chromatin remodeling factor lsh drives cancer progression ... · promoted cancer progression in...

Chromatin remodeling factor LSH drives cancer progression by suppressing the activity of fumarate hydratase

Xiaozhen 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, Desheng Xiao6, Ling Chen1,2,3,4, Shao Liu7, Chao Mao1,2,3, Gang Yin8, Yan Cheng9, Jia Fan10,11, Ya Cao1,2,3,4, Kathrin Muegge12, Yongguang Tao1,2,3,4,+ 1 Center for Medicine Research, Xiangya Hospital, Central South University, Changsha, Hunan,

410008 China 2 Cancer Research Institute, School of Basic Medicine, Central South University, Changsha, Hunan,

410078 China 3 Key Laboratory of Carcinogenesis and Cancer Invasion (Central South University), Ministry of

Education, Hunan, 410078 China 4 Key Laboratory of Carcinogenesis (Central South University), Ministry of Health, Hunan, 410078

China 5 National and Local Joint Engineering Laboratory of High-throughput Molecular Diagnosis

Technology,Translational Medicine Institute,the First People's Hospital of Chenzhou,University of South China, 102 Luojiajing Road, Chenzhou 423000, Hunan, China

6 Department of Pathology, Xiangya Hospital, Central South University, Changsha, Hunan 410078 China

7 Department of Pharmacy, Xiangya Hospital, Central South University, Changsha, Hunan 410078 China

8 Department of Pathology, School of Basic Medicine, Central South University, 172 TongZiPo Rd. Changsha, Hunan, 410013 China

9 Department of pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410078 China

10 Liver Surgery Department, Liver Cancer Institute, Zhongshan Hospital, Key Laboratory of Carcinogenesis and Cancer Invasion (Fudan University), Ministry of Education, Fudan University, Shanghai 200032, China

11 Institute of Biomedical Sciences, Fudan University, Shanghai 200032, China 12 Mouse Cancer Genetics Program, National Cancer Institute, Basic Science Program, Leidos

Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA

* Equal contribution + Corresponding author. Y.T. Email: [email protected] ; Tel. +(86) 731-84805448; Fax. +(86) 731-84470589. Running title: LSH promotes cancer progression by suppressing FH.

Conflict of interest: The authors declare no conflict of interest. This manuscript has

been read and approved by all the authors, and not submitted or under consider for

publication elsewhere.

on March 4, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 14, 2016; DOI: 10.1158/0008-5472.CAN-16-0268

http://cancerres.aacrjournals.org/

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Financial support: This work was supported by the National Basic Research

Program of China [2011CB504300 (Y.Tao); 2015CB553903(Y.Tao)]; the National

Natural Science Foundation of China [81171881 and 81372427(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 with Federal funds

from the Frederick National Laboratory for Cancer Research, National Institutes of

Health, under contract HHSN261200800001E (K.Muegge).




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Abstract

Chromatin modification is pivotal to the epithelial-mesenchymal transition (EMT)

which confers potent metastatic potential to cancer cells. Here we report a role for the

chromatin remodeling factor LSH in nasopharyngeal carcinoma (NPC), a prevalent

cancer in China. LSH expression was increased in NPC, where it was controlled by

the Epstein-Barr virus-encoded protein LMP1. In NPC cells in vitro and in vivo, LSH

promoted cancer progression in part by regulating expression of fumarate hydratase

(FH), a core component of the tricarboxylic acid (TCA) cycle. LSH bound to the FH

promoter, recruiting the epigenetic silencer factor G9a to repress FH transcription.

Clinically, we found that the concentration of TCA intermediates in NPC patient sera

was deregulated in the presence of LSH. RNAi-mediated silending of FH mimicked

LSH overexpression, establishing FH as downstream mediator of LSH effects. The

TCA intermediates α-KG and citrate potentiated the malignant character of NPC cells,

in part by altering IKKα-dependent EMT gene expression. In this manner, LSH

furthered malignant progression of NPC by modifiying cancer cell metabolism to

support EMT.

Keywords: LSH, EMT, E-Cadherin, ZO-1, Vimentin, TCA intermediates, IKKα, G9a,

citrate, α-KG, EBV, LMP1, cancer progression




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Introduction

Chromatin-modifying enzymes utilize co-factors and substrates that are key

components of core metabolic pathways. The cellular concentration of intermediates

fluctuates as a function of the metabolic status and thereby transduces homeostatic

responses via gene expression (1-3). Strikingly, chromatin-modifying enzymes sense

intermediary metabolism products and process this information into gene regulation

which modulate disease progression, including cancer (4). Lymphoid-specific helicase

(LSH), a protein belonging to the SNF2 family of chromatin-remodeling ATPases, is

critical for normal development of plants and mammals by establishing correct DNA

methylation levels and patterns (5-8). LSH maintains genome stability in mammalian

somatic cells (9,10). LSH serves as a target for DeltaNp63alpha driving skin

tumorigenesis in vivo and co-operates with the oncogenic function of E2F3 (11,12).

The role of LSH in metabolism remains unknown.

Epithelial-mesenchymal transition (EMT) is a key mechanism of cancer

progression including metastasis (13-17). The metabolic reprogramming that is

associated with EMT demands fundamental changes of regulatory networks (16).

EMT is a dynamic and reversible process and is provoked by signals from the

microenvironments (17,18). Whether and how metabolic intermediates are involved

in EMT during cancer progression remains poorly understood.

Altered cellular metabolism, in particular the Warburg effect, is a hallmark of

cancer cells, with the tricarboxylic acid (TCA) cycle at the center of oxidative

metabolism serving as a robust source for intermediates required for anabolic




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reactions (19,20). Oncometabolites are defined as metabolites whose abnormal

expression causes metabolic and epigenetic dysregulation and transformation to

malignancy (21). The mitochondrial enzyme fumarate hydratase (FH), a key

component of the TCA cycle, catalyzes the hydration of fumarate to malate and is

essential for cellular energy production and macromolecular biosynthesis. FH has

been identified as a tumor suppressor, and its inactivation by genetic mutations alters

the level of 2-oxoglutarate-dependent oxygenases and leads to epigenetic deregulation

of oncogenes or tumor suppressors (21,22). The molecular mechanisms by which FH

gene expression is controlled remain unclear.

Epstein-Barr virus (EBV) infects more than 90% of the global adult population,

and contributes to several malignancies including nasopharyngeal carcinoma (NPC), a

prevalent cancer in the southern region of China and South-East Asia (23,24).

Epigenetic changes induced by EBV are key events in the viral process of

carcinogenesis. Chromatin remodeling factors are crucial factors of epigenetics and

play a critical part in the development of several malignancies (25,26), but their role

in the progress of NPC remains unknown.

In this study, we examined the physiological role of LSH in NPC by focusing on

cancer progression. We found that LSH was overexpressed in NPC and its expression

correlated with EBV infection. We also demonstrate that LSH directly suppresses FH

in complex with G9a. The downregulation of FH promotes cell migration and

invasion in vitro and tumor growth and metastasis in vivo. Moreover, LSH controls

expression of TCA intermediates that promote cancer progression through activation




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of IKKα, a chromatin modifier and transcriptional activator. Our study reveals a

critical role of LSH in cancer progression that has important implications for the

development of novel therapeutic strategies to treat NPC.




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Material and methods

Cell culture, antibodies, plasmids, siRNAs and chemicals.

Four LMP1-negative NPC cell lines (CNE1, HNE2, HNE3 and HK1) and two

LMP1-positive cell lines (HNE2-LMP1, CNE1-LMP1) were obtained from Cancer

Research Institute of Central South University. The EBV-positive NPC cell line

C666-1 was kindly provided by Professor S.W. Tsao, University of Hong Kong. The

NPC cell lines were cultured in RPMI-1640 (GIBCO, Life Technologies, Basel,

Switzerland) with 10% heat-inactivated FBS (Hyclone, Invitrogen). All cell lines

were maintained at 37°C with 5% CO2. The cell lines tested negative for mycoplasma

contamination. All cell lines were passaged less than 10 times after initial revival

from frozen stocks. All cell lines were authenticated by short tandem repeat profiling

prior to use. The detail in antibodies, plasmids, siRNAs and chemicals was listed into

the supplementary Material and Methods.

Western blot analysis and Co- Immunoprecipitation (Co-IP) assay

Details of the Western blot analysis and Co- Immunoprecipitation (Co-IP) assay

were described previously (27). The detail procedure was listed into the

supplementary Material and Methods. The antibodies used for Western blot detection

were the LSH, G9a antibodies.

Immunohistochemistry (IHC) analysis and in situ hybridization of tumor biopsies

NPC biopsies, validated by pathologist Dr. Desheng Xiao (Xiangya Hospital),

were obtained from the Department of Pathology of Xiangya Hospital. The NPC

tissue array was purchased from Pantomics (Richmond, CA, USA). IHC analysis of




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paraffin sections from NPC patient or xenograft samples was described previously

(28). In situ hydridization was performed using the EBV-encoded RNA (EBER) HRP

conjugated probe and DAB as substrate from the ISH kit (Life technologies),

according to the instructions of the manufacturers.

Quantitative real-time PCR

Details of the procedures were described previously (27,28). The primer

sequences used were used in the supplementary Table S1. The mean± SD of three

independent experiments was shown.

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

assay.

Details of the procedures were described previously (27,28). The detail procedure

was listed into the supplementary Material and Methods.

Immunofluorescence assay and Operetta® High Content Screening and High

Content Analysis

Details of the procedures were described previously (27). The detail procedure


Chromatin immunoprecipitation (ChIP) assay.

ChIP assays were essentially performed as previously described (27,28). ChIP

DNA was analyzed by qPCR with SYBR Green (Bio-Rad) in ABI-7500 (Applied

Biosystems) using the primers as listed in the supplementary Table S2. The antibodies

used are as indicated.




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Targeted GC–MS, 2HG and TCA metabolite measurements

GC/MS assays were essentially performed as described (29). The detail procedure


Nude mice and study approval

A xenograft tumor formation was essentially performed as previously described

(28). The detail procedure was listed into the supplementary Material and Methods.

Statistics

The experiments were repeated at least three times except the nude mice

experiments. Results are expressed as mean ± SD or SEM as indicated. A 2-tailed

Student’s t test was used for intergroup comparisons. A p value less than 0.05 was

considered statistically significant.




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Results

Chromatin remodeling factor LSH is overexpressed in NPC.

To determine the role of LSH in NPC, we performed immunohistochemical

analysis in tissues derived from NPC patients. LSH protein was present in normal

inflamed nasopharyngeal tissues, and its expression greatly increased in NPC tissues

(Figure 1A). Next, NPC tissues were grouped into EBV negative, EBV positive (+)

and strongly positive (++) based on the expression of EBER and EBV-encoded latent

membrane protein 1 (LMP1) using in situ hybridization and immuonhistochemical

analysis, respectively (Figure 1A). LSH expression was elevated in EBV positive

NPCs compared to EBV negative NPCs (Figure 1B). Overall, the expression of the

EBV marker was positively correlated with LSH protein level in EBV infected NPC.

Next, we addressed the question whether LSH is associated with the progression

of NPC (30). The expression of LSH was significantly increased in advanced clinical

stage IV compared to earlier stages of (p<0.05) (Figure 1C). LMP1 is expressed in

many malignancies including NPC (31). We screened LSH expression in a panel of

NPC cells using Western Blot analysis (Figure 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, a cell line that is LMP1 negative, whereas the cell line C666-1, which

expresses the endogenous LMP1 protein, had increased LSH protein levels (Figure

1D). Using two other LMP1-positive NPC cell lines, we could further confirm the

positive relationship between LMP1 expression and LSH expression (Figure 1E).

Overexpression of LSH promotes cancer progression in vitro and in vivo




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To uncover the physiological role of LSH in NPC, we stably overexpressed LSH

in three NPC cell lines, CNE1-LSH, HK1-LSH and HNE3-LSH (inlet of Figure 2A，

Figure 2B and Figure S2A). LSH overexpression increased significantly the growth of

all cell lines in vitro (Figure 2A, Figure 2B and Figure S2A). Stable expression of

LSH in these cell lines enhanced colony formation (Figure 2C and Figure S2B).

Stable expression of LSH in CNE1 and HK1 cells increased migration and invasion in

an in vitro assay (Figure 2D, Figure 2E, and Figure S2C) and migration activity in a

wound healing assay in CNE1 cells (Figure 2F) and HNE3 cells (Figure S2D).

Furthermore, stable expression of LSH impaired the expression level of epithelial

markers (ZO-1 and E-cadherin) and increased the expression level of mesenchymal

markers (Vimentin) in CNE1, HK1 and HNE3 cells (Figure S2E-G), suggesting that

LSH promotes the transition from the epithelial stage to the mesenchymal stage.

Using high content imaging system, stable expression of LSH decreased the staining

level of E-cadherin and increased the staining level of Vimentin in CNE1 cells

(Figure 2G), HK1 and HNE3 cells (Figure S2H,I). Relative intensity of E-cadherin

staining decreased in the presence of LSH in CNE1, HK1 and HNE3 cells (Figure

2H), whereas relative intensity of Vimentin staining increased with over expressing of

LSH (Figure 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 in NPC in vivo, we

applied a xenograft model. First, we tested xenograft tumor formation in nude mice.

We found significantly larger tumors after two months using HEN3-LSH cells as




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compared to HNE3 cells (Figure S3A) whereas the whole body weight remained

unchanged (Figure S4A). The injection of HK1-LSH cells (1 × 107) showed that LSH

overexpression significantly increased the tumor size (Figure S3B), tumor volume

(Figure 2J) and tumor weight (Figure 2K) while the whole body weight remained

unchanged (Figure S4B).

To further extend these observations, we examined EMT markers in biopsies

from xenograft tumors in mice. We found that LSH decreased the expression of the

epithelial marker ZO-1 and increased Vimentin (Figure S3D). Moreover, staining of

tumor sections showed that the level of LSH protein expression was associated with a

decrease in E-cadherin and ZO-1 and an increase in Vimentin (Figure 3E). Similar

changes in EMT marker expression caused by LSH overexpression were detected in

another panel of tumor tissues (HNE3 and HNE3-LSH) (Figure S4C).

To further confirm the role of LSH in vivo, we used an experimental metastasis

model in which the tumor cells (2 × 106) were directly injected into the tail vein of

SCID mice. When animals were euthanized after two months, we found that all

CNE1-LSH recipient mice (6/6) had increased numbers of metastases in the lung and

some metastasis at other sites (chest and abdomen) (Figure 2L and Figure 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 abdomen with

tumor cells. Together, our results demonstrate that LSH expression is linked to cell

migration, invasion, and tumor growth and colonization in vivo, suggesting a critical

function of LSH in tumor growth and metastasis.




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Knockdown of LSH inhibits cancer progression in vitro and in vivo

To further validate the physiological role of LSH in NPC carcinogenesis, we

generated stable LSH knockdown in C666-1 cancer cells. The knockdown approach

successfully reduced LSH protein to less than 10% (inlet of Figure 3A). The

knockdown of LSH resulted in significantly reduced cell growth (Figure 3A) and

impaired the formation of colonies (Figure S5A-B). Furthermore, stable knockdown

of LSH decreased activity for migration and invasion (Figure 3B-C). Stable

knockdown of LSH increased relative intensity of E-cadherin staining and decreased

relative intensity of Vimentin staining (Figure 3D). Next, we injected 3 × 106 of

C666-1 cells into nude mice, and observed that LSH depletion significantly impaired

the tumor volume (Figure 3E), tumor formation and tumor weight (Figure S5C-D),

while the body weight did not change significantly in either groups (Figure S5E).

Finally, we found that reduction of LSH increased ZO-1 and decreased Vimentin

expression in C666-1 cells (Figure S5F), and decreased ZO-1 and E-Cadherin

expression and decreased Vimentin expression in biopsies from tumors generated in

nude mice (Figure S5G). Immunohistochemistry confirmed that LSH knockdown

enhanced E-Cadherin and ZO-1 expression, and diminished Vimentin expression in

tumors that formed after injection into nude mice (Figure 3F). Taken together, these

findings indicate a physiologic role of LSH in the growth, migration and invasion

characteristics of NPC cancer cells in vitro and in vivo.

LSH controls fumarate hydratase expression and LSH binds to the fh promoter

and interacts with G9a.




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To understand more about the molecular mechanism and to identify potential

targets mediating the TCA cycle, we performed a PCR-array in C666-1 cells, HK1

cells and HK1 cells with overexpression of LSH. We noticed that the FH mRNA was

decreased in C666-1 cells compared to the level in HK-1 cells (data not shown).

Moreover, LSH overexpression in HK1 cells repressed specifically mRNA of the FH

gene, whereas genes encoding other components of the TCA cycle were not affected

by LSH overexpression (Figure 4A). The inverse correlation between LSH and FH

expression was further corroborated in NPC cells after stable expression of LSH in

HNE3, HK1 and CNE1 cells using Western analysis (Figure 4B). In addition, after

stable knockdown of LSH in C666-1 cells, FH expression was increased (Figure S6A)

suggesting a repressive regulatory role of LSH in FH expression.

Subsequently, we confirmed the inverse regulation of FH and LSH protein

expression in transplanted tumors from nude mice (Figure S6B-E). Next, we

examined the relationship between FH and LSH expression, in human tumors

performing immunohistochemistry analysis in NPC biopsies. While FH was highly

expressed in normal inflamed nasopharyngeal tissues (NP), FH protein expression

was greatly decreased in NPC tumor samples, and FH had the lowest expression

levels in metastatic tissues of NPC (Figure S6F). The evaluation of LSH and FH

protein levels of all 61 biopsies corroborated the reverse correlation between LSH and

FH (p <0.01) (Figure S6G). Taken together, these results indicate that LSH is a major

regulator of FH expression and downregulates FH protein level in NPC.

Since LSH is localized in the nucleus and acts a chromatin modifier in DNA




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methylation (32), we performed ChIP assay to determine whether LSH could directly

bind to the fh promoter. LSH was directly associated with the fh promoter (Figure 4C).

Bisulfite sequencing revealed no differences in CG methylation at the fh promoter in

dependence of LSH (data not shown), suggesting other mechanisms than DNA

methylation involved in. G9a, also known as euchromatic histone-lysine

N-methyltransferase 2, is an important epigenetic regulator, which mono and

dimethylates Lysine-9 (33). It has been previously detected in a complex with LSH

and implicated as mediator of LSH induced gene repression in mice (8). Since we

noted co-localization of G9a and LSH in C666-1 cells using immunofluorescence

staining (Figure 4D), we further evaluated a possible interaction between LSH and

G9a in human cells. LSH co-immunoprecipitated with G9a and vice versa (Figure 4E),

and Flag-tagged LSH co-precipitated with G9a using a Flag pull-down assay (Figure

4F).

To further address the role of G9a in FH regulation, we performed a ChIP assay

to examine G9a binding to the fh promoter. G9a was associated with the fh promoter

region, and its binding was enhanced in LSH overexpressing cells (Figure 4G).

Knockdown of LSH significantly reduced G9a binding to the fh promoter (Figure 4H),

indicating that LSH controlled G9a association to the fh promoter region. Moreover,

the level of H3K4Me3 modification, a chromatin mark that indicates promoter

activity, was concomitantly deceased in the presence of LSH (Figure 4I). Furthermore,

sequential ChIP assay was performed to determine whether LSH and G9a were

simultaneously present at the fh promoter regions. Successive precipitations of LSH




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followed by G9a precipitation (Figure 4J) and vice versa (Figure 4K), were equally

successful, indicating that both, LSH and G9a, were recruited to the fh promoter as an

intact complex.

Tricarboxylic acid cycle intermediates are dependent on LSH and TCA

intermediates promotes cancer progression in NPC cells

To evaluate the efficiency of the TCA cycle in NPC, we first used GC-MS to

detect the intermediates of TCA cycles. We found that levels of citrate, α-KG,

oxaloacetate (OAA) and cis-aconitate (Cis Aco) were significantly higher in the sera

of patients with NPC than normal controls (Figure 5A). The metabolite levels of

succinate, fumarate, and malate were markedly lower in tumor patients as compared

to healthy controls (Figure 5A).

To address the role of LSH in TCA cycle intermediates, we examined the

concentration of several intermediates in LSH over expressing NPC. We observed that

LSH increased citrate and α-KG concentration in CNE1, HK1 and HNE3 cells

(Figure S7A-B). LSH knockdown in C666-1 cells led to a significant decrease in the

concentration of α-KG and citrate (Figure S7C). Furthermore, we found that the ratio

of α-KG/succinate and α-KG/fumarate increased significantly after stable expression

of LSH in CNE1, HK1 and HNE3 cells (Figure S7D-E). In contrast, LSH knockdown

in C666-1 significantly lowered the ratio of α-KG/succinate and α-KG/fumarate

(Figure S7F). Taken together, the TCA cycle intermediates and the ratio of

α-KG/succinate and α-KG/fumarate are regulated by LSH. There was no correlation

between the EBV status and the intermediates of TCA cycles in NPC patients (Figure




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S8), suggesting that the EBV status does not affect TCA cycle related intermediates in

NPC patients.

To address the role of TCA intermediates in NPC cells, we treated NPC cells with

citrate, cis-Aco and α-KG. The addition of citrate and α-KG promoted plate colony

formation in both HK1 cells (Figure 5B) and HNE3 cells (Figure 5C). Next, we found

that the treatment of citrate and α-KG in CNE1 cells resulted in increased migration

and invasion in an in vitro assay (Figure 5D). Also, the addition of citrate and α-KG in

HK1 and HNE3 cells decreased the concentration of succinate, fumarate and malate

(Figure S9A-B). Furthermore, Western analysis demonstrated a decrease of

E-cadherin and ZO-1 and an increase of Vimentin protein levels after addition of

citrate and α-KG (Figure 5E) in HNE3 cells. Similar findings of E-cadherin, ZO-1

and Vimentin alterations were detected in CNE1 cells after addition of citrate and

α-KG (Figure S9C-D), suggesting that these TCA metabolites can promote EMT.

Interestingly, we also observed a slight decrease in FH protein levels under these

culture conditions, suggesting a feedback loop between TCA metabolites and FH

expression. Lastly, we examined the effect of α-KG on C666-1 tumor characteristics

mediated by LSH. The addition of α-KG increased migration and invasion of C666-1

cells that was reduced after LSH knockdown (Figure 5F), suggesting that the

promoting effect of α-KG on tumor characteristics of NPCs depends in part on the

presence of LSH.

TCA intermediates α-KG and citrate decrease the binding of IKKα to the

epithelial makers




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Finally, we addressed the question how TCA intermediates may regulate gene

expression in NPC. Abnormal levels of TCA intermediates might activate nuclear

factor kappa-B (NF-κB) independent of inhibitor of nuclear factor kappa-B kinase

alpha (IKKα) (34). We first examined whether the TCA intermediates α-KG and

citrate may regulate IKKα in NPC, and we observed that treatment of CNE1 cells

with either citrate or α-KG increased expression of IKKα (Figure 6A). Likewise,

IKKα was enhanced in HNE3 cells after addition of citrate and α-KG (Figure 6B).

Furthermore, IKKα was increased in both HK1 and HNE3 cells after addition of

succinate, fumarate and malate respectively, meanwhile E-cadherin also changed and

FH expression was decreased (Figure S10A-F). We noted that LSH increased IKKα

expression in CNE1, HK1 and HNE3 cells (Figure 6C), whereas LSH knockdown in

C666-1 cells led to a significant decrease in the expression of IKKα (Figure 6D),

consistent with the notion that LSH overexpression leads to higher levels of α-KG and

citrate, which in turn upregulate IKKα levels. In addition, immunohistochemistry

analysis showed that LSH increased IKKα expression in the nucleus in CNE1 cells

after examining transplanted tumors from SCID mice (Figure 6E). Since IKKα can

function as a chromatin modifier (35-37), we examined association to IKKα putative

target genes.

We observed that IKKα was enriched at the promoters for ZO-1 and E-cadherin,

and its association greatly reduced in CNE1 and HK1 cells with LSH overexpression

(Figure 6F-G). In contrast, LSH overexpression enhanced the association of IKKα to

the Vimentin promoter (Figure 6H), consistent with a positive transcriptional activity




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of IKKα (33). Finally, we examined directly the role of TCA intermediates on IKKα

recruitment to the promoter regions of genes involved in EMT. While the addition of

either α-KG or citrate for three days reduced the enrichment of IKKα at the ZO-1 and

E-Cadherin promoter regions (Figure 6I-J), addition of these compounds increased

IKKα recruitment to the Vimentin promoter (Figure 6K). Taken together, our data

suggests that the chromatin regulator and transcriptional activator IKKα may be

involved in the regulation of EMT markers mediating the effect of LSH and TCA

intermediates.

Knockdown of FH promotes cancer progression

Lastly, we detected FH expression in a panel of NPC cells, and we selected

HNE3 cells for the next study (Figure S11). We generated stable FH knockdown in

HNE3 cells, and the knockdown of FH resulted in the decrease of the epithelial

marker ZO-1, whereas LSH protein level did not change (Figure 7A). Furthermore,

FH knockdown decreased significantly the relative intensity of E-cadherin staining

and increased the relative intensity of Vimentin staining in HNE3 cells (Figure 7B).

These findings indicate that FH is directly involved in the regulation of EMT genes

and suggest that LSH acts upstream of the FH pathway. FH knockdown also increased

colony numbers (Figure 7C) and resulted in an increased capacity for migration and

invasion (Figure 7D). Lastly, we examined directly the role of FH in regulation of

TCA cycle intermediates. We observed a decreased in succinate, fumarate and malate

concentrations after knockdown of FH in HNE3 cells (Figure 7E), whereas

knockdown of FH had no effect on α-KG or citrate (Figure S12). However, the ratio




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of α-KG/succinate and α-KG/fumarate increased after depletion of FH (Figure 7F). In

summary, LSH represses FH that in turn affects cancer progression through

de-regulation of TCA intermediates.

Based on our findings, we propose a model for LSH mediated signaling and

enhancement of NPC tumorigenesis (Figure 7G). In this model, LSH acts as a driver

of cancer progression involving EMT, invasion and migration. LSH directly targets

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

progression through the decrease of epithelial markers and the increase of

mesenchymal marker expression. The changes of epithelial marker gene expression

are medicated by IKKα that directly bind to these promoters.




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Discussion

In this study, we provide evidence that LSH plays a critical role in cancer

progression. Our findings suggest that LSH acts as a driver in NPC by promoting cell

growth, migration and invasion that are key characteristics of cancer progression.

Acquired epigenetic abnormalities participate together with chromatin alterations

in the early stages of carcinogenesis. Since the EBV methylome is unchanged

comparing NPC cell lines from southern China and the primary NPCs from southern

Europe (38), additional studies are necessary to address the role of EBV and its

products in epigenetics. Here, we report that LMP1, encoded by the EBV genome,

upregulated the expression level of LSH in NPCs.

LSH is critical for chromatin function and establishment of DNA methylation

(6,7,32,39). Reports show that LSH contributes to the malignant progression of

prostate cancer, melanoma, and head and neck cancer, etc (12,40,41); however, the

molecular mechanisms are not well understood. Here, we demonstrated that LSH was

linked to cancer progression of NPC. We found that FH expression was repressed in

the presence of LSH. FH may serve as a direct target of LSH function, since we found

LSH was associated with the fh promoter. LSH may repress the fh promoter

independent of DNA methylation. LSH can increase nucleosome density (32), cause

RNA polymerase II stalling (42,43) or promotes gene silencing via a G9a/GLP

complex during differentiation and early development (8). Here, we provide evidence

for an interaction of LSH with G9a, recruitment of G9a to the fh promoter in a LSH

dependent manner and subsequent chromatin modification leading to FH promoter




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repression, thus linking epigenetic regulation by LSH with suppression of the

emerging tumor suppressor gene FH (21,44,45).

FH is a key component of the TCA cycle and mediates the reversible conversion

of fumarate to malate. Although FH was discovered as a tumor suppressor (46), the

regulators of this important TCA cycle enzyme and the consequences of FH

downregulation remain unclear. We found that both fumarate and malate were

decreased in the serum of NPC patients, which is consistent with TCA intermediate

changes observed after inactivation of FH caused by genetic mutations (22,44,45).

Moreover, loss of FH leads to cellular senescence due to formation of succinicGSH, a

covalent adduct between fumarate and glutathione(47).

Distinct cancer tissue types may affect the selective accumulation of TCA

metabolite levels in different ways. Metabolite concentrations of colon and stomach

tissues are superimposed on a metabolic pathway map including the TCA cycle. For

example, while malate level decrease and citrate level increase in cancer compared to

normal tissue, the concentrations of cis-aconitate remain unchanged, comparing caner

to normal tissues (48). Also, organ-specific differences were observed in the

metabolite levels of the TCA cycle and other intermediates (48,49). What causes a

selective accumulation of initial metabolites of the TCA cycle during tumorigenesis

while other TCA intermediates show extremely low concentrations is poorly

understood.

Oncometabolite 2-Hydroxyglutarate (2-HG) is a competitive inhibitor of

α-KG-dependent dioxygenases in gliomas and haematological maligancies that carry




23

mutations of isocitrate dehydrogenase genes (IDH1 and IDH2) (50,51). However, we

did not find mutations of IDH1 and IDH2 genes and any accumulation of 2-HG in

NPC (data not shown). We found the ratio of α-KG to succinate and α-KG to fumarate

was increased in NPC patients. Interestingly, an elevated ratio of α-KG to succinate

has been shown to influence the pluripotency state in embryonic stem cells via the

alteration of multiple chromatin modifications (52). It will be interesting to determine

which TCA intermediates alter chromatin, including histone modifications and DNA

methylations in NPC and how these epigenetic changes contribute to cancer

progression.

EMT is not required for metastasis but induces chemoresistance in cancer, and

the disturbance of the epithelial balance is caused by altering several layers of

regulation including epigenetic regulation (18,53-55). The functional interactions

between EMT-inducing transcription factors and the modulators of chromatin

configuration give crucial insights into the underlying mechanism of cancer

progression (18). Metabolic competition can drive cancer progression (56), and this

competition is due to the disturbed balance of TCA intermediates that could trigger

EMT. The reprogramming of gene expression during EMT is initiated and controlled

by signaling pathways that respond to extracellular cues and lead to metabolic

reprogramming. Here we demonstrated that overexpression of LSH is linked to EMT

by increasing migration and invasion ability in NPC. It also indicated that EMT

induction by LMP1 is mediated by LSH. We found that the other many key regulators,

such as TWIST and Snail, that induce EMT is affected by LMP1 (Figure S13).




24

Furthermore, LSH overexpression, as well as de-regulation of TCA intermediates,

leads to IKKα recruitment to the promoters of EMT-related genes. In this way, LSH

induces a cascade of epigenetic and metabolic changes that result in further epigenetic

regulations via IKKα and EMT.

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 a reduction of succinate, fumarate and malate, also

increases in the ratio of α-KG to fumarate. TCA intermediates including α-KG and

citrate decrease E-cadherin and ZO-1 expression and increase Vimentin. The

changes of EMT marker gene expression are controlled by IKKα that binds directly to

these promoters. The pathway leads to EMT, promoting migration, invasion and

cancer progression (Figure 7G). Repression of LSH and its downstream effects may

serve as potential target for the development of novel therapeutic approaches.




25

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Figure Legends

Figure 1 LSH is highly expressed in NPC.

(A) Immunohistochemical analysis was used to determine LSH protein level

(LSH) in an NPC tissue array from NPC patients (upper panel). The level of EBER

was analyzed by in situ hybridization in NPC tissues (lower panel). The level of

LMP1 was further analyzed in NPC tissues. (B) Relative expression of LSH in NP

and NPC tissues with different levels of EBER. n, number of analyzed samples, * p

<0.05, ** p <0.01. (C) Elevated expression of LSH is significantly associated with

late stages of NPC. (D) Western analysis for detection of LSH protein in HK1 and

C666-1 cells. (E) Western analysis for detection of LSH protein in CNE1-LMP1 and

HNE2-LMP1 cells and the parental cells.

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

with overexpression of LSH expression. LSH protein levels are shown in the inlet

figure. (C) Plate colony formation assay was measured in cells as indicated. (D) The

migrating colony number is shown as bar graph (mean ± SD from 3 separate

experiments), 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 for E-cadherin and

Vimentin in CNE1 cells stably expressing a control vector or LSH using High Content

Screening and High Content Analysis. Relative intensity of E-cadherin (H) and




30

Vimentin (I) is shown. (J) Nude mice is shown after injection of HK1 cells stably

expressing control vector or LSH expression plasmids, and tumor volume was

monitored at indicated time points, and (K) tumor weight was recorded. (L)

CNE1-MOCK and CNE1-LSH cells were injected into the tail vein of SCID mice.

Animals (n=6 for each group) were euthanized and the development of lung

metastases was assessed macroscopically or by microscope in paraffin-embedded

sections stained with H&E. * p <0.05, ** p <0.01, *** p <0.001.

Figure 3 Knockdown of LSH inhibits cancer progression in vitro and in vivo.

(A) The MTT assay was performed to assess cell viability in C666-1 NPCs that

were stably transfected with two distinct LSH shRNAs expression vectors (siLSH#1

and siLSH#2) and control cells (siCTRL). LSH protein levels as detected by Western

analysis are shown in the figure within. A representative experiment is shown for the

migration and invasion assay after stable knockdown of LSH in C666-1 cells (B), and

the colony number of migratory and invasive cells is shown (C). (D) Relative

intensity of E-cadherin and Vimentin is shown. (E) A xenograft model of tumor

growth was established in nude mice to evaluate the ability of C666-1 cells with a

stable knockdown of LSH to form tumors within 27 days and tumor volume was

monitored. (F) Immunohistochemical analysis for detection of of E-cadherin and

β-catenin in tumor samples from nude mice. * p <0.05, ** p <0.01.

Figure 4 LSH represses FH expression through the recruitment of G9a to the fh




31

promoter.

(A) RT–PCR analysis for detection of FH mRNA using total RNA derived from

HK1-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

for detection of LSH binding to the fh promoter in CNE1 and HK1 cells. (D) C666-1

Cells were stained with co-localization of LSH and G9a. (E) Co-immunoprecipitation

of LSH and G9a in lysates 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 were immunoprecipitated (IP) with anti-Flag M2 agarose and were

immunoblotted to detect LSH or G9a. (G) ChIP analysis for detection of G9a binding

to the fh promoter 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 detection of 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, *** p <0.001.

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 of NPC

patients (Tumor) and healthy controls (Normal). Growth in plate colony formation

was measured in HK1 (B) and HNE3 (C) cells after the cells were treated with TCA




32

intermediates as indicated. (D) The colony numbers of migration and invasion cells

are shown in CNE1 cells after addition of citrate and α-KG. HNE3 cells (E) were

treated with citrate (Left panel) and α-KG (Right panel) as indicated and Western

analysis performed to assess FH and EMT proteins. (F) The effect of α-KG addition

on migration and invasion ability was measured after depletion of LSH in C666-1

cells. * p <0.05, ** p <0.01, *** p <0.001.

Figure 6 IKKα is a critical regulator of LSH and oncometabolites α-KG and

citrate induced cancer progression.

CNE1 cells (A) and HNE3 (B) cells were treated with citrate (Left) and α-KG

(Right) and the expression of IKKα and LSH proteins was analyzed. (C) Western

analysis was assessed IKKα protein levels in cells as indicated. (D) IKKα expression

was analyzed in C666-1 cells after stable depletion of LSH. (E) Immunohistochemical

analysis was used to analyze the IKKα expression in transplanted tumor tissues after

injection of HK1-LSH cells. ChIP analysis for detection of IKKα binding to the ZO-1

(F), E-Cadherin (G) and Vimentin (H) promoters in CNE1 and HK1 cells in the

presence of LSH. ChIP analysis for detection of IKKα binding to the ZO-1 (I),

E-Cadherin (J) and Vimentin (K) promoters in CNE1 cells after addition of α-KG and

citrate respectively. * p <0.05, ** p <0.01, *** p <0.001.

Figure 7. Kockdown of FH promotes cancer progression and working model.

(A) HNE3 cells were stably transfected with two distinct LSH shRNAs




33

expression vectors (siFH#1 and siFH#2) and control cells (siCTRL) respectively.

Western analysis detected the levels of FH, ZO-1 and LSH protein. (B) Growth in the

plate colony formation assay 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 representative experiment is shown for the migration and invasion assay after

stable knockdown of FH in HNE3 cells. (E) GC-MS to measure the concentration of

indicated TCA metabolites after stable knockdown of FH in HNE3 cells. (F) Ratio of

α-KG to succinate and ratio of α-KG to fumarate are shown in HNE3 cells that stably

knockdown of FH. * p <0.05, ** p <0.01, *** p <0.001. (G) The schematic model of

LSH in cancer progression.




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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