolic competition in the tumormicroenvironment is a driver of cancerprogression. Cell 2015;162:1229–41.






2016;76:5743-5755. Published OnlineFirst June 14, 2016.Cancer Res Xiaozhen He, Bin Yan, Shuang Liu, et al. Suppressing the Activity of Fumarate HydrataseChromatin Remodeling Factor LSH Drives Cancer Progression by

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